TW201643241A - Microfluidic cell culture - Google Patents

Abstract

Systems, methods and kits are described for culturing one or more biological cells in a microfluidic device, including provision of nutrients and gaseous components configured to enhance cell growth, viability, portability, or any combination thereof. In some embodiments, culturing a single cell may produce a clonal population in the microfluidic device.

Description

微流體細胞培養 Microfluidic cell culture

本申請案係根據35 U.S.C.119(e)主張2015年4月22日申請之美國臨時申請案第62/151,325號(其之全部內容以引用的方式併入本文中)之權利的一非臨時申請案。 The present application is based on a non-provisional application of the benefit of US Provisional Application Serial No. 62/151,325, the entire disclosure of which is incorporated herein by reference. case.

在生物科學及相關領域中，培養一或若干細胞可為有用的。本發明之一些實施例包含用於在一微流體器件中培養一細胞或細胞群組之裝置及程序。 It may be useful to culture one or several cells in the biological sciences and related fields. Some embodiments of the invention comprise apparatus and procedures for culturing a cell or group of cells in a microfluidic device.

在一態樣中，提供一種用於培養一或多個生物細胞之微流體器件，其包含：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包含一隔離區域及一連接區域，該隔離區域與該連接區域流體地連接且該連接區域包含至該流動區域之一近端開口，其中該至少一生長室進一步包含經調節以支援該微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。在一些實施例中，該微流體器件之該隔離區域可經構形以含有一第二流體培養基，且其中當該流動區域及該至少一生長室實質上分別填充有該第一流體培養基及該第二流體培養基時，該第二流體培養基之組分可擴散至該第一流體培養基中及/或該第一流體培養基之組分可擴散至該第二流體培養基中，且該第一培養基可實質上不流動至該隔離區域 中。在一些實施例中，該微流體器件可進一步包含具有該流動區域之至少一部分之一微流體通道，且其中該至少一生長室之該連接區域可直接通至該微流體通道中。 In one aspect, a microfluidic device for culturing one or more biological cells is provided, comprising: a flow region configured to flow with one of a first fluid medium; and at least one growth chamber, An isolation region and a connection region are fluidly coupled to the connection region and the connection region includes a proximal opening to the flow region, wherein the at least one growth chamber further includes an adjustment to support the microfluid At least one surface of cell growth, viability, portability, or any combination thereof, within the device. In some embodiments, the isolation region of the microfluidic device can be configured to contain a second fluid medium, and wherein the flow region and the at least one growth chamber are substantially filled with the first fluid medium and In the second fluid medium, components of the second fluid medium may diffuse into the first fluid medium and/or components of the first fluid medium may diffuse into the second fluid medium, and the first medium may be Essentially does not flow to the isolated area in. In some embodiments, the microfluidic device can further comprise a microfluidic channel having at least a portion of the flow region, and wherein the connecting region of the at least one growth chamber can pass directly into the microfluidic channel.

在一些實施例中，可使用支援該微流體器件內之細胞可移植性之一或多個試劑來調節該至少一調節表面。在一些實施例中，可使用包含伸烷基醚部分之一聚合物來調節該至少一調節表面。在其他實施例中，可使用包含糖類部分之一聚合物來調節該至少一調節表面。在一些實施例中，包含糖類部分之該聚合物可包含聚葡萄糖。在其他實施例中，可使用包含胺基酸部分之一聚合物來調節該至少一調節表面。在一些實施例中，該聚合物可為牛血清白蛋白(BSA)或去氧核糖核酸酶1(DNase 1)。在其他實施例中，可使用包含羧酸部分、磺酸部分、核酸部分或膦酸部分之一聚合物來調節該微流體器件之該至少一調節表面。在一些實施例中，可使用包含羧酸部分、磺酸部分、核酸部分或膦酸部分之一聚合物來調節該微流體器件之該至少一調節表面。 In some embodiments, the at least one conditioning surface can be adjusted using one or more reagents that support cell portability within the microfluidic device. In some embodiments, the at least one conditioning surface can be adjusted using a polymer comprising one of the alkylene ether moieties. In other embodiments, the at least one conditioning surface can be adjusted using a polymer comprising one of the carbohydrate moieties. In some embodiments, the polymer comprising a carbohydrate moiety can comprise polydextrose. In other embodiments, the polymer comprising one of the amino acid moieties can be used to adjust the at least one conditioning surface. In some embodiments, the polymer can be bovine serum albumin (BSA) or deoxyribonuclease 1 (DNase 1). In other embodiments, the at least one conditioning surface of the microfluidic device can be adjusted using a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety, or a phosphonic acid moiety. In some embodiments, the at least one conditioning surface of the microfluidic device can be adjusted using a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety, or a phosphonic acid moiety.

在該微流體器件之各種實施例中，該至少一調節表面包含共價鍵聯至該微流體器件之一表面之一鍵聯基團，且該鍵聯基團可鍵聯至經構形以支援該微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之一部分。在一些實施例中，該鍵聯基團可為矽烷氧基鍵聯基團。在其他實施例中，該鍵聯基團可為膦酸酯鍵聯基團。在各種實施例中，該至少一調節表面可包含烷基或氟烷基部分。在一些實施例中，該等氟烷基部分可為全氟烷基部分。在一些實施例中，該等烷基或氟烷基部分可具有大於10個碳之一主鏈長度。該等烷基或氟烷基部分可具有一直鏈結構。在該微流體器件之各種實施例中，該至少一調節表面之該鍵聯基團可直接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在其他實施例中，該鍵聯 基團可間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在一些實施例中，該鍵聯基團可經由一鍵聯子而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在一些實施例中，該鍵聯子可包含伸***基部分。在其他實施例中，該鍵聯子可包含一或多個伸芳基部分。在一些實施例中，該至少一調節表面可包含糖類部分。在其他實施例中，該至少一調節表面可包含伸烷基醚部分。在其他實施例中，該至少一調節表面可包含胺基酸部分。替代地，該至少一調節表面可包含兩性離子。在進一步實施例中，該至少一調節表面可包含膦酸部分或羧酸部分。在其他實施例中，該至少一調節表面包含胺基或胍部分。在一些其他實施例中，該至少一調節表面可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷基化胺、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。 In various embodiments of the microfluidic device, the at least one conditioning surface comprises a covalently bonded to one of the surface of the microfluidic device, and the linking group can be bonded to the conformation Supporting part of any combination of cell growth, viability, portability, or the like within the microfluidic device. In some embodiments, the linking group can be a decyloxy linking group. In other embodiments, the linking group can be a phosphonate linkage group. In various embodiments, the at least one conditioning surface can comprise an alkyl or fluoroalkyl moiety. In some embodiments, the fluoroalkyl moiety can be a perfluoroalkyl moiety. In some embodiments, the alkyl or fluoroalkyl moiety can have a backbone length of greater than 10 carbons. The alkyl or fluoroalkyl moiety can have a straight chain structure. In various embodiments of the microfluidic device, the linking group of the at least one conditioning surface can be directly bonded to the moiety configured to support cell growth, viability, portability, or the like, any combination thereof. . In other embodiments, the linkage The group can be indirectly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linking group can be indirectly linked via a linkage to the portion configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linker can comprise a triazolyl moiety. In other embodiments, the linker can comprise one or more extended aryl moieties. In some embodiments, the at least one conditioning surface can comprise a carbohydrate moiety. In other embodiments, the at least one conditioning surface can comprise an alkyl ether moiety. In other embodiments, the at least one conditioning surface can comprise an amino acid moiety. Alternatively, the at least one conditioning surface can comprise zwitterions. In a further embodiment, the at least one conditioning surface can comprise a phosphonic acid moiety or a carboxylic acid moiety. In other embodiments, the at least one conditioning surface comprises an amine or anthracene moiety. In some other embodiments, the at least one conditioning surface can comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or polysaccharide (which can include, but is not limited to, polydextrose); an alcohol (which includes, but is not limited to, propargyl alcohol); a polyol comprising, but not limited to, a polyvinyl alcohol; an alkyl ether comprising, but not limited to, polyethylene glycol; a polyelectrolyte (which comprises ( But not limited to) polyacrylic acid or polyvinylphosphonic acid); amine groups (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amine groups, phosphonium salts, and containing an unaromatic nitrogen ring) a heterocyclic group of an atom such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid comprising, but not limited to, a propiolic acid (which can provide a carboxylate anion surface); a phosphonic acid comprising But not limited to) ethynylphosphonic acid (which provides a phosphonate anion surface); sulfonate anion; carboxybetaine; sulfobetaine; amine sulfonic acid; or amino acid.

在該微流體器件之各種實施例中，該微流體器件之該至少一調節表面可包含至少一細胞黏著阻斷分子。在一些實施例中，該至少一細胞黏著阻斷分子可破壞肌動蛋白纖維形成，阻斷整合素受體，或減弱細胞與DNA污損表面之結合。在一些實施例中，該至少一細胞黏著阻斷分子可為一含RGD之肽。在其他實施例中，該至少一細胞黏著阻斷分子可為細胞鬆弛素B、一整合素抗體、纖連蛋白(其可包含一小分 子或一DNase 1蛋白)之抑制劑。在其他實施例中，該至少一細胞黏著阻斷分子可包含多於一種類型之細胞黏著阻斷分子之一組合。 In various embodiments of the microfluidic device, the at least one conditioning surface of the microfluidic device can comprise at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule disrupts actin fiber formation, blocks integrin receptors, or attenuates binding of cells to DNA stained surfaces. In some embodiments, the at least one cell adhesion blocking molecule can be an RGD-containing peptide. In other embodiments, the at least one cell adhesion blocking molecule can be cytochalasin B, an integrin antibody, fibronectin (which can comprise a small fraction Inhibitor of a sub- or a DNase 1 protein). In other embodiments, the at least one cell adhesion blocking molecule can comprise a combination of one of more than one type of cell adhesion blocking molecule.

在該微流體器件之各種實施例中，該微流體器件之該至少一調節表面可包含一可裂解部分。在一些實施例中，該可裂解部分可經構形以容許破壞該調節表面，藉此促進該一或多個生物細胞在培養之後之可移植性。 In various embodiments of the microfluidic device, the at least one conditioning surface of the microfluidic device can comprise a cleavable portion. In some embodiments, the cleavable moiety can be configured to permit disruption of the modulating surface, thereby facilitating portability of the one or more biological cells after culture.

在該微流體器件之各種實施例中，該微流體器件之該至少一調節表面可包含哺乳動物血清之一或多個組分。哺乳動物血清之該一或多個組分可包含B27®補充劑、胎牛血清(FBS)或小牛血清(FCS)。 In various embodiments of the microfluidic device, the at least one conditioning surface of the microfluidic device can comprise one or more components of mammalian serum. The one or more components of the mammalian serum may comprise B27® supplements, fetal bovine serum (FBS) or calf serum (FCS).

在該微流體器件之各種實施例中，該微流體器件可進一步包含具有一介電泳(DEP)構形之一基板。在一些實施例中，具有一DEP構形之該基板可經構形以將一或多個生物細胞引入至該生長室中或將該一或多個生物細胞移出該生長室。可光學地致動該DEP構形。 In various embodiments of the microfluidic device, the microfluidic device can further comprise a substrate having a dielectrophoresis (DEP) configuration. In some embodiments, the substrate having a DEP configuration can be configured to introduce one or more biological cells into the growth chamber or to move the one or more biological cells out of the growth chamber. The DEP configuration can be optically actuated.

在該微流體器件之各種實施例中，該微流體器件之該至少一調節表面可經構形以穩定於至少約30℃之一溫度處。 In various embodiments of the microfluidic device, the at least one conditioning surface of the microfluidic device can be configured to stabilize at a temperature of at least about 30 °C.

在該微流體器件之各種實施例中，該微流體器件之該至少一生長室之該隔離區域可具有足以支援細胞擴增至約100個細胞之一範圍之尺寸。在一些實施例中，可在該至少一生長室中維持不超過1×10²個生物細胞，且該至少一生長室之體積可小於或等於約2×10⁶立方微米。在其他實施例中，可在該至少一生長室中維持不超過1×10²個生物細胞，且該至少一生長室之體積可小於或等於約1×10⁷立方微米。 In various embodiments of the microfluidic device, the isolated region of the at least one growth chamber of the microfluidic device can have a size sufficient to support cell expansion to a range of about 100 cells. In some embodiments, can be maintained is not more than 1 × 10 ² cells bio the at least one growth chamber and the growth chamber of a volume of at least be less than or equal to about 2 × 10 ⁶ cubic microns. In other embodiments, can be maintained is not more than 1 × 10 ² cells bio the at least one growth chamber and the growth chamber of a volume of at least be less than or equal to about 1 × 10 ⁷ cubic microns.

在該微流體器件之各種實施例中，該器件可進一步包含：至少一入口，其經構形以將該第一流體培養基或該第二流體培養基輸入至該流動區域中；及至少一出口，其經構形以當該第一流體培養基自該流動區域退出時接收該第一流體培養基。在該微流體器件之各種實施例中，該微流體器件可進一步包含在該至少一生長室或其之該隔離區 域上方之一可變形蓋區域，藉此按壓該可變形蓋區域施加足以使該生物細胞自該隔離區域輸出至該流動區域之一力。在該微流體器件之各種實施例中，該微流體器件可包含一蓋，其中該蓋之至少一部分可為可透氣的，藉此將一氣體分子源提供至定位於該微流體器件中之一流體培養基。該蓋之該可透氣部分可定位於該至少一生長室上方。在其他實施例中，該蓋之該可透氣部分可定位於該流動區域上方。在其他實施例中，該至少一生長室可包含複數個生長室。 In various embodiments of the microfluidic device, the device can further comprise: at least one inlet configured to input the first fluid medium or the second fluid medium into the flow region; and at least one outlet, It is configured to receive the first fluid medium as the first fluid medium exits from the flow region. In various embodiments of the microfluidic device, the microfluidic device can be further included in the at least one growth chamber or the isolation region thereof One of the top regions of the domain deformable cover region whereby pressing the deformable cover region applies a force sufficient to cause the biological cell to output from the isolated region to the flow region. In various embodiments of the microfluidic device, the microfluidic device can include a cover, wherein at least a portion of the cover can be gas permeable, thereby providing a source of gas molecules to one of the microfluidic devices. Fluid medium. The gas permeable portion of the lid can be positioned over the at least one growth chamber. In other embodiments, the gas permeable portion of the cover can be positioned over the flow area. In other embodiments, the at least one growth chamber can comprise a plurality of growth chambers.

在各種實施例中，該一或多個生物細胞可包含複數個生物細胞。在該微流體器件之各種實施例中，該至少一生長室可包含經調節以支援一哺乳動物細胞之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。在其他實施例中，該至少一生長室可包含經調節以支援一免疫細胞之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。在其他實施例中，該免疫細胞可為一淋巴細胞或白血球。在一些其他實施例中，該免疫細胞可為一B細胞、一T細胞、NK細胞、一巨噬細胞或一樹突細胞。 In various embodiments, the one or more biological cells can comprise a plurality of biological cells. In various embodiments of the microfluidic device, the at least one growth chamber can comprise at least one surface conditioned to support cell growth, viability, portability, or the like, of any combination of mammalian cells. In other embodiments, the at least one growth chamber can comprise at least one surface that is adjusted to support cell growth, viability, portability, or the like, of any combination of immune cells. In other embodiments, the immune cell can be a lymphocyte or a white blood cell. In some other embodiments, the immune cell can be a B cell, a T cell, an NK cell, a macrophage, or a dendritic cell.

在該微流體器件之各種實施例中，該至少一生長室可包含經調節以支援一貼附細胞之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 In various embodiments of the microfluidic device, the at least one growth chamber can comprise at least one surface that is adjusted to support cell growth, viability, portability, or the like of any combination of adherent cells.

在該微流體器件之各種實施例中，該至少一生長室可包含經調節以支援一融合瘤細胞之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 In various embodiments of the microfluidic device, the at least one growth chamber can comprise at least one surface conditioned to support cell growth, viability, portability, or the like of any combination of cell growth, viability, or the like.

在該微流體器件之各種實施例中，該至少一生長室可包含經調節以支援一單一細胞及生物細胞之一對應選殖群落之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 In various embodiments of the microfluidic device, the at least one growth chamber can comprise any combination of cell growth, viability, portability, or the like adjusted to support a single cell and one of the biological cells corresponding to the selected colony. At least one surface.

在另一態樣中，提供一種用於在一微流體器件上培養一或多個生物細胞之系統，該系統包含一微流體器件，其具有：一流動區域， 其經構形以含有一第一流體培養基之一流動；及至少一生長室，其中該生長室具有經調節以支援該微流體器件中之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。該至少一生長室可包含一隔離區域及一連接區域，該隔離區域與該連接區域流體地連接且該連接區域具有至該流動區域之一近端開口。在一些實施例中，該微流體器件之該隔離區域可經構形以含有一第二流體培養基，且當該流動區域及該至少一生長室實質上分別填充有該第一流體培養基及該第二流體培養基時，該第二流體培養基之組分可擴散至該第一流體培養基中及/或該第一流體培養基之組分可擴散至該第二流體培養基中，且該第一培養基可實質上不流動至該隔離區域中。在一些實施例中，該微流體器件可進一步包含一微流體通道，其包含該流動區域之至少一部分，且其中該至少一生長室之該連接區域可直接通至該微流體通道中。該微流體器件可為如本文中所描述之任何微流體器件，其具有元件之任何者之任何組合。 In another aspect, a system for culturing one or more biological cells on a microfluidic device is provided, the system comprising a microfluidic device having: a flow region, Constructed to contain one of a first fluid medium flow; and at least one growth chamber, wherein the growth chamber has any adjustments to support cell growth, viability, portability, or the like in the microfluidic device Combining at least one surface. The at least one growth chamber can include an isolation region and a connection region, the isolation region being fluidly coupled to the connection region and having a proximal opening to one of the flow regions. In some embodiments, the isolation region of the microfluidic device can be configured to contain a second fluid medium, and the flow region and the at least one growth chamber are substantially filled with the first fluid medium and the first In the case of a two-fluid medium, components of the second fluid medium may diffuse into the first fluid medium and/or components of the first fluid medium may diffuse into the second fluid medium, and the first medium may be substantially It does not flow into the isolated area. In some embodiments, the microfluidic device can further comprise a microfluidic channel comprising at least a portion of the flow region, and wherein the connection region of the at least one growth chamber can pass directly into the microfluidic channel. The microfluidic device can be any of the microfluidic devices as described herein, with any combination of any of the elements.

在該系統之各種實施例中，該系統可進一步包含經構形以至少灌注該第一流體培養基之一流量控制器。該控制器經構形以非連續地灌注該至少第一流體培養基。 In various embodiments of the system, the system can further comprise a flow controller configured to infuse at least one of the first fluid medium. The controller is configured to non-continuously perfuse the at least first fluid medium.

在該系統之各種實施例中，該系統之該微流體器件可進一步包含具有一介電泳(DEP)構形之一基板，其經構形以將一或多個生物細胞引入至該生長室中或將該一或多個生物細胞移出該生長室。可光學地致動該DEP構形。 In various embodiments of the system, the microfluidic device of the system can further comprise a substrate having a dielectrophoresis (DEP) configuration configured to introduce one or more biological cells into the growth chamber or The one or more biological cells are removed from the growth chamber. The DEP configuration can be optically actuated.

在該系統之各種實施例中，該系統可進一步包含經構形以含有該第一流體培養基之一儲液器，其中該儲液器流體地連接至該微流體器件。該儲液器可經構形以與能夠使該第一流體培養基飽含溶解氣體分子之一氣體環境相通。 In various embodiments of the system, the system can further comprise a reservoir configured to contain the first fluid medium, wherein the reservoir is fluidly coupled to the microfluidic device. The reservoir can be configured to communicate with a gaseous environment that is capable of saturating the first fluid medium with a dissolved gas molecule.

在該系統之各種實施例中，該系統可進一步包含連接至該微流 體器件之至少一入口之一感測器，其中該感測器可經構形以偵測該第一流體培養基之一pH。在該系統之各種實施例中，該系統可進一步包含連接至至少一出口之一感測器，其中該感測器經構形以在該第一流體培養基離開該微流體器件時偵測該第一流體培養基之一pH。在一些實施例中，該感測器可為一光學感測器。 In various embodiments of the system, the system can further include connecting to the microflow One of the at least one inlet of the body device, wherein the sensor is configurable to detect a pH of the first fluid medium. In various embodiments of the system, the system can further include a sensor coupled to the at least one outlet, wherein the sensor is configured to detect the first fluid medium as it exits the microfluidic device One of a fluid medium pH. In some embodiments, the sensor can be an optical sensor.

在該系統之各種實施例中，該系統可進一步包含經構形以擷取該至少一生長室及含於其內之任何生物細胞之一影像之一偵測器。在一些實施例中，該一或多個生物細胞可包含一或多個哺乳動物細胞。在其他實施例中，該一或多個生物細胞可包含一或多個融合瘤細胞。在其他實施例中，該一或多個生物細胞可包含一或多個淋巴細胞或白血球細胞。替代地，該一或多個生物細胞可包含一或多個貼附細胞。 In various embodiments of the system, the system can further include a detector configured to capture the at least one growth chamber and one of the images of any of the biological cells contained therein. In some embodiments, the one or more biological cells can comprise one or more mammalian cells. In other embodiments, the one or more biological cells can comprise one or more fusion tumor cells. In other embodiments, the one or more biological cells can comprise one or more lymphocytes or white blood cells. Alternatively, the one or more biological cells may comprise one or more adherent cells.

在該系統之各種實施例中，該生長室中之該一或多個生物細胞可為一單一細胞且該群落可為生物細胞之一選殖群落。 In various embodiments of the system, the one or more biological cells in the growth chamber can be a single cell and the colony can be a colony of one of the biological cells.

在另一態樣中，提供一種組合物，其包含：一基板，其具有一介電泳(DEP)構形及一表面；及一調節表面，其共價鍵聯至該基板之該表面之氧化物部分。該組合物可具有式1或式2之一結構，且可具有式1或式2之元素之任何值，如本文中所界定： In another aspect, a composition is provided comprising: a substrate having a dielectrophoresis (DEP) configuration and a surface; and an conditioning surface covalently bonded to the oxide of the surface of the substrate section. The composition may have a structure of Formula 1 or Formula 2 and may have any value for the element of Formula 1 or Formula 2, as defined herein:

在該組合物之一些實施例中，該調節表面可包含共價鍵聯至該表面之該等氧化物部分之一鍵聯基團，且該鍵聯基團可鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之一部分。在 一些實施例中，該鍵聯基團可為矽烷氧基鍵聯基團。在其他實施例中，該鍵聯基團可為膦酸酯基。在一些實施例中，該鍵聯基團可直接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在一些實施例中，該鍵聯基團可間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在一些實施例中，該鍵聯基團可經由連接至一鍵聯子而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在一些實施例中，該鍵聯基團可經由連接至一鍵聯子之一第一端而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯子可進一步包含一直鏈部分，其中該直鏈部分之一主鏈包括選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之任何組合之1個至200個非氫原子。在一些實施例中，該鍵聯子可進一步包含伸***基部分。在一些實施例中，該伸***基部分可中斷該鍵聯子之該直鏈部分或可連接至該鍵聯子之該直鏈部分之一第二端。在其他實施例中，該直鏈部分之該主鏈可包含伸芳基部分。 In some embodiments of the composition, the conditioning surface can comprise a linkage group covalently bonded to one of the oxide moieties of the surface, and the linkage group can be bonded to the conformation to support Part of any combination of cell growth, viability, portability, or the like. in In some embodiments, the linking group can be a decyloxy linking group. In other embodiments, the linking group can be a phosphonate group. In some embodiments, the linking group can be directly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linking group can be indirectly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linking group can be indirectly linked to the portion configured to support cell growth, viability, portability, or the like, via attachment to a linkage. In some embodiments, the linking group can be indirectly linked to a conformation to support cell growth, viability, portability, or the like, via attachment to one of the first ends of a linkage. This part. The linker may further comprise a straight chain moiety, wherein one of the linear chain backbones comprises from 1 to 200 non-selective groups selected from the group consisting of a ruthenium atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. A hydrogen atom. In some embodiments, the linker can further comprise a triazolyl moiety. In some embodiments, the triazole moiety can interrupt the linear portion of the bond or can be attached to the second end of one of the linear portions of the bond. In other embodiments, the backbone of the linear moiety can comprise an extended aryl moiety.

在各種實施例中，經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分包括烷基部分、氟烷基部分、單糖或多糖、乙醇部分、多元醇部分、伸烷基醚部分、聚電解質部分、胺基部分、羧酸部分、膦酸部分、磺酸鹽陰離子部分、羧基甜菜鹼部分、磺基甜菜鹼部分、胺磺酸部分或胺基酸部分。在一些實施例中，該至少一調節表面可包含胺基酸、烷基部分、全氟烷基部分、聚葡萄糖部分及/或伸烷基醚部分。在一些實施例中，該至少一調節表面可包含烷基或全氟烷基部分。在一些實施例中，該等烷基或全氟烷基部分具有大於10個碳之一主鏈長度。在各種實施例中，該調節表面可進一步包含一或多個可裂解部分。該可裂解部分可經構形以容許破壞該調節表面，藉此促進該一或多個生物細胞在培養之後之可移植性。 In various embodiments, the moiety configured to support cell growth, viability, portability, or any combination thereof, etc., includes an alkyl moiety, a fluoroalkyl moiety, a monosaccharide or polysaccharide, an ethanol moiety, a polyol moiety. And an alkyl ether moiety, a polyelectrolyte moiety, an amine moiety, a carboxylic acid moiety, a phosphonic acid moiety, a sulfonate anion moiety, a carboxybetaine moiety, a sulfobetaine moiety, an aminesulfonic acid moiety or an amino acid moiety. In some embodiments, the at least one conditioning surface can comprise an amino acid, an alkyl moiety, a perfluoroalkyl moiety, a polydextrose moiety, and/or an alkylene ether moiety. In some embodiments, the at least one conditioning surface can comprise an alkyl or perfluoroalkyl moiety. In some embodiments, the alkyl or perfluoroalkyl moiety has a backbone length of greater than 10 carbons. In various embodiments, the conditioning surface can further comprise one or more cleavable portions. The cleavable moiety can be configured to permit disruption of the modulating surface, thereby facilitating portability of the one or more biological cells after culture.

在另一態樣中，提供一種在一微流體器件中培養至少一生物細胞之方法，該微流體器件具有一流動區域(其經構形以含有一第一流體培養基之一流動)及至少一生長室，該方法包括下列步驟：將該至少一生物細胞引入至該至少一生長室中，其中該至少一生長室經構形以具有經調節以支援細胞生長、存活性、可移植性或其等之任何組合之至少一表面；及在至少足以使該至少一生物細胞擴增而產生生物細胞之一群落的一長時間週期內培育該至少一生物細胞。該至少一生長室可包含一隔離區域及一連接區域，該隔離區域與該連接區域流體地連接且該連接區域具有至該流動區域之一近端開口。在一些實施例中，該微流體器件之該隔離區域可經構形以含有一第二流體培養基，且其中當該流動區域及該至少一生長室實質上分別填充有該第一流體培養基及該第二流體培養基時，該第二流體培養基之組分可擴散至該第一流體培養基中及/或該第一流體培養基之組分可擴散至該第二流體培養基中，且該第一培養基可實質上不流動至該隔離區域中。在一些實施例中，該微流體器件可進一步包含具有該流動區域之至少一部分之一微流體通道，且其中該至少一生長室之該連接區域可直接通至該微流體通道中。該微流體器件可為如本文中所描述之任何微流體器件，其具有元件之任何者之任何組合。 In another aspect, a method of culturing at least one biological cell in a microfluidic device having a flow region configured to contain a flow of a first fluid medium and at least one a growth chamber, the method comprising the steps of: introducing the at least one biological cell into the at least one growth chamber, wherein the at least one growth chamber is configured to have a modulation to support cell growth, viability, portability or At least one surface of any combination; and cultivating the at least one biological cell for a long period of time at least sufficient to cause the at least one biological cell to expand to produce a colony of biological cells. The at least one growth chamber can include an isolation region and a connection region, the isolation region being fluidly coupled to the connection region and having a proximal opening to one of the flow regions. In some embodiments, the isolation region of the microfluidic device can be configured to contain a second fluid medium, and wherein the flow region and the at least one growth chamber are substantially filled with the first fluid medium and In the second fluid medium, components of the second fluid medium may diffuse into the first fluid medium and/or components of the first fluid medium may diffuse into the second fluid medium, and the first medium may be It does not substantially flow into the isolated area. In some embodiments, the microfluidic device can further comprise a microfluidic channel having at least a portion of the flow region, and wherein the connecting region of the at least one growth chamber can pass directly into the microfluidic channel. The microfluidic device can be any of the microfluidic devices as described herein, with any combination of any of the elements.

在該方法之一些實施例中，該至少一調節表面可包含共價鍵聯至該表面之一鍵聯基團，且其中該鍵聯基團進一步鍵聯至經構形以支援該微流體器件內之該一或多個生物細胞之細胞生長、存活性、可移植性或其等之任何組合之一部分。在一些其他實施例中，經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質 (其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷基化胺、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。在一些實施例中，該至少一調節表面可包含烷基或全氟烷基部分。在其他實施例中，該至少一調節表面可包含伸烷基醚部分或聚葡萄糖部分。 In some embodiments of the method, the at least one conditioning surface can comprise a covalently bonded to one of the surface linking groups, and wherein the linking group is further bonded to the conformation to support the microfluidic device Part of any combination of cell growth, viability, portability, or the like of the one or more biological cells. In some other embodiments, the portion configured to support cell growth, viability, portability, or any combination thereof, etc., can comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group) moiety; a sugar or polysaccharide (which may include, but is not limited to, polydextrose); an alcohol (including but not limited to, propargyl alcohol); a polyol comprising, but not limited to, a polyvinyl alcohol; an alkyl ether, It includes, but is not limited to, polyethylene glycol; polyelectrolyte (which includes, but is not limited to, polyacrylic acid or polyvinylphosphonic acid); amine groups (which include derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amine groups, phosphonium salts, and one containing no a heterocyclic group of an aromatized nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid comprising, but not limited to, a propiolic acid (which provides a carboxylate anion surface); a phosphonic acid , which includes, but is not limited to, ethynylphosphonic acid (which provides a phosphonate anion surface); a sulfonic acid anion; a carboxybetaine; a sulfobetaine; an amine sulfonic acid; or an amino acid. In some embodiments, the at least one conditioning surface can comprise an alkyl or perfluoroalkyl moiety. In other embodiments, the at least one conditioning surface can comprise an alkyl ether moiety or a polydextrose moiety.

在該方法之一些實施例中，該方法可包含下列步驟：調節該至少一生長室之至少一表面。在一些實施例中，調節可包含：使用包含一聚合物之一調節試劑來處理該至少一生長室之該至少一表面。在其他實施例中，調節可包含：使用哺乳動物血清之一或多個組分來處理該至少一生長室之至少一表面。在其他實施例中，調節可包含：使用至少一細胞黏著阻斷分子來處理該至少一生長室之至少一表面。 In some embodiments of the method, the method can include the step of adjusting at least one surface of the at least one growth chamber. In some embodiments, adjusting can include treating the at least one surface of the at least one growth chamber with a conditioning reagent comprising a polymer. In other embodiments, the adjusting can comprise treating at least one surface of the at least one growth chamber with one or more components of the mammalian serum. In other embodiments, the adjusting can include treating at least one surface of the at least one growth chamber with at least one cell adhesion blocking molecule.

在該方法之一些實施例中，將該至少一生物細胞引入至該至少一生長室中可包含：使用具有足以移動該至少一生物細胞之力量之一介電泳(DEP)力。在一些實施例中，使用一DEP力可包含：光學地致動該DEP力。 In some embodiments of the method, introducing the at least one biological cell into the at least one growth chamber can comprise: using a dielectrophoresis (DEP) force having a force sufficient to move the at least one biological cell. In some embodiments, using a DEP force can include optically actuating the DEP force.

在該方法之一些實施例中，該方法可進一步包含下列步驟：在該培育步驟期間灌注該第一流體培養基，其中該第一流體培養基經由該微流體器件之至少一入口而引入且經由該微流體器件之至少一出口而輸出，其中在輸出之後，該第一流體培養基視情況包括來自該第二流體培養基之組分。 In some embodiments of the method, the method may further comprise the step of: priming the first fluid medium during the incubating step, wherein the first fluid medium is introduced via the at least one inlet of the microfluidic device and via the micro The fluid device is outputted from at least one outlet, wherein after the output, the first fluid medium optionally includes components from the second fluid medium.

在該方法之一些實施例中，該方法可進一步包含下列步驟：在該培育步驟之後，使該調節表面之一或多個可裂解部***解，藉此促進該一或多個生物細胞自該生長室或其隔離區域輸出至該流動區域 中。 In some embodiments of the method, the method may further comprise the step of, after the incubating step, lysing one or more cleavable moieties of the conditioning surface, thereby promoting the growth of the one or more biological cells from the growth The chamber or its isolated area is output to the flow area in.

在該方法之一些實施例中，該方法可進一步包含下列步驟：使一或多個生物細胞自該生長室或其隔離區域輸出至該流動區域中。 In some embodiments of the method, the method can further comprise the step of outputting one or more biological cells from the growth chamber or an isolated region thereof into the flow region.

在該方法之一些實施例中，該至少一生物細胞可包含一哺乳動物細胞。在該方法之其他實施例中，該至少一生物細胞可包含至少一免疫細胞。在該方法之其他實施例中，該至少一免疫細胞可包含一淋巴細胞或白血球。在該方法之一些其他實施例中，該至少一免疫細胞可包含一B細胞、一T細胞、NK細胞、一巨噬細胞或一樹突細胞。在其他實施例中，該至少一生物細胞可包含一貼附細胞。替代地，該至少一生物細胞可包含一融合瘤細胞。 In some embodiments of the method, the at least one biological cell can comprise a mammalian cell. In other embodiments of the method, the at least one biological cell can comprise at least one immune cell. In other embodiments of the method, the at least one immune cell can comprise a lymphocyte or a white blood cell. In some other embodiments of the method, the at least one immune cell can comprise a B cell, a T cell, an NK cell, a macrophage, or a dendritic cell. In other embodiments, the at least one biological cell can comprise an attached cell. Alternatively, the at least one biological cell can comprise a fusion tumor cell.

在該方法之一些實施例中，將該至少一生物細胞引入至該至少一生長室中之該步驟可包含將一單一細胞引入至該生長室中，且由該培育步驟產生之生物細胞之該群落可為一選殖群落。 In some embodiments of the method, the step of introducing the at least one biological cell into the at least one growth chamber can comprise introducing a single cell into the growth chamber, and the biological cell produced by the incubation step The community can be a colonization community.

在另一態樣中，提供一種用於培養一生物細胞之套組，其包含一微流體器件，該微流體器件具有：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包含經調節以支援該微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。該至少一生長室可包含一隔離區域及一連接區域，該隔離區域與該連接區域流體地連接且該連接區域具有至該流動區域之一近端開口。該微流體器件可為如本文中所描述之任何微流體器件，其具有元件之任何組合。在一些實施例中，該微流體器件之該至少一調節表面可包含烷基部分、氟烷基部分、單糖或多糖部分、乙醇部分、多元醇部分、伸烷基醚部分；聚電解質部分、胺基部分、羧酸部分、膦酸部分、磺酸鹽部分、羧基甜菜鹼部分、磺基甜菜鹼部分、胺磺酸部分或胺基酸部分。在一些實施例中，該微流體器件之該至少一調節表面包括下列之至少一者：糖類部分、伸烷基醚部分、烷基部分、氟烷 基部分或胺基酸部分。在一些實施例中，該等烷基或氟烷基部分具有大於10個碳之一主鏈長度。 In another aspect, a kit for culturing a biological cell is provided, comprising a microfluidic device having: a flow region configured to contain a flow of a first fluid medium And at least one growth chamber comprising at least one surface conditioned to support cell growth, viability, portability, or the like in the microfluidic device. The at least one growth chamber can include an isolation region and a connection region, the isolation region being fluidly coupled to the connection region and having a proximal opening to one of the flow regions. The microfluidic device can be any microfluidic device as described herein having any combination of elements. In some embodiments, the at least one conditioning surface of the microfluidic device can comprise an alkyl moiety, a fluoroalkyl moiety, a monosaccharide or polysaccharide moiety, an ethanol moiety, a polyol moiety, an alkyl ether moiety, a polyelectrolyte moiety, An amine moiety, a carboxylic acid moiety, a phosphonic acid moiety, a sulfonate moiety, a carboxybetaine moiety, a sulfobetaine moiety, an aminesulfonic acid moiety or an amino acid moiety. In some embodiments, the at least one conditioning surface of the microfluidic device comprises at least one of: a sugar moiety, an alkyl ether moiety, an alkyl moiety, a fluorocarbon a base moiety or an amino acid moiety. In some embodiments, the alkyl or fluoroalkyl moiety has a backbone length of greater than 10 carbons.

在該套組之各種實施例中，該微流體器件之該至少一調節表面可包含共價鍵聯至該微流體器件之一表面之一鍵聯基團，且該鍵聯基團可鍵聯至經構形以支援該微流體器件內之該一或多個生物細胞之生長、存活性、可移植性或其等之任何組合之一部分。在一些實施例中，該鍵聯基團可為矽烷氧基鍵聯基團。在其他實施例中，該鍵聯基團可為膦酸酯鍵聯基團。在一些實施例中，該鍵聯基團可直接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在其他實施例中，該鍵聯基團可間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯基團可經由一鍵聯子而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯基團可經由連接至一鍵聯子之一第一端而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在各種實施例中，該鍵聯子可進一步包含一直鏈部分，其中該直鏈部分之一主鏈包括選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之任何組合之1個至200個非氫原子。在一些實施例中，該鍵聯子可進一步包含伸***基部分。 In various embodiments of the kit, the at least one conditioning surface of the microfluidic device can comprise a linkage group covalently bonded to one of the surfaces of the microfluidic device, and the linkage group can be linked To a portion configured to support growth, viability, portability, or the like of the one or more biological cells within the microfluidic device. In some embodiments, the linking group can be a decyloxy linking group. In other embodiments, the linking group can be a phosphonate linkage group. In some embodiments, the linking group can be directly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. In other embodiments, the linking group can be indirectly linked to that portion that is configured to support cell growth, viability, portability, or the like, in any combination. The linking group can be indirectly linked via a linkage to the portion configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly linked to the portion configured to support cell growth, viability, portability, or the like, via attachment to one of the first ends of a linkage. In various embodiments, the linker may further comprise a straight chain moiety, wherein one of the backbone portions comprises a bond selected from the group consisting of a ruthenium atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. 1 to 200 non-hydrogen atoms. In some embodiments, the linker can further comprise a triazolyl moiety.

在該套組之各種實施例中，該套組可進一步包含一表面調節試劑。在一些實施例中，該表面調節試劑可包含一聚合物，其包括下列之至少一者：伸烷基醚部分、羧酸部分、磺酸部分、膦酸部分、胺基酸部分、核酸部分或糖類部分。在一些實施例中，該表面調節試劑可包含一聚合物，其包含下列之至少一者：伸烷基醚部分、胺基酸部分及/或糖類部分。 In various embodiments of the kit, the kit can further comprise a surface conditioning agent. In some embodiments, the surface conditioning agent can comprise a polymer comprising at least one of: an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety, or Sugar part. In some embodiments, the surface conditioning agent can comprise a polymer comprising at least one of the alkyl ether moiety, the amino acid moiety, and/or the carbohydrate moiety.

在其他實施例中，該表面調節試劑可包含至少一細胞黏著阻斷分子。在一些實施例中，該至少一細胞黏著阻斷分子可破壞肌動蛋白 纖維形成，阻斷整合素受體，或減弱細胞與DNA污損表面之結合。在一些實施例中，該至少一細胞黏著阻斷分子可為細胞鬆弛素B、一含RGD之肽、纖連蛋白之一抑制劑、一整合素抗體或一DNase 1蛋白。在一些實施例中，該表面調節試劑可包含一個以上細胞黏著阻斷分子之一組合。 In other embodiments, the surface conditioning agent can comprise at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule disrupts actin Fibrosis forms, blocks integrin receptors, or attenuates the binding of cells to DNA-stained surfaces. In some embodiments, the at least one cell adhesion blocking molecule can be cytochalasin B, an RGD-containing peptide, an inhibitor of fibronectin, an integrin antibody, or a DNase 1 protein. In some embodiments, the surface conditioning agent can comprise a combination of one or more cell adhesion blocking molecules.

在其他實施例中，該表面調節試劑可包含哺乳動物血清之一或多個組分。在一些實施例中，該哺乳動物血清可為胎牛血清(FBS)或小牛血清(FCS)。 In other embodiments, the surface conditioning agent can comprise one or more components of a mammalian serum. In some embodiments, the mammalian serum can be fetal bovine serum (FBS) or calf serum (FCS).

在該套組之各種實施例中，該套組可進一步包含一培養基添加物，其包含經構形以加強生長室之該至少一表面之該調節之一試劑。該培養基添加物可包含一Pluronics®聚合物。 In various embodiments of the kit, the kit can further comprise a media supplement comprising one of the conditioning agents configured to enhance the at least one surface of the growth chamber. The medium supplement may comprise a Pluronics® polymer.

在該套組之各種實施例中，該微流體器件之該至少一調節表面可包含一可裂解部分。在一些實施例中，該套組可進一步包含經構形以使該調節表面之該可裂解部***解之一試劑。 In various embodiments of the kit, the at least one conditioning surface of the microfluidic device can comprise a cleavable portion. In some embodiments, the kit can further comprise an agent configured to lyse the cleavable portion of the conditioning surface.

在該套組之各種實施例中，該套組可進一步包含用以偵測該生物細胞之一狀態之至少一試劑。 In various embodiments of the kit, the kit can further comprise at least one reagent for detecting the state of one of the biological cells.

100‧‧‧微流體器件 100‧‧‧Microfluidic devices

102‧‧‧封閉體/蓋 102‧‧‧Closed/cover

104‧‧‧支撐結構/基底 104‧‧‧Support structure/base

106‧‧‧流動路徑 106‧‧‧Flow path

107‧‧‧端口 107‧‧‧Port

108‧‧‧微流體線路結構 108‧‧‧Microfluidic line structure

109‧‧‧內表面 109‧‧‧ inner surface

110‧‧‧蓋 110‧‧‧ Cover

114‧‧‧框架 114‧‧‧Frame

116‧‧‧微流體線路材料 116‧‧‧Microfluidic circuit materials

120‧‧‧微流體線路 120‧‧‧Microfluidic lines

122‧‧‧微流體通道 122‧‧‧Microfluidic channel

124‧‧‧微流體生長室/入口 124‧‧‧Microfluidic growth chamber/inlet

124'‧‧‧出口 124'‧‧‧Export

126‧‧‧微流體生長室 126‧‧‧Microfluidic growth chamber

128‧‧‧微流體生長室 128‧‧‧Microfluidic growth chamber

130‧‧‧微流體生長室 130‧‧‧Microfluidic growth chamber

132‧‧‧微小物體捕集器 132‧‧‧Micro Object Collector

134‧‧‧側通路/流動通道/流動區域 134‧‧‧Side access/flow channel/flow area

150‧‧‧系統 150‧‧‧ system

152‧‧‧控制及監測設備 152‧‧‧Control and monitoring equipment

154‧‧‧主控制器 154‧‧‧Master controller

156‧‧‧控制模組 156‧‧‧Control Module

158‧‧‧記憶體 158‧‧‧ memory

160‧‧‧培養基模組 160‧‧‧ medium module

162‧‧‧動力模組 162‧‧‧Power Module

164‧‧‧成像模組 164‧‧‧ imaging module

166‧‧‧傾斜模組 166‧‧‧ tilt module

168‧‧‧其他模組 168‧‧‧Other modules

170‧‧‧顯示器件 170‧‧‧Display devices

172‧‧‧輸入/輸出器件/控制模組 172‧‧‧Input/Output Devices/Control Modules

178‧‧‧培養基源 178‧‧‧ medium source

180‧‧‧流體培養基/控制/監測設備 180‧‧‧Fluid medium/control/monitoring equipment

190‧‧‧傾斜器件 190‧‧‧ tilting device

192‧‧‧電源 192‧‧‧Power supply

194‧‧‧成像器件 194‧‧‧ imaging device

200‧‧‧微流體器件 200‧‧‧Microfluidic devices

202‧‧‧區域/室/流體培養基 202‧‧‧Regional/Room/Fluid Medium

202'‧‧‧廢流體培養基 202'‧‧‧Waste fluid medium

202a'‧‧‧染色流體培養基 202a'‧‧‧Dyeing fluid medium

202a"‧‧‧染色出流流體培養基 202a"‧‧‧Dyeing outflow fluid medium

204‧‧‧底部電極/流體培養基 204‧‧‧Bottom electrode/fluid medium

206‧‧‧電極啟動基板 206‧‧‧Electrode starter substrate

208‧‧‧內表面 208‧‧‧ inner surface

210‧‧‧頂部電極 210‧‧‧Top electrode

212‧‧‧電源/流體流 212‧‧‧Power/fluid flow

214‧‧‧未經照射之介電泳(DEP)電極區域 214‧‧‧Unilluminated dielectrophoresis (DEP) electrode area

214a‧‧‧經照射之介電泳(DEP)電極區域 214a‧‧‧Determinated Dielectrophoresis (DEP) Electrode Region

220‧‧‧光源 220‧‧‧Light source

222‧‧‧光圖案 222‧‧‧Light pattern

224‧‧‧正方形圖案 224‧‧‧ square pattern

226‧‧‧控制器 226‧‧‧ Controller

240‧‧‧微流體器件 240‧‧‧Microfluidic devices

242‧‧‧端口 242‧‧‧port

244‧‧‧生長室 244‧‧‧Growing room

246‧‧‧生長室 246‧‧‧ Growth room

248‧‧‧生長室 248‧‧‧Growing room

250‧‧‧隔離結構 250‧‧‧Isolation structure

252‧‧‧近端開口 252‧‧‧ proximal opening

254‧‧‧連接區域 254‧‧‧Connected area

256‧‧‧遠端開口 256‧‧‧ distal opening

258‧‧‧隔離區域 258‧‧‧Isolated area

260‧‧‧流 260‧‧‧ flow

262‧‧‧副流 262‧‧‧ Vicestream

270‧‧‧微小物體 270‧‧‧ tiny objects

280‧‧‧第二培養基 280‧‧‧Second medium

290‧‧‧微流體器件 290‧‧‧Microfluidic devices

300‧‧‧結構/巢套 300‧‧‧Structure / Nest

302‧‧‧插座 302‧‧‧ socket

304‧‧‧電信號產生子系統 304‧‧‧Electrical Signal Generation Subsystem

306‧‧‧熱控制子系統 306‧‧‧ Thermal Control Subsystem

308‧‧‧控制器 308‧‧‧ Controller

310‧‧‧介面 310‧‧‧ interface

320‧‧‧印刷電路板總成(PCBA) 320‧‧‧ Printed Circuit Board Assembly (PCBA)

330‧‧‧流體路徑 330‧‧‧ Fluid path

332‧‧‧入口 332‧‧‧ entrance

334‧‧‧出口 334‧‧‧Export

340‧‧‧罩殼 340‧‧‧Shell

350‧‧‧串列埠 350‧‧‧Chain

360‧‧‧微流體器件 360‧‧‧Microfluidic devices

400‧‧‧微流體器件 400‧‧‧Microfluidic devices

402‧‧‧第一培養基 402‧‧‧First medium

404‧‧‧第二培養基 404‧‧‧Second medium

412‧‧‧微流體線路結構 412‧‧‧Microfluidic line structure

414‧‧‧框架 414‧‧‧Frame

416‧‧‧微流體線路材料 416‧‧‧Microfluidic line materials

420‧‧‧第一光源 420‧‧‧first light source

422‧‧‧光調變子系統 422‧‧‧Light modulation subsystem

424‧‧‧分光器 424‧‧ ‧ splitter

426‧‧‧置物台 426‧‧‧Stores

428‧‧‧樣本平面 428‧‧‧sample plane

430‧‧‧第二光源 430‧‧‧second light source

432‧‧‧微流體線路 432‧‧‧Microfluidic lines

434‧‧‧流動通道 434‧‧‧Flow channel

436‧‧‧生長室 436‧‧‧Growing room

440‧‧‧偵測器 440‧‧‧Detector

442‧‧‧連接區域 442‧‧‧Connected area

444‧‧‧隔離區域 444‧‧‧Isolated area

446‧‧‧隔離結構 446‧‧‧Isolation structure

448‧‧‧二向色濾光器 448‧‧‧ dichroic filter

450‧‧‧顯微鏡 450‧‧‧Microscope

452‧‧‧二向色濾光器 452‧‧‧ dichroic filter

454‧‧‧物鏡 454‧‧‧ objective lens

472‧‧‧近端開口 472‧‧‧ proximal opening

474‧‧‧遠端開口 474‧‧‧ distal opening

482‧‧‧流體培養基流 482‧‧‧ Fluid medium flow

484‧‧‧副流 484‧‧‧ Vicestream

500A‧‧‧微流體器件 500A‧‧‧Microfluidic devices

500B‧‧‧微流體器件 500B‧‧‧Microfluidic devices

500C‧‧‧微流體器件 500C‧‧‧Microfluidic devices

500D‧‧‧微流體器件 500D‧‧‧Microfluidic devices

500E‧‧‧微流體器件 500E‧‧‧Microfluidic devices

502‧‧‧微流體器件/儲液器 502‧‧‧Microfluidic devices/reservoir

502'‧‧‧儲液器 502'‧‧‧Liquid

504‧‧‧泵連接導管 504‧‧‧ pump connection catheter

504'‧‧‧可透氣管道 504'‧‧‧ breathable pipe

506‧‧‧轉移連接導管 506‧‧‧Transfer connection catheter

508‧‧‧出口連接導管 508‧‧‧Export connection catheter

510‧‧‧封閉體 510‧‧‧Closed

512‧‧‧基底 512‧‧‧Base

514‧‧‧泵/泵組件 514‧‧‧ pump/pump assembly

516‧‧‧室/封閉體 516‧‧‧room/enclosure

518‧‧‧可透氣塊 518‧‧‧ breathable block

518'‧‧‧可透氣區段/可透氣塊 518'‧‧‧ breathable section / breathable block

520‧‧‧閥 520‧‧‧ valve

522‧‧‧轉移連接導管 522‧‧‧Transfer connection catheter

524‧‧‧氣體環境源 524‧‧‧ gas environment source

526‧‧‧連接供給/連接導管 526‧‧‧Connecting supply/connection conduit

600‧‧‧微流體器件 600‧‧‧Microfluidic devices

606‧‧‧流體培養基入口管道 606‧‧‧Fluid medium inlet pipe

608‧‧‧流體培養基出口管道 608‧‧‧ Fluid medium outlet pipe

610‧‧‧感測器 610‧‧‧ sensor

610'‧‧‧感測器 610'‧‧‧ Sensor

612‧‧‧整合比色感測器 612‧‧‧Integrated colorimetric sensor

612'‧‧‧整合比色感測器 612'‧‧‧ integrated colorimetric sensor

614‧‧‧發光二極體(LED) 614‧‧‧Light Emitting Diode (LED)

614'‧‧‧發光二極體(LED) 614'‧‧‧Light Emitting Diode (LED)

700‧‧‧方法/灌注程序 700‧‧‧Method/Perfusion Procedure

800‧‧‧方法 800‧‧‧ method

900‧‧‧微流體器件 900‧‧‧Microfluidic devices

902‧‧‧封閉區域 902‧‧‧closed area

904‧‧‧第一介電泳(DEP)基板 904‧‧‧First Dielectrophoresis (DEP) Substrate

906‧‧‧第二介電泳(DEP)基板 906‧‧‧Second Dielectrophoresis (DEP) Substrate

910‧‧‧內表面 910‧‧‧ inner surface

912‧‧‧內表面 912‧‧‧ inner surface

914‧‧‧矽烷氧基鍵聯基團 914‧‧‧ 矽 alkoxy linkage group

916‧‧‧調節表面 916‧‧‧Adjustment surface

1002‧‧‧細胞 1002‧‧‧ cells

1004‧‧‧細胞 1004‧‧‧ cells

1006‧‧‧細胞 1006‧‧‧ cells

1008‧‧‧細胞 1008‧‧‧ cells

1010‧‧‧細胞 1010‧‧‧ cells

1012‧‧‧細胞 1012‧‧‧ cells

1014‧‧‧細胞 1014‧‧‧ cells

1016‧‧‧細胞 1016‧‧‧ cells

1018‧‧‧細胞 1018‧‧‧ cells

1102‧‧‧細胞 1102‧‧‧ cells

1202‧‧‧細胞群組 1202‧‧‧cell group

1204‧‧‧進一步增殖細胞群體 1204‧‧‧ further proliferating cell population

1206‧‧‧經擴增之OKT3細胞 1206‧‧‧Amplified OKT3 cells

1302‧‧‧光阱 1302‧‧‧Light trap

1304‧‧‧光阱 1304‧‧‧Light trap

1306‧‧‧釋放擴增細胞 1306‧‧‧ release of expanded cells

7002‧‧‧步驟 7002‧‧‧Steps

7004‧‧‧步驟 7004‧‧‧Steps

8002‧‧‧步驟 8002‧‧‧ steps

8004‧‧‧步驟 8004‧‧‧Steps

8006‧‧‧步驟 8006‧‧‧Steps

8008‧‧‧步驟 8008‧‧‧Steps

D_p‧‧‧副流之滲透深度 D _p ‧‧‧ penetration depth of the secondary stream

L_c1‧‧‧連接區域之長度 L _c1 ‧‧‧The length of the connection area

L_c2‧‧‧連接區域之長度 L _c2 ‧‧‧ Length of the connection area

W_ch‧‧‧通道寬度 W _ch ‧‧‧ channel width

W_con‧‧‧連接區域之寬度 W _con ‧‧‧Width of the connection area

W_con1‧‧‧近端開口之寬度 W _con1 ‧‧‧ width of the proximal opening

W_con2‧‧‧遠端開口之寬度 W _con2 ‧‧‧Width of the distal opening

圖1繪示根據本發明之一些實施例之用於與一微流體器件及相關聯控制設備一起使用之一系統之一實例。 1 illustrates an example of one system for use with a microfluidic device and associated control device in accordance with some embodiments of the present invention.

圖2A及圖2B繪示根據本發明之一些實施例之一微流體器件。 2A and 2B illustrate a microfluidic device in accordance with some embodiments of the present invention.

圖2C及圖2D繪示根據本發明之一些實施例之生長室。 2C and 2D illustrate a growth chamber in accordance with some embodiments of the present invention.

圖2E繪示根據本發明之一些實施例之一詳細生長室。 2E depicts a detailed growth chamber in accordance with some embodiments of the present invention.

圖2F繪示根據本發明之一實施例之一微流體器件。 2F illustrates a microfluidic device in accordance with an embodiment of the present invention.

圖3A繪示根據本發明之一些實施例之用於與一微流體器件及相關聯控制設備一起使用之一系統之一特定實例。 3A illustrates a particular example of one of the systems for use with a microfluidic device and associated control device in accordance with some embodiments of the present invention.

圖3B繪示根據本發明之一些實施例之一成像器件。 FIG. 3B illustrates an imaging device in accordance with some embodiments of the present invention.

圖4A至圖4C展示一微流體器件之另一實施例，其包含用於其內之一生長室之另一實例。 4A-4C illustrate another embodiment of a microfluidic device that includes another example for a growth chamber therein.

圖5A至圖5E各表示能夠將調節培養基提供至一微流體器件以支援細胞生長、存活性、可移植性或其等之任何組合之系統組件之一實施例。 5A-5E each illustrate one embodiment of a system component capable of providing a conditioning medium to a microfluidic device to support cell growth, viability, portability, or the like.

圖6係具有一或多個感測器之一微流體器件之一圖示，該一或多個感測器能夠偵測進入及/或離開該微流體器件之培養基之pH。 6 is an illustration of one of the microfluidic devices having one or more sensors capable of detecting the pH of the medium entering and/or exiting the microfluidic device.

圖7係用於灌注將一流體培養基於一微流體器件中之一程序之一實施例之一實例。 Figure 7 is an illustration of one example of one of the procedures for perfusing a fluid medium into a microfluidic device.

圖8係用於將一流體培養基灌注於一微流體器件中之一程序之另一實施例之一實例。 Figure 8 is an example of another embodiment of a procedure for infusing a fluid medium into a microfluidic device.

圖9係提供進一步支援細胞生長、存活性、可移植性或其等之任何組合之一調節表面之一示意圖。 Figure 9 is a schematic illustration of one of the regulatory surfaces that provides further support for cell growth, viability, portability, or any combination thereof.

圖10A至圖10E係根據本文中所描述之方法之一培養實驗之一實施例之攝影圖。 10A-10E are photographic images of one embodiment of a culture experiment in accordance with one of the methods described herein.

圖11A係根據本文中所描述之方法之一培養實驗之另一實施例之一攝影圖，其展示將一細胞放置於一微流體器件之生長室中之前之該器件。 Figure 11A is a photographic representation of another embodiment of a culture experiment in accordance with one of the methods described herein, showing the device prior to placing a cell in a growth chamber of a microfluidic device.

圖11B係將一細胞放置於微流體器件之一生長室中之後之圖11A之培養實驗之一實施例之一攝影圖。 Figure 11B is a photographic representation of one of the embodiments of the culture experiment of Figure 11A after placing a cell in a growth chamber of a microfluidic device.

圖12A至圖12C係一稍後時間點處之圖11A及圖11B之培養實驗之一實施例之攝影圖，其展示圖11B之細胞之培育期間之細胞擴增。 Figures 12A through 12C are photographic images of one embodiment of the culture experiment of Figures 11A and 11B at a later time point showing cell expansion during incubation of the cells of Figure 11B.

圖13A至圖13C係一稍後時間點處之圖11A至圖11B及圖12A至圖12C之培養實驗之一實施例之攝影圖，其展示培育期結束之後之擴增細胞之輸出。 13A to 13C are photographs of an embodiment of the culture experiment of Figs. 11A to 11B and Figs. 12A to 12C at a later time point, showing the output of the expanded cells after the end of the incubation period.

圖14A及圖14B係具有至少一調節表面之一微流體器件中之另一 培養實驗之一實施例之攝影圖。 14A and 14B are another of the microfluidic devices having at least one adjustment surface A photographic image of one of the examples of the culture experiment was carried out.

微流體環境提供對一細胞或細胞群組提供一局部環境之機會，該局部環境依一時間相依方式及位置相依濃度將營養素及/或可溶細胞生長傳物種提供至該細胞或細胞群組。此等條件可表示更像活體內之生長條件或替代地容許擾動此等典型條件以容許非標準條件下之研究及生長。無法使用標準化大規模細胞培養方法來滿足此等要求。然而，需要改良方案來使一或若干細胞之操縱更容易以：a)將該(等)細胞放置至有益於支援細胞生長、存活性、可移植性或其等之任何組合之一微流體環境中；b)成功地維持該(等)細胞及/或擴增該(等)細胞之群體；及/或c)界定導致成功生長及/或維持之條件。本文中所描述之系統及方法允許用於微流體細胞培養之更精確細胞處置、環境控制及細胞隔離技術，且可用以產生(例如)選殖細胞群體。 The microfluidic environment provides an opportunity to provide a local environment to a cell or group of cells that provides nutrients and/or soluble cell growth species to the cell or group of cells in a time dependent manner and in a position dependent concentration. Such conditions may indicate more like growth conditions in vivo or alternatively allow for perturbation of such typical conditions to permit research and growth under non-standard conditions. Standardized large-scale cell culture methods cannot be used to meet these requirements. However, there is a need for improved protocols to make manipulation of one or several cells easier: a) placing the cells in a microfluidic environment that is beneficial for supporting cell growth, viability, portability, or any combination thereof. And b) defining a condition that results in successful growth and/or maintenance. The systems and methods described herein allow for more precise cell handling, environmental control, and cell isolation techniques for microfluidic cell culture, and can be used to generate, for example, a population of conserved cells.

本說明書描述本發明之例示性實施例及應用。然而，本發明不受限於此等例示性實施例及應用或不受限於該等例示性實施例及應用之操作方式或其在本文中之描述方式。而且，圖式可展示簡化或局部視圖，且為清楚起見，圖式中之元件之尺寸可被放大或否則可不成比例。另外，當本文中使用術語「在...上」、「附接至」或「耦合至」時，一元件(例如一材料、一層、一基板等等)可「在另一元件上」、「附接至另一元件」或「耦合至另一元件」，無論該元件是否直接在該另一元件上、直接附接至該另一元件或直接耦合至該另一元件或在該元件與該另一元件之間存在一或多個介入元件。同樣地，方向(例如上方、下方、頂部、底部、側、向上、向下、下面、上面、上、下、水平、垂直、「x」、「y」、「z」等等)(若提供)係相對的，僅供例示的，用於使繪示及討論方便，且絕非意在限制。另外，當參考一元件系列(例如元件a、b、c)時，此參考意欲包含所列元件本身之任何者、 非全部所列元件之任何組合及/或全部所列元件之一組合。本說明書中之章節劃分僅為了使檢查方便且不限制所討論之元件之任何組合。 This specification describes exemplary embodiments and applications of the invention. However, the invention is not limited by the exemplary embodiments and applications or the manner of operation of the exemplary embodiments and applications or the manners described herein. Furthermore, the drawings may be simplified or partial, and the dimensions of the elements in the figures may be exaggerated or otherwise disproportionate. In addition, when the terms "on", "attached to" or "coupled to" are used herein, an element (eg, a material, a layer, a substrate, etc.) can be "on another element", "Attached to another element" or "coupled to another element" whether or not the element is directly attached to the other element or directly coupled to the other element or directly coupled to or in the element There are one or more intervening elements between the other elements. Similarly, direction (eg top, bottom, top, bottom, side, up, down, down, top, up, down, horizontal, vertical, "x", "y", "z", etc.) (if provided) The terms are relative and are for illustrative purposes only and are not intended to be limiting. In addition, when referring to a series of elements (eg, elements a, b, c), this reference is intended to include any of the listed elements themselves, Any combination of any of the listed components and/or all of the listed components. The divisions in this specification are merely for ease of inspection and do not limit any combination of the elements discussed.

如本文中所使用，「實質上」意謂足以達成所欲目的。因此，術語「實質上」允許相對於一絕對或完美狀態、尺寸、量測、結果或其類似者之小幅、微不足道變動，諸如將由一般技術者預期且不會明顯地影響總體效能之變動。當與數值或參數或可表達為數值之特性一起使用時，「實質上」意謂在10%內。 As used herein, "substantially" means sufficient to achieve the desired purpose. Thus, the term "substantially" allows for small, insignificant changes relative to an absolute or perfect state, size, measurement, result, or the like, such as would be expected by a person of ordinary skill and would not significantly affect the overall performance. When used in conjunction with a numerical value or parameter or characteristic that can be expressed as a numerical value, "substantially" means within 10%.

術語「若干」意謂一個以上。如本文中所使用，術語「複數個」可為2個、3個、4個、5個、6個、7個、8個、9個、10個或更多。 The term "a number" means more than one. As used herein, the term "plurality" can be two, three, four, five, six, seven, eight, nine, ten or more.

如本文中所使用，「空氣」係指在地球之大氣中占主導地位之氣體之組合物。四種最豐富氣體係氮氣(通常存在約78體積%之一濃度，例如，在自約70%至約80%之一範圍內)、氧氣(在海平面處通常存在約20.95體積%，例如，在自約10%至約25%之一範圍內)、氬氣(通常存在約1.0體積%，例如，在自約0.1%至約3%之一範圍內)及二氧化碳(通常存在約0.04%，例如，在自約0.01%至約0.07%之一範圍內)。空氣可具有其他微量氣體，諸如甲烷、氧化亞氮或臭氧、微量污染物及有機材料，諸如花粉、柴油顆粒及其類似者。空氣可包含水蒸氣(通常存在約0.25%，或可存在自約10ppm至約5體積%之一範圍)。空氣可經提供以在培養實驗中用作為一經過濾之受控組合物且可如本文中所描述被調節。 As used herein, "air" refers to a composition of gases that predominate in the atmosphere of the earth. The four most abundant gas systems nitrogen (typically present at a concentration of about 78% by volume, for example, in the range from about 70% to about 80%), oxygen (usually present at about 20.95 vol% at sea level, for example, In the range from about 10% to about 25%, argon (typically present at about 1.0% by volume, for example, in the range from about 0.1% to about 3%) and carbon dioxide (usually present at about 0.04%, For example, in a range from about 0.01% to about 0.07%). The air may have other trace gases such as methane, nitrous oxide or ozone, trace contaminants and organic materials such as pollen, diesel particulates and the like. The air may comprise water vapor (typically present at about 0.25%, or may be present in a range from about 10 ppm to about 5% by volume). Air can be provided for use as a filtered controlled composition in a culture experiment and can be adjusted as described herein.

如本文中所使用，術語「安置」涵蓋於其含義「定位」內。 As used herein, the term "placement" is used within its meaning "location."

如本文中所使用，一「微流體器件」或「微流體裝置」係包含經構形以保持一流體之一或多個離散微流體線路之一器件，各微流體線路由流體地互連之線路元件(其包含(但不限於)(若干)區域、(若干)流動路徑、(若干)通道、(若干)室及/或(若干)圍欄)及至少兩個端口(其經構形以允許流體(及視情況，懸浮於流體中之微小物體)流動至微 流體器件中及/或流出微流體器件)組成。通常，一微流體器件之一微流體線路將包含至少一微流體通道及至少一室，且將保持小於約1毫升(例如，小於約750微升、約500微升、約250微升、約200微升、約150微升、約100微升、約75微升、約50微升、約25微升、約20微升、約15微升、約10微升、約9微升、約8微升、約7微升、約6微升、約5微升、約4微升、約3微升或約2微升)之一流體體積。在某些實施例中，微流體線路保持約1微升至約2微升、約1微升至約3微升、約1微升至約4微升、約1微升至約5微升、約2微升至約5微升、約2微升至約8微升、約2微升至約10微升、約2微升至約12微升、約2微升至約15微升、約2微升至約20微升、約5微升至約20微升、約5微升至約30微升、約5微升至約40微升、約5微升至約50微升、約10微升至約50微升、約10微升至約75微升、約10微升至約100微升、約20微升至約100微升、約20微升至約150微升、約20微升至約200微升、約50微升至約200微升、約50微升至約250微升或約50微升至約300微升。 As used herein, a "microfluidic device" or "microfluidic device" includes a device configured to hold one or more discrete microfluidic circuits of a fluid, each microfluidic line being fluidly interconnected. a line component (including but not limited to) (several) zones, (several) flow paths, (several) channels, (several) rooms and/or (several) fences, and at least two ports (configured to allow Fluid (and, as the case may be, tiny objects suspended in the fluid) flow to the micro Composed of a fluid device and/or an outflow microfluidic device. Typically, a microfluidic circuit of a microfluidic device will comprise at least one microfluidic channel and at least one chamber, and will remain less than about 1 milliliter (eg, less than about 750 microliters, about 500 microliters, about 250 microliters, about 200 microliters, about 150 microliters, about 100 microliters, about 75 microliters, about 50 microliters, about 25 microliters, about 20 microliters, about 15 microliters, about 10 microliters, about 9 microliters, about One fluid volume of 8 microliters, about 7 microliters, about 6 microliters, about 5 microliters, about 4 microliters, about 3 microliters, or about 2 microliters. In certain embodiments, the microfluidic line maintains from about 1 microliter to about 2 microliters, from about 1 microliter to about 3 microliters, from about 1 microliter to about 4 microliters, from about 1 microliter to about 5 microliters. From about 2 microliters to about 5 microliters, from about 2 microliters to about 8 microliters, from about 2 microliters to about 10 microliters, from about 2 microliters to about 12 microliters, from about 2 microliters to about 15 microliters From about 2 microliters to about 20 microliters, from about 5 microliters to about 20 microliters, from about 5 microliters to about 30 microliters, from about 5 microliters to about 40 microliters, from about 5 microliters to about 50 microliters From about 10 microliters to about 50 microliters, from about 10 microliters to about 75 microliters, from about 10 microliters to about 100 microliters, from about 20 microliters to about 100 microliters, from about 20 microliters to about 150 microliters From about 20 microliters to about 200 microliters, from about 50 microliters to about 200 microliters, from about 50 microliters to about 250 microliters or from about 50 microliters to about 300 microliters.

如本文中所使用，一「奈流體器件」或「奈流體裝置」係具有一微流體線路之一類型之微流體器件，該微流體線路含有經構形以保持小於約1微升(例如，小於約750奈升、約500奈升、約250奈升、約200奈升、約150奈升、約100奈升、約75奈升、約50奈升、約25奈升、約20奈升、約15奈升、約10奈升、約9奈升、約8奈升、約7奈升、約6奈升、約5奈升、約4奈升、約3奈升、約2奈升、約1奈升或更小)之一流體體積之至少一線路元件。通常，一奈流體器件將包括複數個線路元件(例如至少2個、3個、4個、5個、6個、7個、8個、9個、10個、15個、20個、25個、50個、75個、100個、150個、200個、250個、300個、400個、500個、600個、700個、800個、900個、1000個、1500個、2000個、2500個、3000個、3500個、4000個、4500個、5000個、6000個、7000個、8000個、9000個、10000個或更多)。 在某些實施例中，該至少一線路元件之一或多者(例如全部)經構形以保持約100皮升至約1奈升、約100皮升至約2奈升、約100皮升至約5奈升、約250皮升至約2奈升、約250皮升至約5奈升、約250皮升至約10奈升、約500皮升至約5奈升、約500皮升至約10奈升、約500皮升至約15奈升、約750皮升至約10奈升、約750皮升至約15奈升、約750皮升至約20奈升、約1奈升至約10奈升、約1奈升至約15奈升、約1奈升至約20奈升、約1奈升至約25奈升或約1奈升至約50奈升之一流體體積。在其他實施例中，該至少一線路元件之一或多者(例如全部)經構形以保持約100奈升至約200奈升、約100奈升至約300奈升、約100奈升至約400奈升、約100奈升至約500奈升、約200奈升至約300奈升、約200奈升至約400奈升、約200奈升至約500奈升、約200奈升至約600奈升、約200奈升至約700奈升、約250奈升至約400奈升、約250奈升至約500奈升、約250奈升至約600奈升或約250奈升至約750奈升之一流體體積。 As used herein, a "nanofluidic device" or "nanofluidic device" has a microfluidic device of one type of microfluidic circuit that is configured to remain less than about 1 microliter (eg, Less than about 750 nanoliters, about 500 nanoliters, about 250 nanoliters, about 200 nanoliters, about 150 nanoliters, about 100 nanoliters, about 75 nanoliters, about 50 nanoliters, about 25 nanoliters, about 20 nanoliters. , about 15 liters, about 10 liters, about 9 liters, about 8 liters, about 7 liters, about 6 liters, about 5 liters, about 4 liters, about 3 liters, about 2 liters At least one line element of one fluid volume, about 1 nanoliter or less. Typically, a one-way fluid device will include a plurality of line elements (eg, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25) , 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 , 3000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 or more). In certain embodiments, one or more (eg, all) of the at least one line component are configured to maintain from about 100 picoliters to about 1 nanoliter, from about 100 picoliters to about 2 nanoliters, and about 100 picoliters. Up to about 5 nanoliters, about 250 picoliters to about 2 nanoliters, about 250 picoliters to about 5 nanoliters, about 250 picoliters to about 10 nanoliters, about 500 picoliters to about 5 nanoliters, about 500 picolitres Up to about 10 nanoliters, about 500 picoliters to about 15 nanoliters, about 750 picoliters to about 10 nanoliters, about 750 picoliters to about 15 nanoliters, about 750 picoliters to about 20 nanoliters, about 1 nanoliter Up to about 10 nanoliters, about 1 nanoliter to about 15 nanoliters, about 1 nanoliter to about 20 nanoliters, about 1 nanoliter to about 25 nanoliters, or about 1 nanoliter to about 50 nanoliters of fluid volume. In other embodiments, one or more (eg, all) of the at least one line component are configured to maintain from about 100 nanoliters to about 200 nanoliters, from about 100 nanoliters to about 300 nanoliters, and about 100 nanoliters to About 400 liters, about 100 liters to about 500 liters, about 200 liters to about 300 liters, about 200 liters to about 400 liters, about 200 liters to about 500 liters, about 200 liters to About 600 liters, about 200 liters to about 700 liters, about 250 liters to about 400 liters, about 250 liters to about 500 liters, about 250 liters to about 600 liters or about 250 liters to A fluid volume of about 750 nanoliters.

如本文中所使用，一「微流體通道」或「流動通道」係指一微流體器件之流動區域，其具有顯著地長於水平尺寸及垂直尺寸兩者之一長度。例如，流動通道可為水平尺寸或垂直尺寸之至少5倍長度，例如至少10倍長度、至少25倍長度、至少100倍長度、至少200倍長度、至少300倍長度、至少400倍長度、至少500倍長度或更長。在一些實施例中，一流動通道之長度係在自約20,000微米至約100,000微米之範圍內，其包含其間之任何範圍。在一些實施例中，水平尺寸係在自約100微米至約1000微米(例如約150微米至約500微米)之範圍內且垂直尺寸係在自約25微米至約200微米(例如自約40微米至約150微米)之範圍內。應注意，一流動通道可在一微流體器件中具有各種不同空間構形，且因此不受限於一完全線性元件。例如，一流動通道可為或包含具有下列構形之一或多個區段：曲線、彎曲、螺旋、傾斜、下 傾、分叉(例如多個不同流動路徑)及其等之任何組合。另外，一流動通道可具有沿其路徑之不同加寬及收縮橫截面積以在其內提供一所要流體流動。 As used herein, a "microfluidic channel" or "flow channel" refers to a flow region of a microfluidic device that has a length that is significantly longer than either a horizontal dimension and a vertical dimension. For example, the flow channel can be at least 5 times the length of the horizontal or vertical dimension, such as at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 300 times the length, at least 400 times the length, at least 500 Multiple length or longer. In some embodiments, a flow channel has a length ranging from about 20,000 microns to about 100,000 microns, including any range therebetween. In some embodiments, the horizontal dimension ranges from about 100 microns to about 1000 microns (eg, from about 150 microns to about 500 microns) and the vertical dimension ranges from about 25 microns to about 200 microns (eg, from about 40 microns). Up to about 150 microns). It should be noted that a flow channel can have a variety of different spatial configurations in a microfluidic device, and thus is not limited to a fully linear element. For example, a flow channel can be or include one or more segments having the following configuration: curve, bend, spiral, tilt, down Tilting, bifurcation (eg, multiple different flow paths), and any combination thereof. Additionally, a flow channel can have a different widened and contracted cross-sectional area along its path to provide a desired fluid flow therein.

如本文中所使用，術語「障礙」一般係指一凸塊或類似類型之結構，其足夠大以便部分(但非完全)阻礙目標微小物體在一微流體器件中之兩個不同區域或線路元件之間的移動。該兩個不同區域/線路元件可為(例如)一微流體培育室及一微流體通道或一微流體培育室之一連接區域及一隔離區域。 As used herein, the term "barrier" generally refers to a bump or similar type of structure that is large enough to partially (but not completely) obstruct two small areas or line elements of a target micro-object in a microfluidic device. The movement between. The two different zone/line elements can be, for example, a microfluidic incubation chamber and a microfluidic channel or a microfluidic incubation chamber connection region and an isolation region.

如本文中所使用，術語「收縮」一般係指一微流體器件中之一線路元件(或兩個線路元件之間的一界面)之一寬度之一變窄。收縮可定位於(例如)一微流體培育室與一微流體通道之間的界面處或定位於一微流體培育室之一隔離區域與一連接區域之間的界面處。 As used herein, the term "shrinkage" generally refers to a narrowing of one of the widths of one of the line elements (or an interface between two line elements) in a microfluidic device. The contraction can be located, for example, at an interface between a microfluidic incubation chamber and a microfluidic channel or at an interface between an isolated region of a microfluidic incubation chamber and a connection region.

如本文中所使用，「透明」係指允許可見光穿過且實質上不會在該光穿過時更改該光之一材料。 As used herein, "transparent" refers to a material that allows visible light to pass through and does not substantially alter the light as it passes.

如本文中所使用，術語「微小物體」一般係指可根據本發明而隔離及收集之任何微觀物體。微小物體之非限制性實例包含：無生命微小物體，諸如微粒；微珠(例如聚苯乙烯珠粒、Luminex^TM珠粒或其類似者)；磁珠；微桿；微絲；量子點及其類似者；生物微小物體，諸如細胞(例如胚胎細胞、卵母細胞、***細胞、自一組織解離之細胞、真核細胞、原生生物細胞、動物細胞、哺乳動物細胞、人類細胞、免疫細胞(其包含(但不限於)T細胞、B細胞、自然殺手細胞、巨噬細胞、樹突細胞及其類似者)、融合瘤細胞、培養細胞、來自一細胞系之細胞、癌細胞(其包含(但不限於)循環腫瘤細胞)、受感染細胞、轉染及/或轉形細胞(其包含(但不限於)CHO細胞)、報導體細胞、原核細胞及其類似者)；生物細胞器(例如細胞核)；囊泡或複合物；合成囊泡；脂質體(例如，合成的或衍生自膜配製物)；脂質奈筏(如 Ritchie等人(2009)之「Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs」(Methods Enzymol.，464：211-231)中所描述)及其類似者；或無生命微小物體及生物微小物體之一組合(例如附接至細胞之微珠、經脂質體塗佈之微珠、經脂質體塗佈之磁珠或其類似者)。珠粒可進一步具有共價或非共價鍵聯之其他部分/分子，諸如螢光標記、蛋白質、小分子傳信部分、抗原或能夠用於一化驗中之化學/生物物種。 As used herein, the term "microscopic object" generally refers to any microscopic object that can be isolated and collected in accordance with the present invention. Non-limiting examples of the fine objects comprising: fine inanimate objects, such as fine particles; beads (e.g., polystyrene beads, Luminex ^TM beads or the like); beads; micro rod; microfilament; quantum dots and Similar to; biological microscopic objects, such as cells (eg, embryonic cells, oocytes, sperm cells, cells dissociated from a tissue, eukaryotic cells, protist cells, animal cells, mammalian cells, human cells, immune cells (its Including (but not limited to) T cells, B cells, natural killer cells, macrophages, dendritic cells and the like), fusion tumor cells, cultured cells, cells from a cell line, cancer cells (which contain (but Not limited to) circulating tumor cells), infected cells, transfected and/or transformed cells (including but not limited to CHO cells), reporter cells, prokaryotic cells and the like); biological organelles (eg, nuclei) a vesicle or complex; a synthetic vesicle; a liposome (eg, synthetic or derived from a membrane formulation); a lipid nep (such as Ritchie et al. (2009) "Reconstitution of Membrane Proteins in Phospholipid Bila Yer Nanodiscs" (described in Methods Enzymol., 464:211-231) and the like; or a combination of inanimate tiny objects and biological microscopic objects (eg, microbeads attached to cells, liposomes coated) Microbeads, liposomes coated magnetic beads or the like). The beads may further have other moieties/molecules that are covalently or non-covalently linked, such as fluorescent labels, proteins, small molecule signaling moieties, antigens, or chemical/biological species that can be used in an assay.

如本文中所使用，術語「細胞」係指一生物細胞，其可為一植物細胞、一動物細胞(例如一哺乳動物細胞)、一細菌細胞、一真菌細胞或其類似者。一哺乳動物細胞可來自(例如)一人、一小鼠、一大鼠、一馬、一山羊、一綿羊、一牛、一靈長類動物或其類似者。 As used herein, the term "cell" refers to a biological cell which may be a plant cell, an animal cell (eg, a mammalian cell), a bacterial cell, a fungal cell, or the like. A mammalian cell can be derived, for example, from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, a primate or the like.

若能夠生殖之一生物細胞群落中之全部活細胞係衍生自一單一母細胞之子細胞，則該群落係「選殖的」。術語「選殖細胞」係指相同選殖群落之細胞。 A community is "selected" if it is capable of reproducing all living cell lines in one of the biological cell populations derived from a single parent cell. The term "selected cells" refers to cells of the same selected colony.

如本文中所使用，生物細胞之「群落」係指2個或2個以上細胞(例如2個至20個、4個至40個、6個至60個、8個至80個、10個至100個、20個至200個、40個至400個、60個至600個、80個至800個、100個至1000個或大於1000個細胞)。 As used herein, a "community" of a biological cell refers to two or more cells (eg, 2 to 20, 4 to 40, 6 to 60, 8 to 80, 10 to 100, 20 to 200, 40 to 400, 60 to 600, 80 to 800, 100 to 1000 or more than 1000 cells).

如本文中所使用，術語「維持(一或若干)細胞」係指提供包括流體組分及氣體組分兩者(其等提供使細胞保持存活及/或擴增所需之條件)之一環境。 As used herein, the term "maintaining (one or several) cells" refers to providing an environment that includes both fluid components and gas components (which provide the conditions needed to maintain cells for survival and/or expansion). .

如本文中所使用，術語「擴增」在涉及細胞時係指細胞數目之增加。 As used herein, the term "amplification" when referring to a cell refers to an increase in the number of cells.

如本文中所涉及，「可透氣」意謂材料或結構可滲透氧氣、二氧化碳或氮氣之至少一者。在一些實施例中，可透氣材料或結構可滲透氧氣、二氧化碳及氮氣之一者以上且可進一步滲透此等氣體之全部三 者。 As used herein, "breathable" means that the material or structure is permeable to at least one of oxygen, carbon dioxide, or nitrogen. In some embodiments, the gas permeable material or structure can penetrate more than one of oxygen, carbon dioxide, and nitrogen and can further penetrate all three of the gases. By.

一流體培養基之一「組分」係存在於該培養基中之任何化學或生化分子，其包含溶劑分子、離子、小分子、抗生素、核苷酸及核苷、核酸、胺基酸、肽、蛋白質、糖、碳水化合物、脂質、脂肪酸、膽固醇、代謝物或其類似者。 A "component" of a fluid medium is any chemical or biochemical molecule present in the medium, which comprises solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins. , sugar, carbohydrates, lipids, fatty acids, cholesterol, metabolites or the like.

如本文中參考一流體培養基所使用，「擴散」係指該流體培養基之一組分依一濃度梯度之熱力移動。 As used herein with reference to a fluid medium, "diffusion" means that one of the components of the fluid medium moves according to the thermal force of a concentration gradient.

片語「一培養基之流動」意謂主要歸因於除擴散之外之任何機制之一流體培養基之整體移動。例如，一培養基之流動可涉及該流體培養基自一點至另一點之移動(歸因於該等點之間的壓力差)。此流動可包含液體之一連續、脈衝、週期性、隨機、間歇或往復流動或其等之任何組合。當一流體培養基流動至另一流體培養基中時，可導致該等培養基之紊流及混合。 The phrase "flow of a medium" means primarily due to the overall movement of the fluid medium, which is one of any mechanism other than diffusion. For example, the flow of a medium may involve movement of the fluid medium from one point to another (due to the pressure difference between the points). This flow may comprise one of a continuous, pulsed, periodic, random, intermittent or reciprocating flow of liquid or any combination thereof. When a fluid medium flows into another fluid medium, it can cause turbulence and mixing of the medium.

片語「實質上無流動」係指一流體培養基之一流動速率，其當隨時間平均化時小於一材料(例如所關注之一分析物)之組分擴散至該流體培養基中或該流體培養基內之速率。此一材料之組分之擴散速率可取決於(例如)溫度、該等組分之大小及該等組分與該流體培養基之間的相互作用之強度。 The phrase "substantially no flow" refers to a flow rate of a fluid medium that, when averaged over time, is less than a component of a material (eg, one of the analytes of interest) diffuses into the fluid medium or the fluid medium The rate inside. The rate of diffusion of the components of such a material may depend, for example, on the temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

如本文中參考一微流體器件內之不同區域所使用，片語「流體地連接」意謂：當該等不同區域實質上填充有流體(諸如流體培養基)時，該等區域之各者中之流體經連接以便形成一單一流體。此不意謂該等不同區域中之流體(或流體培養基)需要具有相同組合物。確切而言，一微流體器件之不同流體連接區域中之流體可具有不同組合物(例如不同濃度之溶質，諸如蛋白質、碳水化合物、離子或其他分子)，其等隨著溶質依其各自濃度梯度移動及/或流體流動通過該器件而變化。 As used herein with reference to different regions within a microfluidic device, the phrase "fluidly connected" means that when the different regions are substantially filled with a fluid, such as a fluid medium, each of the regions The fluid is connected to form a single fluid. This does not mean that the fluid (or fluid medium) in the different regions needs to have the same composition. Specifically, the fluids in different fluid connection regions of a microfluidic device can have different compositions (eg, different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules), such as the solute according to their respective concentration gradients. Movement and/or fluid flow varies through the device.

一微流體(或奈流體)器件可包括「掃及」區域及「未掃及」區域。如本文中所使用，一「掃及」區域由一微流體線路之一或多個流體互連線路元件組成，該一或多個流體互連線路元件之各者在流體流動通過該微流體線路時經歷一培養基之流動。一掃及區域之該等線路元件可包含(例如)區域、通道及全部或部分室。如本文中所使用，一「未掃及」區域由一微流體線路之一或多個流體互連線路元件組成，該一或多個流體互連線路元件之各者在流體流動通過該微流體線路時實質上未經歷流體通量。一未掃及區域可流體地連接至一掃及區域，前提是：該等流體連接經結構化以實現培養基在該掃及區域與該未掃及區域之間的擴散但實質上未實現培養基在該掃及區域與該未掃及區域之間的流動。因此，該微流體器件可經結構化以實質上使一未掃及區域與一掃及區域中之培養基之一流動隔離，同時實質上僅實現該掃及區域與該未掃及區域之間的擴散性流體連通。例如，一微流體器件之一流動通道係一掃及區域之一實例，而一微流體器件之一隔離區域(下文將進一步詳細描述)係一未掃及區域之一實例。 A microfluidic (or nanofluidic) device can include a "sweep" region and an "unswept" region. As used herein, a "sweep" region is comprised of one or a plurality of fluid interconnecting circuit elements, each of which is in fluid flow through the microfluidic circuit. It flows through a medium. The line elements of the swept area may include, for example, zones, channels, and all or part of the chamber. As used herein, an "unswept" region is comprised of one or a plurality of fluid interconnecting circuit elements, each of which is in fluid flow through the microfluidic fluid. The fluid flux is not substantially experienced at the time of the line. An unswept region is fluidly connectable to the sweep and region, provided that the fluid connections are structured to effect diffusion of the medium between the sweep region and the unswept region but substantially no material is achieved Sweep the flow between the area and the unswept area. Thus, the microfluidic device can be structured to substantially isolate an unswept region from one of the culture medium in a sweep region while substantially only achieving diffusion between the sweep region and the unswept region Sexual fluid communication. For example, one of the flow channels of a microfluidic device is an example of a sweep and region, and an isolated region of a microfluidic device (described in further detail below) is an example of an unswept region.

如本文中所使用，流體培養基流之一「非掃及」速率意謂足以容許生長室之一隔離區域中之一第二流體培養基之組分擴散至流動區域中之第一流體培養基中及/或容許第一流體培養基之組分擴散至隔離區域中之第二流體培養基中之一流動速率；且其中第一培養基實質上未進一步流動至隔離區域中。 As used herein, a "non-sweep" rate of a fluid medium stream is sufficient to allow diffusion of one of the second fluid medium components in one of the isolation chambers of the growth chamber into the first fluid medium in the flow region and/or Or allowing a component of the first fluid medium to diffuse to a flow rate in the second fluid medium in the isolated region; and wherein the first medium does not substantially flow further into the isolated region.

如本文中所使用，一「流動路徑」係指一或多個流體連接線路元件(例如(若干)通道、(若干)區域、(若干)室及其類似者)，其等界定培養基之一流動之軌跡且經受培養基之一流動。因此，一流動路徑係一微流體器件之一掃及區域之一實例。其他線路元件(例如未掃及區域)可與包括該流動路徑但未經受該流動路徑之培養基之流動的線路元件流體地連接。 As used herein, a "flow path" refers to one or more fluid connection line elements (eg, (several) channels, (several) regions, (several) chambers, and the like) that define the flow of one of the media The trajectory is followed by one of the media flows. Thus, a flow path is one example of a region of a microfluidic device that sweeps. Other line components (e.g., unswept areas) may be fluidly coupled to line elements that include the flow path but are not subject to flow of the medium of the flow path.

如本文中所使用，「伸芳基」係指具有6個至10個環原子之芳香族基(例如，C6-C10芳香族或C6-C10芳基)，其具有至少一環(其具有呈碳環形之一共軛π電子系統(例如苯基、茀基及萘基))且具有附接至一分子之其他部分之一或兩個點。每當一數值範圍(諸如「6至10」)出現於本文中時，其係指該給定範圍內之各整數；例如，「6個至10個環原子」意謂芳基可由6個環原子、7個環原子等等(直至且包含10個環原子)組成。該術語包含單環或稠環多環(即，共用環原子之相鄰對之環)基。伸芳基之實例包含(但不限於)伸苯基、伸萘基及其類似者)。伸芳基部分可進一步經取代或可僅具有附接至分子之其他部分之一或兩點之取代。 As used herein, "extended aryl" refers to an aromatic radical having from 6 to 10 ring atoms (eg, C6-C10 aromatic or C6-C10 aryl) having at least one ring (which has carbon One of the rings is a conjugated π-electron system (eg, phenyl, fluorenyl, and naphthyl) and has one or two points attached to other moieties of one molecule. Whenever a numerical range (such as "6 to 10") appears herein, it refers to each integer within the given range; for example, "6 to 10 ring atoms" means that the aryl group can be 6 rings An atom, 7 ring atoms, etc. (up to and including 10 ring atoms). The term encompasses a monocyclic or fused-ring polycyclic ring (ie, a ring of adjacent pairs of shared ring atoms). Examples of aryl groups include, but are not limited to, phenylene, naphthyl, and the like. The aryl moiety can be further substituted or can have only one or two substitutions attached to other moieties of the molecule.

如本文中所使用，「伸雜芳基」係指一5員至18員芳香族基(例如C3-C13雜芳基)，其包含選自氮、氧及硫之一或多個環雜原子，且可包含一單環、雙環、三環或四環系統且前綴「伸」指示雜芳基環系統具有附接至一分子之其他部分之一或兩個點。每當一數值範圍(諸如「5至18」)出現於本文中時，其係指該給定範圍內之各整數；例如，「5個至18個環原子」意謂雜芳基可由5個環原子、6個環原子等等(直至且包含18個環原子)組成。一含N之「雜芳香族」或「雜芳基」部分係指芳香基，其中環之骨骼原子之至少一者係一氮原子。多環雜芳基可為稠合的或非稠合的。可視情況氧化雜芳基中之(若干)雜原子。可視情況季銨化一或多個氮原子(若存在)。雜芳基透過(若干)環之任何原子而附接至分子之剩餘部分。伸雜芳基之實例包含(但不限於)伸苯并咪唑基、伸苯吲哚基、伸異噁唑基、伸噻唑基、伸***基、伸四唑基及伸苯硫基(即，伸噻吩基)。伸雜芳基部分可進一步經取代或可僅具有附接至該分子之其他部分之一或兩個點之取代。 As used herein, "heteroaryl" refers to a 5 to 18 membered aromatic group (eg, C3-C13 heteroaryl) containing one or more ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. And may comprise a monocyclic, bicyclic, tricyclic or tetracyclic system and the prefix "extension" indicates that the heteroaryl ring system has one or two points attached to one other moiety of a molecule. Whenever a range of values (such as "5 to 18") is used herein, it refers to each integer within the given range; for example, "5 to 18 ring atoms" means that the heteroaryl group can be 5 It consists of a ring atom, 6 ring atoms, etc. (up to and including 18 ring atoms). A "heteroaromatic" or "heteroaryl" moiety containing N refers to an aryl group wherein at least one of the skeletal atoms of the ring is a nitrogen atom. Polycyclic heteroaryl groups can be fused or non-fused. The (several) heteroatoms in the heteroaryl group may be oxidized as appropriate. One or more nitrogen atoms, if any, may be quaternized as appropriate. The heteroaryl is attached to the remainder of the molecule through any atom of the ring(s). Examples of heteroaryl groups include, but are not limited to, benzimidazolyl, benzoyl, exoisoxazole, thiazolyl, triazolyl, tetrazolyl, and phenylthio (ie, Extending thienyl). The heteroaryl moiety can be further substituted or can have only one or two points attached to other moieties of the molecule.

如本文中所使用，術語「雜環」係指：一經或未經3員、4員、5員、6員或7員取代之飽和或部分不飽和環，其含有1個、2個或3個雜 原子，較佳地，含有獨立地選自氧、氮及硫之1個或2個雜原子；或一雙環系統，其含有高達10個原子，該等原子包含獨立地選自氧、氮及硫之至少一雜原子，其中含有雜原子之環係飽和的。雜環基之實例包含(但不限於)四氫呋喃基、四氫糠基、吡咯啶基、六氫吡啶基、4-吡喃基、四氫吡喃基、硫基、嗎啉基、六氫吡嗪基、二氧戊環基、二氧己環基、吲哚啉基及5-甲基-6-苯并二氫吡喃基。雜環基具有附接至分子之其他部分之一或兩個點或可進一步經取代或可不進一步經取代。 As used herein, the term "heterocycle" refers to a saturated or partially unsaturated ring that has been substituted with 3, 4, 5, 6 or 7 members, and contains 1, 2 or 3 a hetero atom, preferably containing one or two heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur; or a bicyclic system containing up to 10 atoms, the atoms comprising independently selected from the group consisting of oxygen and nitrogen And at least one hetero atom of sulfur, wherein the ring containing the hetero atom is saturated. Examples of heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydroindenyl, pyrrolidinyl, hexahydropyridyl, 4-pyranyl, tetrahydropyranyl, sulfur Methyl, morpholinyl, hexahydropyrazinyl, dioxolanyl, dioxocyclo, porphyrin and 5-methyl-6-chroman. The heterocyclic group has one or two points attached to other moieties of the molecule or may be further substituted or may not be further substituted.

系統system

本發明提供一種用於在一微流體器件中培養一或多個生物細胞之系統，其包含一微流體器件，該微流體器件包括：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其中該生長室具有經調節以支援細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 The present invention provides a system for culturing one or more biological cells in a microfluidic device, comprising a microfluidic device comprising: a flow region configured to contain a first fluid medium One of the flows; and at least one growth chamber, wherein the growth chamber has at least one surface that is adjusted to support cell growth, viability, portability, or any combination thereof.

微流體器件及用於操作及觀測此等器件之系統Microfluidic devices and systems for operating and observing such devices

圖1繪示可用於本發明之實踐中之一微流體器件100及一系統150之一實例。圖中展示微流體器件100之一透視圖，其使其蓋110經部分切除以提供微流體器件100之一部分視圖。微流體器件100大體上包括一微流體線路120，其包括一流動路徑106，一流體培養基180可流動通過流動路徑106以視情況運送一或多個微小物體(圖中未展示)至微流體線路120中及/或通過微流體線路120。雖然圖1中繪示一單一微流體線路120，但適合微流體器件可包含複數個(例如2個或3個)此等微流體線路。無論如何，微流體器件100可經構形為一奈流體器件。在圖1所繪示之實施例中，微流體線路120包括複數個微流體生長室124、126、128及130，其等各具有與流動路徑106流體連通之一或多個開口。如下文將進一步討論，微流體生長室包括各種特徵及結構， 其等已經最佳化以即使在一培養基180流動通過流動路徑106時亦使微小物體保留於微流體器件(諸如微流體器件100)中。然而，在開始描述上述各者之前，提供微流體器件100及系統150之一簡要描述。 1 illustrates an example of a microfluidic device 100 and a system 150 that can be used in the practice of the present invention. A perspective view of one of the microfluidic devices 100 is shown, with its cover 110 partially cut away to provide a partial view of the microfluidic device 100. The microfluidic device 100 generally includes a microfluidic line 120 that includes a flow path 106 through which a fluid medium 180 can flow to transport one or more tiny objects (not shown) to the microfluidic line as appropriate. 120 and/or through microfluidic line 120. Although a single microfluidic line 120 is illustrated in FIG. 1, suitable microfluidic devices can include a plurality (eg, 2 or 3) of such microfluidic circuits. In any event, the microfluidic device 100 can be configured as a one-way fluid device. In the embodiment illustrated in FIG. 1, microfluidic line 120 includes a plurality of microfluidic growth chambers 124, 126, 128, and 130, each having one or more openings in fluid communication with flow path 106. As will be discussed further below, the microfluidic growth chamber includes various features and structures, It has been optimized to retain tiny objects in the microfluidic device (such as the microfluidic device 100) even as it flows through the flow path 106. However, a brief description of one of the microfluidic device 100 and system 150 is provided prior to beginning the description of each of the above.

如圖1中大體上所繪示，微流體線路120由一封閉體102界定。雖然可將封閉體102實體地結構化成不同構形，但在圖1所展示之實例中，將封閉體102描繪為包括一支撐結構104(例如一基底)、一微流體線路結構108及一蓋110。支撐結構104、微流體線路結構108及蓋110可彼此附接。例如，微流體線路結構108可安置於支撐結構104之一內表面109上，且蓋110可安置於微流體線路結構108上方。微流體線路結構108可與支撐結構104及蓋110一起界定微流體線路120之元件。 As generally illustrated in FIG. 1, the microfluidic line 120 is defined by an enclosure 102. Although the enclosure 102 can be physically structured into different configurations, in the example shown in FIG. 1, the enclosure 102 is depicted as including a support structure 104 (eg, a substrate), a microfluidic circuit structure 108, and a cover. 110. The support structure 104, the microfluidic line structure 108, and the cover 110 can be attached to each other. For example, the microfluidic line structure 108 can be disposed on an inner surface 109 of the support structure 104 and the cover 110 can be disposed over the microfluidic line structure 108. The microfluidic line structure 108 can define elements of the microfluidic line 120 with the support structure 104 and the cover 110.

支撐結構104可位於微流體線路120之底部處且蓋110可位於微流體線路120之頂部處，如圖1中所繪示。替代地，可使支撐結構104及蓋110依其他定向構形。例如，支撐結構104可位於微流體線路120之頂部處且蓋110可位於微流體線路120之底部處。無論如何，可存在各包括進入或離開封閉體102之一通路之一或多個端口107。一通路之實例包含一閥、一閘、一貫通孔或其類似者。如圖中所繪示，端口107係由微流體線路結構108中之一間隙產生之一貫通孔。然而，端口107可位於封閉體102之其他組件(諸如蓋110)中。雖然圖1中僅繪示一個端口107，但微流體線路120可具有兩個或兩個以上端口107。例如，可存在用作使流體進入微流體線路120之一入口之一第一端口107，且可存在用作使流體退出微流體線路120之一出口之一第二端口107。一端口107是否用作一入口或一出口可取決於流體流動通過流體路徑106之方向。 The support structure 104 can be located at the bottom of the microfluidic line 120 and the cover 110 can be located at the top of the microfluidic line 120, as depicted in FIG. Alternatively, support structure 104 and cover 110 can be configured in other orientations. For example, the support structure 104 can be located at the top of the microfluidic line 120 and the cover 110 can be located at the bottom of the microfluidic line 120. In any event, there may be one or more ports 107 each including one of the passages into or out of the enclosure 102. An example of a passage includes a valve, a gate, a through hole, or the like. As depicted in the figure, port 107 is one of the through holes created by one of the gaps in microfluidic line structure 108. However, port 107 can be located in other components of enclosure 102, such as cover 110. Although only one port 107 is illustrated in FIG. 1, the microfluidic line 120 can have two or more ports 107. For example, there may be a first port 107 that serves to bring fluid into one of the inlets of the microfluidic line 120, and there may be a second port 107 that serves to withdraw fluid from one of the outlets of the microfluidic line 120. Whether a port 107 acts as an inlet or an outlet may depend on the direction of fluid flow through the fluid path 106.

支撐結構104可包括一或多個電極(圖中未展示)及一基板或複數個互連基板。例如，支撐結構104可包括一或多個半導體基板，其等之各者電連接至一電極(例如，該等半導體基板之全部或一子集可電 連接至一單一電極)。支撐結構104可進一步包括一印刷電路板總成(「PCBA」)。例如，該(等)半導體基板可安裝於一PCBA上。 The support structure 104 can include one or more electrodes (not shown) and a substrate or a plurality of interconnect substrates. For example, the support structure 104 can include one or more semiconductor substrates, each of which is electrically coupled to an electrode (eg, all or a subset of the semiconductor substrates can be electrically Connect to a single electrode). The support structure 104 can further include a printed circuit board assembly ("PCBA"). For example, the (etc.) semiconductor substrate can be mounted on a PCBA.

微流體線路結構108可界定微流體線路120之線路元件。此等線路元件可包括可在微流體線路120填充有流體時流體地互連之空間或區域，諸如流動通道、室、圍欄、捕集器及其類似者。在圖1所繪示之微流體線路120中，微流體線路結構108包括一框架114及一微流體線路材料116。框架114可部分或完全圍封微流體線路材料116。框架114可為(例如)實質上包圍微流體線路材料116之一相對剛性結構。例如，框架114可包括一金屬材料。 The microfluidic line structure 108 can define the line elements of the microfluidic line 120. Such line elements can include spaces or areas that can be fluidly interconnected when the microfluidic line 120 is filled with fluid, such as flow channels, chambers, fences, traps, and the like. In the microfluidic circuit 120 illustrated in FIG. 1, the microfluidic line structure 108 includes a frame 114 and a microfluidic line material 116. The frame 114 may partially or completely enclose the microfluidic line material 116. The frame 114 can be, for example, substantially surrounding a relatively rigid structure of the microfluidic line material 116. For example, the frame 114 can include a metallic material.

可使用空穴或其類似者來圖案化微流體線路材料116以界定微流體線路120之線路元件及互連件。微流體線路材料116可包括可透氣之一可撓性材料，諸如一可撓性聚合物(例如橡膠、塑膠、彈性體、聚矽氧、聚二甲矽氧烷(「PDMS」)或其類似者)。可構成微流體線路材料116之材料之其他實例包含模製玻璃、一可蝕刻材料(諸如聚矽氧(例如光可圖案化聚矽氧或「PPS」)、光阻劑(例如SU8)或其類似者。在一些實施例中，此等材料(及因此微流體線路材料116)可為剛性的及/或實質上可不透氣的。無論如何，微流體線路材料116可安置於支撐結構104上及框架114內。 The microfluidic line material 116 can be patterned using holes or the like to define the line elements and interconnects of the microfluidic line 120. The microfluidic line material 116 can comprise a gas permeable one of a flexible material such as a flexible polymer (eg, rubber, plastic, elastomer, polyoxyxene, polydimethyloxane ("PDMS"), or the like. By). Other examples of materials that may form the microfluidic line material 116 include molded glass, an etchable material such as polyfluorene (eg, photopatternable polyoxyl or "PPS"), a photoresist (eg, SU8), or Similarly, in some embodiments, the materials (and thus the microfluidic line material 116) can be rigid and/or substantially gas impermeable. In any event, the microfluidic line material 116 can be disposed on the support structure 104 and Inside the frame 114.

蓋110可為框架114及/或微流體線路材料116之一整合部分。替代地，蓋110可為一不同結構元件，如圖1中所繪示。蓋110可包括相同於或不同於框架114及/或微流體線路材料116之材料。類似地，支撐結構104可為與框架114或微流體線路材料116分離之一結構(如圖中所繪示)或為框架114或微流體線路材料116之一整合部分。同樣地，框架114及微流體線路材料116可為分離結構(如圖1中所展示)或為相同結構之整合部分。 The cover 110 can be an integral part of the frame 114 and/or the microfluidic line material 116. Alternatively, cover 110 can be a different structural element, as depicted in FIG. Cover 110 may comprise the same material as or different from frame 114 and/or microfluidic line material 116. Similarly, the support structure 104 can be one of the structures separate from the frame 114 or the microfluidic line material 116 (as illustrated) or one of the frame 114 or the microfluidic line material 116. Likewise, frame 114 and microfluidic line material 116 can be separate structures (as shown in Figure 1) or an integral part of the same structure.

在一些實施例中，蓋110可包括一剛性材料。該剛性材料可為玻 璃或具有類似性質之一材料。在一些實施例中，蓋110可包括一可變形材料。該可變形材料可為一聚合物，諸如PDMS。在一些實施例中，蓋110可包括剛性材料及可變形材料兩者。例如，蓋110之一或多個部分(例如定位於生長室124、126、128、130上方之一或多個部分)可包括與蓋110之剛性材料界接之一可變形材料。在一些實施例中，蓋110可進一步包含一或多個電極。該一或多個電極可包括可塗佈於玻璃或一類似絕緣材料上之一導電氧化物，諸如氧化銦錫(ITO)。替代地，該一或多個電極可為嵌入於一可變形材料(諸如一聚合物(例如PDMS))中之可撓性電極，諸如單壁奈米管、多壁奈米管、奈米線、導電奈米粒子之叢集或其等之組合。例如，U.S.2012/0325665(Chiou等人)中已描述可用於微流體器件中之可撓性電極，該案之內容以引用的方式併入本文中。在一些實施例中，蓋110可經改質(例如，藉由調節向內面向微流體線路120之一表面之全部或部分)以支援細胞黏著、存活性及/或生長。該改質可包含塗佈一合成或天然聚合物。在一些實施例中，蓋110及/或支撐結構104可為透光的。蓋110亦可包含至少一可透氣材料，例如PDMS或PPS。 In some embodiments, the cover 110 can include a rigid material. The rigid material can be glass Glass or a material with similar properties. In some embodiments, the cover 110 can include a deformable material. The deformable material can be a polymer such as PDMS. In some embodiments, the cover 110 can include both a rigid material and a deformable material. For example, one or more portions of the cover 110 (eg, positioned one or more portions above the growth chambers 124, 126, 128, 130) can include one of the deformable materials that interface with the rigid material of the cover 110. In some embodiments, the cover 110 can further include one or more electrodes. The one or more electrodes can comprise a conductive oxide, such as indium tin oxide (ITO), that can be coated on glass or a similar insulating material. Alternatively, the one or more electrodes may be flexible electrodes embedded in a deformable material such as a polymer (eg, PDMS), such as single-walled nanotubes, multi-walled nanotubes, nanowires , a cluster of conductive nanoparticles or a combination thereof. Flexible electrodes that can be used in microfluidic devices have been described, for example, in U.S. Patent Application Serial No. 2012/032566, the entire disclosure of which is incorporated herein by reference. In some embodiments, the cover 110 can be modified (eg, by adjusting all or a portion of the surface of one of the microfluidic lines 120 inwardly) to support cell adhesion, viability, and/or growth. The modification can comprise coating a synthetic or natural polymer. In some embodiments, the cover 110 and/or the support structure 104 can be light transmissive. Cover 110 can also include at least one gas permeable material, such as PDMS or PPS.

圖1亦展示用於操作及控制微流體器件(諸如微流體器件100)之一系統150。如圖中所繪示，系統150包含一電源192、一成像器件194及一傾斜器件190。 FIG. 1 also shows a system 150 for operating and controlling a microfluidic device, such as microfluidic device 100. As shown in the figure, system 150 includes a power source 192, an imaging device 194, and a tilting device 190.

電源192可將電力提供至微流體器件100及/或傾斜器件190以根據需要提供偏壓電壓或電流。電源192可(例如)包括一或多個交流(AC)及/或直流(DC)電壓源或電流源。成像器件194可包括用於擷取微流體線路120內之影像之一器件，諸如一數位攝影機。在一些例項中，成像器件194進一步包括具有一快圖框速率及/或高敏感度(例如，用於弱光應用)之一偵測器。成像器件194亦可包含用於將刺激輻射及/或光束導引至微流體線路120中且收集自微流體線路120(或含於其內之 微小物體)反射或發射之輻射及/或光束之一機構。發射光束可在可見光譜中且可(例如)包含螢光發射。反射光束可包含源自一LED或一寬光譜燈(諸如一汞燈(例如一高壓汞燈)或一氙弧燈)之反射發射。如關於圖3所討論，成像器件194可進一步包含一顯微鏡(或一光學元件串)，其可或可不包含一目鏡。 Power source 192 can provide power to microfluidic device 100 and/or tilt device 190 to provide a bias voltage or current as desired. Power source 192 can, for example, include one or more alternating current (AC) and/or direct current (DC) voltage sources or current sources. Imaging device 194 can include a device for capturing images within microfluidic line 120, such as a digital camera. In some examples, imaging device 194 further includes a detector having a fast frame rate and/or high sensitivity (eg, for low light applications). Imaging device 194 can also include for directing stimulating radiation and/or light beams into microfluidic line 120 and collected from (or contained within) microfluidic line 120 A mechanism that reflects or emits radiation and/or light. The emitted light beam can be in the visible spectrum and can, for example, comprise a fluorescent emission. The reflected beam may comprise a reflected emission originating from an LED or a broad spectrum lamp such as a mercury lamp (e.g., a high pressure mercury lamp) or a xenon arc lamp. As discussed with respect to FIG. 3, imaging device 194 can further include a microscope (or a string of optical elements) that may or may not include an eyepiece.

系統150可進一步包括經構形以使一微流體器件100圍繞一或多個旋轉軸旋轉之一傾斜器件190。在一些實施例中，傾斜器件190經構形以圍繞至少一軸支撐及/或保持包括微流體線路120之封閉體102，使得微流體器件100(及因此微流體線路120)可保持於一水平定向(即，相對於x軸及y軸成0°)、一垂直定向(即，相對於x軸及/或y軸成90°)或其間之任何定向上。微流體器件100(及微流體線路120)相對於一軸之定向在本文中指稱微流體器件100(及微流體線路120)之「傾角」。例如，傾斜器件190可使微流體器件100相對於x軸傾斜0.1°、0.2°、0.3°、0.4°、0.5°、0.6°、0.7°、0.8°、0.9°、1°、2°、3°、4°、5°、10°、15°、20°、25°、30°、35°、40°、45°、50°、55°、60°、65°、70°、75°、80°、90°或其間之任何度數。水平定向(及因此x軸及y軸)經界定為法向於由重力界定之一垂直軸。傾斜器件亦可使微流體器件100(及微流體線路120)相對於x軸及/或y軸傾斜大於90°之任何度數，或使微流體器件100(及微流體線路120)相對於x軸或y軸傾斜180°以使微流體器件100(及微流體線路120)完全倒置。類似地，在一些實施例中，傾斜器件190使微流體器件100(及微流體線路120)圍繞由微流體線路120之流動路徑106或某一其他部分界定之一旋轉軸傾斜。 System 150 can further include a tilting device 190 configured to rotate a microfluidic device 100 about one or more axes of rotation. In some embodiments, the tilting device 190 is configured to support and/or retain the enclosure 102 including the microfluidic line 120 about at least one axis such that the microfluidic device 100 (and thus the microfluidic line 120) can remain in a horizontal orientation. (ie, 0° with respect to the x-axis and y-axis), a vertical orientation (ie, 90° with respect to the x-axis and/or y-axis), or any orientation therebetween. The orientation of microfluidic device 100 (and microfluidic line 120) relative to one axis is referred to herein as the "tilt angle" of microfluidic device 100 (and microfluidic line 120). For example, tilting device 190 can tilt microfluidic device 100 by 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3 with respect to the x-axis. °, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80°, 90° or any degree between them. The horizontal orientation (and thus the x-axis and the y-axis) is defined as normal to one of the vertical axes defined by gravity. The tilting device can also tilt the microfluidic device 100 (and the microfluidic line 120) by any degree greater than 90° relative to the x-axis and/or y-axis, or to cause the microfluidic device 100 (and the microfluidic line 120) to be relative to the x-axis. Or the y-axis is tilted by 180° to completely invert the microfluidic device 100 (and the microfluidic line 120). Similarly, in some embodiments, tilting device 190 tilts microfluidic device 100 (and microfluidic line 120) about one of the axes of rotation defined by flow path 106 or some other portion of microfluidic line 120.

在一些例項中，使微流體器件100傾斜成一垂直定向，使得流動路徑106定位於一或多個生長室上方或一或多個生長室下方。如本文中所使用，術語「上方」指示：在由重力界定之一垂直軸上，流動路徑106定位成高於一或多個生長室(即，一流動路徑106上方之一生長 室中之一物體將具有比流動路徑中之一物體高之一重力勢能)。如本文中所使用，術語「下方」指示：在由重力界定之一垂直軸上，流動路徑106定位成低於一或多個生長室(即，一流動路徑106下方之一生長室中之一物體將具有比流動路徑中之一物體低之一重力勢能)。 In some examples, the microfluidic device 100 is tilted into a vertical orientation such that the flow path 106 is positioned above one or more growth chambers or below one or more growth chambers. As used herein, the term "above" indicates that on one of the vertical axes defined by gravity, the flow path 106 is positioned higher than one or more growth chambers (ie, one of the flow paths 106 is grown above) One of the objects in the chamber will have a gravitational potential energy that is higher than one of the objects in the flow path). As used herein, the term "below" indicates that on one of the vertical axes defined by gravity, the flow path 106 is positioned lower than one or more growth chambers (ie, one of the growth chambers below one of the flow paths 106) The object will have a gravitational potential energy that is lower than one of the objects in the flow path).

在一些例項中，傾斜器件190使微流體器件100圍繞平行於流動路徑106之一軸傾斜。而且，可使微流體器件100傾斜小於90°之一角度，使得流動路徑106定位於一或多個生長室上方或一或多個生長室下方且非直接定位於生長室上方或生長室下方。在其他例項中，傾斜器件190使微流體器件100圍繞垂直於流動路徑106之一軸傾斜。在其他例項中，傾斜器件190使微流體器件100圍繞既不平行於流動路徑106且不垂直於流動路徑106之一軸傾斜。 In some examples, tilting device 190 tilts microfluidic device 100 about an axis that is parallel to flow path 106. Moreover, the microfluidic device 100 can be tilted by an angle of less than 90° such that the flow path 106 is positioned above or below one or more growth chambers and is not directly positioned above the growth chamber or below the growth chamber. In other examples, tilting device 190 tilts microfluidic device 100 about an axis that is perpendicular to flow path 106. In other examples, tilting device 190 causes microfluidic device 100 to tilt about an axis that is neither parallel to flow path 106 and that is not perpendicular to one of flow paths 106.

系統150可進一步包含一培養基源178。培養基源178(例如一容器、儲液器或其類似者)可包括各用於保持一不同流體培養基180之多個區段或容器。因此，培養基源178可為位於微流體器件100外且與微流體器件100分離之一器件，如圖1中所繪示。替代地，培養基源178可全部或部分定位於微流體器件100之封閉體102內。例如，培養基源178可包括儲液器，其係微流體器件100之部分。 System 150 can further include a media source 178. Media source 178 (e.g., a container, reservoir, or the like) can include a plurality of sections or containers each for holding a different fluid medium 180. Thus, the medium source 178 can be one that is external to the microfluidic device 100 and separate from the microfluidic device 100, as depicted in FIG. Alternatively, the medium source 178 can be positioned wholly or partially within the enclosure 102 of the microfluidic device 100. For example, media source 178 can include a reservoir that is part of microfluidic device 100.

圖1亦繪示控制及監測設備152之實例之簡化方塊圖描述，控制及監測設備152構成系統150之部分且可與一微流體器件100一起使用。如圖中所展示，此控制及監測設備152之實例包含：一主控制器154，其包括：用於控制培養基源178之一培養基模組160、用於控制微流體線路120中之微小物體(圖中未展示)及/或培養基(例如培養基之液滴)之移動及/或選擇之一動力模組162、用於控制一成像器件194(例如一攝影機、顯微鏡、光源或其等之任何組合)來擷取影像(例如數位影像)之一成像模組164、及用於控制一傾斜器件190之一傾斜模組166。控制設備152亦可包含用於控制、監測或執行關於微流體器件 100之其他功能之其他模組168。如圖中所展示，設備152可進一步包含一顯示器件170及一輸入/輸出器件172。 1 also illustrates a simplified block diagram depiction of an example of a control and monitoring device 152 that forms part of system 150 and that can be used with a microfluidic device 100. As shown in the figure, an example of such a control and monitoring device 152 includes a main controller 154 that includes a medium module 160 for controlling a medium source 178 for controlling minute objects in the microfluidic line 120 ( Moving and/or selecting one of the power modules 162 for controlling the movement of a medium (eg, a droplet of a medium) for controlling an imaging device 194 (eg, a camera, microscope, light source, or the like, any combination thereof) An imaging module 164 for capturing an image (eg, a digital image) and a tilting module 166 for controlling a tilting device 190. Control device 152 can also include control, monitoring, or execution of the microfluidic device Other modules 168 of other functions of 100. As shown in the figure, device 152 can further include a display device 170 and an input/output device 172.

主控制器154可包括一控制模組156及一數位記憶體158。控制模組156可包括(例如)一數位處理器，其經構形以根據作為非暫時性資料或信號儲存於記憶體158中之機械可執行指令(例如軟體、韌體、原始程式碼或其類似者)而操作。替代地或另外，控制模組156可包括固線式數位電路及/或類比電路。可類似地構形培養基模組160、動力模組162、成像模組164、傾斜模組166及/或其他模組168。因此，可由如上文所討論般構形之主控制器154、培養基模組160、動力模組162、成像模組164、傾斜模組166及/或其他模組168之任何一或多者執行如關於微流體器件100或任何其他微流體裝置而執行之本文中所討論之功能、程序、行動、動作或一程序之步驟。類似地，主控制器154、培養基模組160、動力模組162、成像模組164、傾斜模組166及/或其他模組168可經通信地耦合以傳輸及接收用於本文中所討論之任何功能、程序、行動、動作或步驟中之資料。 The main controller 154 can include a control module 156 and a digital memory 158. Control module 156 can include, for example, a digital processor configured to store mechanically executable instructions (eg, software, firmware, source code, or the like, stored in memory 158 as non-transitory data or signals Similar to). Alternatively or in addition, control module 156 can include a fixed line digital circuit and/or analog circuit. The culture medium module 160, the power module 162, the imaging module 164, the tilt module 166, and/or other modules 168 can be similarly configured. Thus, any one or more of the main controller 154, the culture module 160, the power module 162, the imaging module 164, the tilt module 166, and/or other modules 168 configured as discussed above can be implemented as The steps, functions, procedures, acts, actions, or procedures discussed herein with respect to the microfluidic device 100 or any other microfluidic device. Similarly, main controller 154, culture module 160, power module 162, imaging module 164, tilt module 166, and/or other modules 168 can be communicatively coupled for transmission and reception for use in the discussion herein. Information in any function, procedure, action, action or step.

培養基模組160控制培養基源178。例如，培養基模組160可控制培養基源178將一選定流體培養基180輸入至封閉體102中(例如，透過一入口107)。培養基模組160亦可控制自封閉體102移除培養基(例如，透過一出口(圖中未展示))。因此，可將一或多個培養基選擇性地輸入至微流體線路120中及自微流體線路120移除。培養基模組160亦可控制流體培養基180在微流體線路120內之流動路徑106中之流動。例如，在一些實施例中，在傾斜模組166致使傾斜器件190使微流體器件100傾斜一所要傾斜角之前，培養基模組160停止培養基180在流動路徑106中流動且停止培養基180流動通過封閉體102。 The medium module 160 controls the medium source 178. For example, the culture medium module 160 can control the culture medium source 178 to input a selected fluid culture medium 180 into the enclosure 102 (eg, through an inlet 107). The media module 160 can also control the removal of media from the enclosure 102 (e.g., through an outlet (not shown)). Thus, one or more culture media can be selectively input into and removed from the microfluidic line 120. The media module 160 can also control the flow of the fluid medium 180 in the flow path 106 within the microfluidic line 120. For example, in some embodiments, before the tilt module 166 causes the tilting device 190 to tilt the microfluidic device 100 to a desired tilt angle, the media module 160 stops the flow of the medium 180 in the flow path 106 and stops the flow of the medium 180 through the enclosure. 102.

動力模組162可經構形以控制微流體線路120中之微小物體(圖中未展示)之選擇、捕集及移動。如下文將關於圖2A及圖2B而討論，封 閉體102可包括一介電泳(DEP)、光電鑷子(OET)及/或光電潤濕(OEW)構形(圖1中未展示)，且動力模組162可控制啟動電極及/或電晶體(例如光電晶體)來選擇及移動流動路徑106及/或生長室124、126、128、130中之微小物體(圖中未展示)及/或培養基之液滴(圖中未展示)。 Power module 162 can be configured to control the selection, capture, and movement of tiny objects (not shown) in microfluidic line 120. As will be discussed below with respect to Figures 2A and 2B, The closed body 102 can include a dielectrophoresis (DEP), an optoelectronic tweezers (OET), and/or an opto-wetting (OEW) configuration (not shown in FIG. 1), and the power module 162 can control the actuation electrode and/or the transistor ( For example, a photonic crystal) selects and moves droplets (not shown) of the flow path 106 and/or growth chambers 124, 126, 128, 130 and/or droplets of the culture medium (not shown).

成像模組164可控制成像器件194。例如，成像模組164可接收及處理來自成像器件194之影像資料。來自成像器件194之影像資料可包括由成像器件194擷取之任何類型之資訊(例如，存在或不存在微小物體、培養基之液滴、標記(諸如螢光標記)之累積等等)。成像模組164可使用由成像器件194擷取之資訊來進一步計算物體(例如微小物體、培養基之液滴)之位置及/或此等物體在微流體器件100內之運動速率。 Imaging module 164 can control imaging device 194. For example, imaging module 164 can receive and process image data from imaging device 194. Image data from imaging device 194 can include any type of information captured by imaging device 194 (eg, presence or absence of microscopic objects, droplets of culture medium, accumulation of labels (such as fluorescent markers), etc.). Imaging module 164 can use information captured by imaging device 194 to further calculate the location of objects (eg, tiny objects, droplets of culture medium) and/or the rate of movement of such objects within microfluidic device 100.

傾斜模組166可控制傾斜器件190之傾斜運動。替代地或另外，傾斜模組166可控制傾斜速率及時序以最佳化微小物體經由重力而轉移至一或多個生長室。傾斜模組166與成像模組164通信地耦合以接收描述微小物體及/或培養基之液滴在微流體線路120中之運動之資料。傾斜模組166可使用此資料來調整微流體線路120之傾角以調整微小物體及/或培養基之液滴在微流體線路120中之移動速率。傾斜模組166亦可使用此資料來反覆地調整一微小物體及/或培養基之液滴在微流體線路120中之位置。 The tilt module 166 can control the tilting motion of the tilting device 190. Alternatively or in addition, the tilt module 166 can control the tilt rate and timing to optimize the transfer of tiny objects to one or more growth chambers via gravity. Tilt module 166 is communicatively coupled to imaging module 164 to receive information describing the movement of droplets of microscopic objects and/or culture medium in microfluidic circuitry 120. The tilt module 166 can use this information to adjust the tilt of the microfluidic line 120 to adjust the rate of movement of droplets of fine objects and/or media in the microfluidic line 120. The tilt module 166 can also use this information to repeatedly adjust the position of a droplet of a fine object and/or medium in the microfluidic line 120.

在圖1所展示之實例中，將微流體線路120繪示為包括一微流體通道122及生長室124、126、128、130。各室包括至通道122之一開口，但其他部分經封閉使得室可實質上使室內之微小物體與通道122之流動路徑106中或其他室中之流體培養基180及/或微小物體隔離。在一些實施例中，室124、126、128、130經構形以實體地圈住微流體線路120內之一或多個微小物體。根據本發明之生長室可包括各種形狀、表面及特徵，其等經最佳化以與DEP、OET、OEW及/或重力一 起使用，如將在下文詳細討論及展示。 In the example shown in FIG. 1, microfluidic line 120 is depicted as including a microfluidic channel 122 and growth chambers 124, 126, 128, 130. Each chamber includes an opening to one of the channels 122, but the other portions are closed such that the chamber can substantially isolate the tiny objects in the chamber from the fluid medium 180 and/or tiny objects in the flow path 106 of the channel 122 or in other chambers. In some embodiments, the chambers 124, 126, 128, 130 are configured to physically enclose one or more tiny objects within the microfluidic line 120. Growth chambers in accordance with the present invention can include a variety of shapes, surfaces, and features that are optimized for DEP, OET, OEW, and/or gravity Use as will be discussed and illustrated in detail below.

微流體線路120可包括任何數目個微流體生長室。雖然圖中展示5個生長室，但微流體線路120可具有更少或更多生長室。在一些實施例中，微流體線路120包括複數個微流體生長室，其中該等生長室之兩者或兩者以上包括不同結構及/或特徵。 Microfluidic line 120 can include any number of microfluidic growth chambers. Although five growth chambers are shown in the figures, the microfluidic line 120 can have fewer or more growth chambers. In some embodiments, the microfluidic line 120 includes a plurality of microfluidic growth chambers, wherein two or more of the growth chambers comprise different structures and/or features.

在圖1所繪示之實施例中，展示一單一通道122及流動路徑106。然而，其他實施例可含有各經構形以包括一流動路徑106之多個通道122。微流體線路120進一步包括與流動路徑106及流體培養基180流體連通之一入口閥或入口107，藉此流體培養基180可經由入口107而進入通道122。在一些例項中，流動路徑106包括一單一路徑。在一些例項中，將該單一路徑配置成一Z字形圖案，藉此流動路徑106在交替方向上橫跨微流體器件100行進兩次或兩次以上。 In the embodiment illustrated in FIG. 1, a single channel 122 and flow path 106 are shown. However, other embodiments may include a plurality of channels 122 each configured to include a flow path 106. The microfluidic line 120 further includes an inlet valve or inlet 107 in fluid communication with the flow path 106 and the fluid medium 180, whereby the fluid medium 180 can enter the channel 122 via the inlet 107. In some examples, flow path 106 includes a single path. In some examples, the single path is configured in a zigzag pattern whereby flow path 106 travels across microfluidic device 100 two or more times in alternating directions.

在一些例項中，微流體線路120包括複數個並行通道122及流動路徑106，其中各流動路徑106內之流體培養基180在相同方向上流動。在一些例項中，各流動路徑106內之流體培養基在一向前方向或一相反方向之至少一者上流動。在一些例項中，複數個生長室經構形(例如，相對於一通道122)使得其等可並行地裝載有目標微小物體。 In some examples, the microfluidic line 120 includes a plurality of parallel channels 122 and flow paths 106, wherein the fluid medium 180 within each flow path 106 flows in the same direction. In some embodiments, the fluid medium within each flow path 106 flows in at least one of a forward direction or an opposite direction. In some instances, the plurality of growth chambers are configured (e.g., relative to a channel 122) such that they can be loaded with target microscopic objects in parallel.

在一些實施例中，微流體線路120進一步包括一或多個微小物體捕集器132。捕集器132一般形成於形成一通道122之邊界之一壁中，且可經定位成相對於微流體生長室124、126、128、130之一或多者之一開口。在一些實施例中，捕集器132經構形以接收或捕獲來自流動路徑106之一單一微小物體。在一些實施例中，捕集器132經構形以接收或捕獲來自流動路徑106之複數個微小物體。在一些例項中，捕集器132包括近似等於一單一目標微小物體之體積的一體積。 In some embodiments, the microfluidic line 120 further includes one or more micro object traps 132. The trap 132 is generally formed in one of the walls forming a boundary of the channel 122 and can be positioned to open relative to one of the microfluidic growth chambers 124, 126, 128, 130. In some embodiments, the trap 132 is configured to receive or capture a single tiny object from one of the flow paths 106. In some embodiments, the trap 132 is configured to receive or capture a plurality of tiny objects from the flow path 106. In some instances, the trap 132 includes a volume that is approximately equal to the volume of a single target micro-object.

捕集器132可進一步包括經構形以有助於目標微小物體流動至捕集器132中之一開口。在一些例項中，捕集器132包括具有近似等於一 單一目標微小物體之尺寸之一高度及寬度之一開口，藉此防止較大微小物體進入至微小物體捕集器中。捕集器132可進一步包括經構形以有助於目標微小物體保留於捕集器132內之其他特徵。在一些例項中，捕集器132與一微流體生長室之開口對準且位於與一微流體生長室之開口相對之一通道122之側上，使得在使微流體器件100圍繞平行於通道122之一軸傾斜之後，所捕集之微小物體依致使微小物體降落至生長室之開口中之一軌跡退出捕集器132。在一些例項中，捕集器132包括一側通路134，其小於目標微小物體以促進流動通過捕集器132且藉此增加捕獲捕集器132中之一微小物體之可能性。 The trap 132 can further include a configuration to facilitate flow of the target minute object to one of the openings in the trap 132. In some examples, the trap 132 includes approximately one equal to one One of the heights and widths of one of the dimensions of the single target micro-object is opened, thereby preventing larger tiny objects from entering the micro-object trap. The trap 132 can further include other features configured to facilitate retention of the target minute object within the trap 132. In some embodiments, the trap 132 is aligned with the opening of a microfluidic growth chamber and is located on a side of the channel 122 opposite the opening of a microfluidic growth chamber such that the microfluidic device 100 is placed parallel to the channel. After tilting one of the axes 122, the trapped minute object exits the trap 132 in a trajectory that causes the tiny object to land into the opening of the growth chamber. In some examples, the trap 132 includes a side passage 134 that is smaller than the target minute object to facilitate flow through the trap 132 and thereby increase the likelihood of capturing one of the tiny objects in the trap 132.

在一些實施例中，經由一或多個電極(圖中未展示)而橫跨流體培養基180(例如，在流動路徑中及/或在生長室中)施加介電泳(DEP)力以操縱、運輸、分離及分類定位於流動路徑及/或生長室中之微小物體。例如，在一些實施例中，將DEP力施加於微流體線路120之一或多個部分以將一單一微小物體自流動路徑106轉移至一所要微流體生長室中。在一些實施例中，DEP力用以防止一生長室(例如生長室124、126、128或130)內之一微小物體自該生長室排出。進一步言之，在一些實施例中，DEP力用以自一生長室選擇性地移除先前根據本發明之教示而收集之一微小物體。在一些實施例中，DEP力包括光電鑷子(OET)力。 In some embodiments, a dielectrophoresis (DEP) force is applied across a fluid medium 180 (eg, in a flow path and/or in a growth chamber) via one or more electrodes (not shown) for manipulation, transportation, Separating and classifying tiny objects positioned in the flow path and/or growth chamber. For example, in some embodiments, a DEP force is applied to one or more portions of the microfluidic line 120 to transfer a single tiny object from the flow path 106 into a desired microfluidic growth chamber. In some embodiments, the DEP force is used to prevent a tiny object within a growth chamber (eg, growth chamber 124, 126, 128, or 130) from exiting the growth chamber. Further, in some embodiments, the DEP force is used to selectively remove a small object previously collected from a growth chamber in accordance with the teachings of the present invention. In some embodiments, the DEP force includes an optoelectronic force (OET) force.

在其他實施例中，經由一或多個電極(圖中未展示)而將光電潤濕(OEW)力施加於微流體器件100之支撐結構104(及/或蓋110)中之一或多個位置(例如有助於界定流動路徑及/或生長室之位置)以操縱、運輸、分離及分類定位於微流體線路120中之液滴。例如，在一些實施例中，將OEW力施加於支撐架構104(及/或蓋110)中之一或多個位置以將一單一液滴自流動路徑106轉移至一所要微流體生長室中。在一些實施例中，OEW力用以防止一生長室(例如生長室124、126、128或 130)內之一液滴自該生長室排出。進一步言之，在一些實施例中，OEW力用以自一生長室選擇性地移除先前根據本發明之教示而收集之一液滴。 In other embodiments, a photo-wetting (OEW) force is applied to one or more of the support structures 104 (and/or cover 110) of the microfluidic device 100 via one or more electrodes (not shown). The location (e.g., helps define the location of the flow path and/or growth chamber) to manipulate, transport, separate, and classify droplets positioned in the microfluidic line 120. For example, in some embodiments, an OEW force is applied to one or more locations in support structure 104 (and/or cover 110) to transfer a single drop from flow path 106 into a desired microfluidic growth chamber. In some embodiments, the OEW force is used to prevent a growth chamber (eg, growth chambers 124, 126, 128 or One of the drops in 130) is discharged from the growth chamber. Further, in some embodiments, the OEW force is used to selectively remove one of the droplets previously collected from a growth chamber in accordance with the teachings of the present invention.

在一些實施例中，DEP力及/或OEW力與其他力(諸如流動力及/或重力)組合以便操縱、運輸、分離及分類微流體線路120內之微小物體及/或液滴。例如，封閉體102可經傾斜(例如，藉由傾斜器件190)以將流動路徑106及定位於流動路徑106內之微小物體定位於微流體生長室上方，且重力可將微小物體及/或液滴運輸至室中。在一些實施例中，可在其他力之前施加DEP力及/或OEW力。在其他實施例中，可在其他力之後施加DEP力及/或OEW力。在其他例項中，DEP力及/或OEW力可與其他力同時被施加或與其他力依一交替方式被施加。 In some embodiments, the DEP force and/or OEW force is combined with other forces, such as flow forces and/or gravity, to manipulate, transport, separate, and classify tiny objects and/or droplets within the microfluidic line 120. For example, the enclosure 102 can be tilted (eg, by tilting the device 190) to position the flow path 106 and the tiny objects positioned within the flow path 106 above the microfluidic growth chamber, and gravity can move the tiny objects and/or liquids The drops are transported to the chamber. In some embodiments, the DEP force and/or OEW force can be applied before other forces. In other embodiments, the DEP force and/or the OEW force may be applied after other forces. In other examples, the DEP force and/or OEW force may be applied simultaneously with other forces or alternated with other forces.

圖2A至圖2F繪示可用於本發明之實踐中之微流體器件之各種實施例。圖2A描繪其中微流體器件200經構形為一光學致動之電動器件之一實施例。此項技術中已知各種光學致動之電動器件，其包含具有一光電鑷子(OET)構形之器件及具有一光電潤濕(OEW)構形之器件。下列美國專利文件(其等之各者之全文以引用的方式併入本文中)中繪示適合OET構形之實例：美國專利第RE 44,711號(Wu等人)(最初發表為美國專利第7,612,355號)及美國專利第7,956,339號(Ohta等人)。美國專利第6,958,132號(Chiou等人)及美國專利申請公開案第2012/0024708號(Chiou等人)中繪示OEW構形之實例，該兩個專利之全文以引用的方式併入本文中。一光學致動之電動器件之又一實例包含一組合之OET/OEW構形，美國專利公開案第20150306598號(Khandros等人)及第20150306599號(Khandros等人)及其對應PCT公開案WO2015/164846及WO2015/164847中展示該組合之OET/OEW構形之實例，全部該等案之全文以引用的方式併入本文中。 2A-2F illustrate various embodiments of microfluidic devices that can be used in the practice of the present invention. 2A depicts an embodiment in which the microfluidic device 200 is configured as an optically actuated electromechanical device. Various optically actuated electromechanical devices are known in the art that include a device having an optoelectronic force (OET) configuration and a device having a photo-wetting (OEW) configuration. An example of a suitable OET configuration is shown in the following U.S. Patent Application, the entire disclosure of each of which is hereby incorporated by reference: U.S. Pat. No. No. No. 44,711 (Wu et al.) (published as U.S. Patent No. 7,612,355 No. 7,956,339 (Ohta et al.). Examples of OEW configurations are illustrated in U.S. Patent No. 6,958,132 (Chiou et al.) and U.S. Patent Application Publication No. 2012/0024708 (Chiou et al.). A further example of an optically actuated electromechanical device comprises a combined OET/OEW configuration, U.S. Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599 (Khandros et al.) and their corresponding PCT publication WO2015/ Examples of OET/OEW configurations of this combination are shown in 164,846 and WO 2015/164847, the entireties of each of which are incorporated herein by reference.

動力微流體器件構形Dynamic microfluidic device configuration

如上文所描述，系統之控制及監測設備可包括用於選擇及移動一微流體器件之微流體線路中之物體(諸如微小物體或液滴)之一動力模組。微流體器件可具有取決於所移動之物體之類型及其他考量之各種動力構形。例如，可利用一介電泳(DEP)構形來選擇及移動微流體線路中之微小物體。因此，微流體器件100之支撐結構104及/或蓋110可包括用於選擇性地誘發微流體線路120中之一流體培養基180中之微小物體上之DEP力之一DEP構形且藉此選擇、捕獲及/或移動個別微小物體或微小物體群組。替代地，微流體器件100之支撐結構104及/或蓋110可包括用於選擇性地誘發微流體線路120中之一流體培養基180中之液滴上之電潤濕(EW)力之一EW構形且藉此選擇、捕獲及/或移動個別液滴或液滴群組。 As described above, the control and monitoring device of the system can include a power module for selecting and moving an object (such as a small object or droplet) in a microfluidic circuit of a microfluidic device. The microfluidic device can have a variety of dynamic configurations depending on the type of object being moved and other considerations. For example, a dielectrophoresis (DEP) configuration can be utilized to select and move tiny objects in a microfluidic circuit. Accordingly, the support structure 104 and/or the cover 110 of the microfluidic device 100 can include a DEP configuration for selectively inducing a DEP force on a tiny object in the fluid medium 180 in the microfluidic line 120 and thereby selecting Capture, and/or move individual tiny objects or groups of tiny objects. Alternatively, the support structure 104 and/or cover 110 of the microfluidic device 100 can include one of the electrowetting (EW) forces on the droplets in one of the fluid medium 180 in the microfluidic line 120. The configuration and thereby select, capture and/or move individual droplets or groups of droplets.

圖2A及圖2B中繪示包括一DEP構形之一微流體器件200之一實例。雖然為簡單起見，圖2A及圖2B分別展示具有一敞開區域/室202之微流體器件200之一封閉體102之一部分之一側視橫截面圖及一俯視橫截面圖，但應瞭解，區域/室202可為具有一更詳細結構(諸如一生長室、一流動區域或一流動通道)之一流體線路元件之部分。此外，微流體器件200可包含其他流體線路元件。例如，微流體器件200可包含複數個生長室及/或一或多個流動區域或流動通道，諸如本文中關於微流體器件100所描述之生長室及/或流動區域或流動通道。可將一DEP構形併入至微流體器件200之任何此等流體線路元件或其選擇部分中。應進一步瞭解，上文所描述或下文將描述之微流體器件組件及系統組件之任何者可併入於微流體器件200中及/或與微流體器件200一起使用。例如，包含上文所描述之控制及監測設備152之系統150可與包含培養基模組160、動力模組162、成像模組164、傾斜模組166及其他模組168之一或多者之微流體器件200一起使用。 An example of a microfluidic device 200 that includes a DEP configuration is illustrated in Figures 2A and 2B. Although for simplicity, FIGS. 2A and 2B respectively show a side cross-sectional view and a top cross-sectional view of one of the portions of one of the enclosures 102 of the microfluidic device 200 having an open region/chamber 202, it will be appreciated that The zone/chamber 202 can be part of a fluid circuit component having a more detailed structure, such as a growth chamber, a flow region, or a flow channel. Additionally, the microfluidic device 200 can include other fluid circuit components. For example, the microfluidic device 200 can include a plurality of growth chambers and/or one or more flow regions or flow channels, such as the growth chambers and/or flow regions or flow channels described herein with respect to the microfluidic device 100. A DEP configuration can be incorporated into any of these fluid circuit elements or selected portions of the microfluidic device 200. It should be further appreciated that any of the microfluidic device components and system components described above or described below can be incorporated into and/or used with the microfluidic device 200. For example, the system 150 including the control and monitoring device 152 described above can be combined with one or more of the media module 160, the power module 162, the imaging module 164, the tilt module 166, and other modules 168. The fluidic device 200 is used together.

如圖2A中所見，微流體器件200包含：一支撐結構104，其具有 一底部電極204及上覆於底部電極204上之一電極啟動基板206；及一蓋110，其具有一頂部電極210，其中頂部電極210與底部電極204間隔開。頂部電極210及電極啟動基板206界定區域/室202之相對表面。因此，含於區域/室202中之一培養基180提供頂部電極210與電極啟動基板206之間的一電阻連接。圖中亦展示一電源212，其經構形以連接至底部電極204及頂部電極210且在該等電極之間產生產生區域/室202中之DEP力所需之一偏壓電壓。電源212可為(例如)一交流(AC)電源。 As seen in Figure 2A, the microfluidic device 200 comprises: a support structure 104 having A bottom electrode 204 and an electrode starting substrate 206 overlying the bottom electrode 204; and a cover 110 having a top electrode 210, wherein the top electrode 210 is spaced apart from the bottom electrode 204. The top electrode 210 and the electrode activation substrate 206 define opposing surfaces of the region/chamber 202. Thus, one of the media 180 contained in the zone/chamber 202 provides a resistive connection between the top electrode 210 and the electrode activation substrate 206. Also shown is a power source 212 that is configured to connect to the bottom electrode 204 and the top electrode 210 and to create a bias voltage between the electrodes that produces the DEP force in the region/chamber 202. Power source 212 can be, for example, an alternating current (AC) power source.

在某些實施例中，圖2A及圖2B中所繪示之微流體器件200可具有一光學致動之DEP構形。據此，改變來自光源220之光圖案222(其可由動力模組162控制)可選擇性地啟動及不啟動改變電極啟動基板206之內表面208之區域214處之DEP電極之圖案。(在下文中，具有一DEP構形之一微流體器件之區域214指稱「DEP電極區域」)。如圖2B中所繪示，導引至電極啟動基板206之內表面208上之一光圖案222可照射呈一圖案(諸如一正方形)之選定DEP電極區域214a(展示為白色)。未經照射之DEP電極區域214(畫有交叉影線)在下文中指稱「暗」DEP電極區域214。通過DEP電極啟動基板206(即，自底部電極204直至與流動區域106中之培養基180界接之電極啟動基板206之內表面208)之相對電阻抗大於通過各暗DEP電極區域214處之區域/室202中之培養基180(即，自電極啟動基板206之內表面208至蓋110之頂部電極210)之相對電阻抗。然而，一經照射之DEP電極區域214a展現通過電極啟動基板206之一減小相對阻抗，其小於通過各經照射DEP電極區域214a處之區域/室202中之培養基180之相對阻抗。 In some embodiments, the microfluidic device 200 illustrated in Figures 2A and 2B can have an optically actuated DEP configuration. Accordingly, changing the light pattern 222 from the light source 220 (which can be controlled by the power module 162) can selectively activate and not initiate a pattern of DEP electrodes at regions 214 that change the inner surface 208 of the electrode activation substrate 206. (In the following, a region 214 of a microfluidic device having a DEP configuration refers to a "DEP electrode region"). As shown in FIG. 2B, a light pattern 222 directed onto the inner surface 208 of the electrode activation substrate 206 can illuminate a selected DEP electrode region 214a (shown in white) in a pattern, such as a square. The unexposed DEP electrode region 214 (cross-hatched) is referred to hereinafter as the "dark" DEP electrode region 214. The relative electrical impedance through the DEP electrode activation substrate 206 (i.e., the inner surface 208 of the electrode activation substrate 206 from the bottom electrode 204 to the medium 180 in the flow region 106) is greater than the area through each of the dark DEP electrode regions 214. The relative electrical impedance of the culture medium 180 in the chamber 202 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the cover 110). However, once illuminated DEP electrode region 214a exhibits a reduced relative impedance through one of electrode activation substrates 206 that is less than the relative impedance of medium 180 in region/chamber 202 through each irradiated DEP electrode region 214a.

在啟動電源212之後，前述DEP構形在經照射之DEP電極區域214a與相鄰暗DEP電極區域214之間的流體培養基180中產生一電場梯度，其繼而產生吸引或排斥流體培養基180中之鄰近微小物體(圖中未展示)之局部DEP力。因此，可藉由改變自一光源220投射至微流體器 件200中之光圖案222而在區域/室202之內表面208處之諸多不同此等DEP電極區域214處選擇性地啟動及不啟動吸引或排斥流體培養基180中之微小物體之DEP電極。DEP力是否吸引或排斥鄰近微小物體可取決於諸如電源212之頻率及培養基180及/或微小物體(圖中未展示)之介電性質之參數。 After the power source 212 is activated, the aforementioned DEP configuration creates an electric field gradient in the fluid medium 180 between the irradiated DEP electrode region 214a and the adjacent dark DEP electrode region 214, which in turn creates an attraction or repulsive proximity in the fluid medium 180. The local DEP force of a tiny object (not shown). Therefore, it can be projected from the light source 220 to the microfluidizer by changing The light pattern 222 in the member 200 selectively activates and does not activate the DEP electrode that attracts or repels tiny objects in the fluid medium 180 at a plurality of different DEP electrode regions 214 at the inner surface 208 of the region/chamber 202. Whether the DEP force attracts or repels adjacent tiny objects may depend on parameters such as the frequency of the power source 212 and the dielectric properties of the medium 180 and/or tiny objects (not shown).

圖2B中所繪示之經照射DEP電極區域214a之正方形圖案224僅係一實例。可由投影至器件200中之光圖案222照射(且藉此啟動)DEP電極區域214之任何圖案，且可藉由改變或移動光圖案222而重複地改變經照射/經啟動DEP電極區域214之圖案。 The square pattern 224 of the illuminated DEP electrode region 214a depicted in Figure 2B is merely an example. Any pattern of DEP electrode regions 214 can be illuminated (and thereby activated) by light pattern 222 projected into device 200, and the pattern of illuminated/activated DEP electrode regions 214 can be repeatedly altered by changing or moving light pattern 222. .

在一些實施例中，電極啟動基板206可包括一光導材料或由一光導材料組成。在此等實施例中，電極啟動基板206之內表面208可為無特徵的。例如，電極啟動基板206可包括一層氫化非晶矽(a-Si：H)或由一層氫化非晶矽(a-Si：H)組成。a-Si：H可包括(例如)約8%至約40%氫(經計算為100*氫原子之數目/氫原子及矽原子之總數目)。a-Si：H層可具有約500奈米至約2.0微米之一厚度。在此等實施例中，DEP電極區域214可產生於電極啟動基板206之內表面208上之任何位置處且在電極啟動基板206之內表面208上呈根據光圖案222之任何圖案。因此，DEP電極區域214之數目及圖案無需固定，而是可對應於光圖案222。例如，美國專利第RE 44,711號(Wu等人)(最初發表為美國專利第7,612,355號)中已描述具有一DEP構形(其包括諸如上文所討論之一光導層)之微流體器件之實例，該專利之全部內容以引用的方式併入本文中。 In some embodiments, the electrode activation substrate 206 can comprise or consist of a photoconductive material. In such embodiments, the inner surface 208 of the electrode activation substrate 206 can be featureless. For example, the electrode start substrate 206 may comprise a layer of hydrogenated amorphous germanium (a-Si:H) or a layer of hydrogenated amorphous germanium (a-Si:H). a-Si:H may include, for example, from about 8% to about 40% hydrogen (calculated as the number of 100* hydrogen atoms / the total number of hydrogen atoms and helium atoms). The a-Si:H layer may have a thickness of from about 500 nm to about 2.0 microns. In such embodiments, the DEP electrode region 214 can be produced at any location on the inner surface 208 of the electrode activation substrate 206 and in any pattern according to the light pattern 222 on the inner surface 208 of the electrode activation substrate 206. Therefore, the number and pattern of the DEP electrode regions 214 need not be fixed, but may correspond to the light pattern 222. An example of a microfluidic device having a DEP configuration comprising a photoconductive layer such as one discussed above has been described in U.S. Patent No. 4,612,355, issued to U.S. Patent No. 7,612,355. The entire content of this patent is incorporated herein by reference.

在其他實施例中，電極啟動基板206可包括一基板，其包括形成半導體積體電路之複數個摻雜層、電絕緣層(或區域)及導電層，諸如半導體領域中所知。例如，電極啟動基板206可包括複數個光電晶體(其包含(例如)橫向雙極光電晶體)，各光電晶體對應於一DEP電極區 域214。替代地，電極啟動基板206可包括由光電晶體開關控制之電極(例如導電金屬電極)，其中各此類電極對應於一DEP電極區域214。電極啟動基板206可包含此等光電晶體或光電晶體控制電極之一圖案。例如，該圖案可為配置成列及行之實質上呈正方形之光電晶體或光電晶體控制電極之一陣列，諸如圖2B中所展示。替代地，該圖案可為形成一六方晶格之實質上呈六邊形之光電晶體或光電晶體控制電極之一陣列。無論圖案如何，電路元件可形成電極啟動基板206之內表面208處之DEP電極區域214與底部電極210之間的電連接，且該等電連接(即，光電晶體或電極)可由光圖案222選擇性地啟動及不啟動。當未啟動各電連接時，各電連接可具有高阻抗，使得通過電極啟動基板206(即，自底部電極204至與區域/室202中之培養基180界接之電極啟動基板206之內表面208)之相對阻抗大於通過對應DEP電極區域214處之培養基180(即，自電極啟動基板206之內表面208至蓋110之頂部電極210)之相對阻抗。然而，當由光圖案222中之光啟動時，通過電極啟動基板206之相對阻抗小於通過各經照射DEP電極區域214處之培養基180之相對阻抗，藉此啟動對應DEP電極區域214處之DEP電極，如上文所討論。因此，可依由光圖案222判定之一方式在區域/室202中之電極啟動基板206之內表面208處之諸多不同DEP電極區域214處選擇性地啟動及不啟動吸引或排斥培養基180中之微小物體(圖中未展示)之DEP電極。 In other embodiments, the electrode enable substrate 206 can include a substrate that includes a plurality of doped layers, electrically insulating layers (or regions), and conductive layers that form a semiconductor integrated circuit, such as is known in the semiconductor arts. For example, the electrode enable substrate 206 can include a plurality of photocrystals (including, for example, lateral bipolar optoelectronic crystals), each photocrystal corresponding to a DEP electrode region Field 214. Alternatively, the electrode activation substrate 206 can include electrodes (eg, conductive metal electrodes) that are controlled by a photodiode switch, wherein each such electrode corresponds to a DEP electrode region 214. The electrode enable substrate 206 can comprise a pattern of one of such optoelectronic crystals or photonic crystal control electrodes. For example, the pattern can be an array of substantially square photoelectric crystal or optoelectronic crystal control electrodes arranged in columns and rows, such as shown in Figure 2B. Alternatively, the pattern can be an array of substantially hexagonal photovoltaic or photocrystalline control electrodes forming a hexagonal lattice. Regardless of the pattern, the circuit components can form an electrical connection between the DEP electrode region 214 and the bottom electrode 210 at the inner surface 208 of the electrode enable substrate 206, and the electrical connections (ie, optoelectronic crystals or electrodes) can be selected by the light pattern 222. Start and do not start. When the electrical connections are not activated, each electrical connection can have a high impedance such that the substrate 206 is activated by the electrodes (i.e., the inner surface 208 of the substrate 206 is activated from the bottom electrode 204 to the electrode interfaced with the medium 180 in the region/chamber 202). The relative impedance is greater than the relative impedance through the medium 180 at the corresponding DEP electrode region 214 (ie, from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the cover 110). However, when activated by light in the light pattern 222, the relative impedance through the electrode-activated substrate 206 is less than the relative impedance through the medium 180 at each of the irradiated DEP electrode regions 214, thereby activating the DEP electrode at the corresponding DEP electrode region 214. , as discussed above. Accordingly, the attraction or rejection medium 180 can be selectively activated and deactivated at a plurality of different DEP electrode regions 214 at the inner surface 208 of the electrode activation substrate 206 in the region/chamber 202 in a manner determined by the light pattern 222. A DEP electrode of a tiny object (not shown).

例如，美國專利第7,956,339號(Ohta等人)中已描述具有包括光電晶體之電極啟動基板之微流體器件之實例(例如，參閱圖21及圖22中所繪示之器件300及其描述)，該專利之全部內容以引用的方式併入本文中。例如，美國專利申請案第2014/0124370號(Short等人)中已描述具有包括由光電晶體開關控制之電極之電極啟動基板之微流體器件之實例(例如，參閱全部圖式中所繪示之器件200、400、500、600及900 及其描述)，該案之全部內容以引用的方式併入本文中。 For example, an example of a microfluidic device having an electrode-activated substrate comprising a photovoltaic crystal has been described in U.S. Patent No. 7,956,339 (Ohta et al.), for example, the device 300 and its description as illustrated in Figures 21 and 22, The entire content of this patent is incorporated herein by reference. An example of a microfluidic device having an electrode-initiating substrate comprising an electrode controlled by a photo-crystal switch has been described in, for example, U.S. Patent Application Serial No. 2014/0124370 (Short et al.). Devices 200, 400, 500, 600, and 900 And its description), the entire contents of which is incorporated herein by reference.

在一DEP構形之微流體器件之一些實施例中，頂部電極210係封閉體102之一第一壁(或蓋110)之部分，且電極啟動基板206及底部電極204係封閉體102之一第二壁(或支撐結構104)之部分。區域/室202可介於第一壁與第二壁之間。在其他實施例中，電極210係第二壁(或支撐結構104)之部分且電極啟動基板206及/或電極210之一或兩者係第一壁(或蓋110)之部分。而且，光源220可替代地用以自下方照射封閉體102。 In some embodiments of a DEP-configured microfluidic device, the top electrode 210 is part of a first wall (or cover 110) of the enclosure 102, and the electrode activation substrate 206 and the bottom electrode 204 are one of the enclosures 102. Part of the second wall (or support structure 104). The zone/chamber 202 can be between the first wall and the second wall. In other embodiments, electrode 210 is part of a second wall (or support structure 104) and one or both of electrode activation substrate 206 and/or electrode 210 are part of a first wall (or cover 110). Moreover, light source 220 can alternatively be used to illuminate enclosure 102 from below.

就具有一DEP構形之圖2A至圖2B之微流體器件200而言，動力模組162可藉由將一光圖案222投影至器件200中以啟動一圖案(例如正方形圖案224)(其包圍且捕獲之區域/室202中之培養基180中之一微小物體(圖中未展示))中之電極啟動基板206之內表面208之DEP電極區域214a處之一第一組之一或多個DEP電極而選擇該微小物體。接著，動力模組162可藉由相對於器件200移動光圖案222以啟動DEP電極區域214處之一第二組之一或多個DEP電極而移動該捕獲之微小物體。替代地，可相對於光圖案222移動器件200。 For a microfluidic device 200 of FIGS. 2A-2B having a DEP configuration, the power module 162 can be activated by projecting a light pattern 222 into the device 200 to initiate a pattern (eg, a square pattern 224) And one of the first group or the plurality of DEPs at one of the DEP electrode regions 214a of the inner surface 208 of the electrode activation substrate 206 in one of the mediums 180 in the captured region/chamber 202 (not shown) The electrode is selected for the electrode. Next, the power module 162 can move the captured minute object by moving the light pattern 222 relative to the device 200 to activate one of the second set or one of the DEP electrodes at the DEP electrode region 214. Alternatively, device 200 can be moved relative to light pattern 222.

在其他實施例中，微流體器件200可具有一DEP構形，其不依賴於電極啟動基板206之內表面208處之DEP電極之光啟動。例如，電極啟動基板206可包括可選擇性地定址及供能之電極，其經定位成相對於包含至少一電極(例如蓋110)之一表面。開關(例如一半導體基板中之電晶體開關)可選擇性地接通及切斷以啟動或不啟動DEP電極區域214處之DEP電極，藉此在啟動DEP電極附近產生作用於區域/室202中之一微小物體(圖中未展示)之一凈DEP力。取決於諸如電源212之頻率及區域/室202中之培養基(圖中未展示)及/或微小物體之介電性質之特性，DEP力可吸引或排斥一鄰近微小物體。可藉由選擇性地啟動及不啟動一組DEP電極(例如，在形成一正方形圖案224之一組DEP電極區 域214處)而捕集區域/室202中之一或多個微小物體且使其在區域/室202內移動。圖1中之動力模組162可控制此等開關且因此啟動及不啟動DEP電極之個別者以選擇、捕集及移動區域/室202周圍之特定微小物體(圖中未展示)。此項技術中已知且(例如)美國專利第6,294,063號(Becker等人)及第6,942,776號(Medoro)中已描述具有包含可選擇性定址及供能之電極之一DEP構形之微流體器件，該等專利之全部內容以引用的方式併入本文中。 In other embodiments, the microfluidic device 200 can have a DEP configuration that is independent of the light initiation of the DEP electrode at the inner surface 208 of the electrode activation substrate 206. For example, electrode activation substrate 206 can include an electrode that can be selectively addressed and energized that is positioned relative to a surface that includes at least one electrode (eg, cover 110). A switch, such as a transistor switch in a semiconductor substrate, can be selectively turned "on" and "off" to activate or deactivate the DEP electrode at the DEP electrode region 214, thereby acting in the region/chamber 202 adjacent the activated DEP electrode. One of the tiny objects (not shown) has a net DEP force. Depending on the nature of the frequency of the power source 212 and the medium in the zone/chamber 202 (not shown) and/or the nature of the dielectric properties of the tiny objects, the DEP force can attract or repel a neighboring minute object. A set of DEP electrodes can be selectively activated and deactivated (eg, in a group of DEP electrodes forming a square pattern 224) At region 214) one or more of the micro-objects in the region/chamber 202 are captured and moved within the region/chamber 202. The power module 162 of FIG. 1 can control such switches and thus activate and deactivate individual DEP electrodes to select, capture, and move specific small objects (not shown) around the area/chamber 202. Microfluidic devices having a DEP configuration comprising one of selectively addressable and energizable electrodes have been described in the art and are described in, for example, U.S. Patent Nos. 6,294,063 (Becker et al.) and 6,942,776 (Medoro). The entire contents of these patents are incorporated herein by reference.

作為又一實例，微流體器件200可具有一電潤濕(EW)構形，其可替換DEP構形或可定位於與具有DEP構形之部分分離之微流體器件200之一部分中。EW構形可為一光電潤濕構形或一介電潤濕潤(EWOD)構形，該兩個構形在此項技術中係已知的。在一些EW構形中，支撐結構104具有夾於一介電層(圖中未展示)與底部電極204之間的一電極啟動基板206。該介電層可包括一疏水材料及/或可塗佈有一疏水材料。針對具有一EW構形之微流體器件200，支撐結構104之內表面208係該介電層或其疏水塗層之內表面。 As yet another example, the microfluidic device 200 can have an electrowetting (EW) configuration that can be substituted for the DEP configuration or can be positioned in a portion of the microfluidic device 200 that is separate from the portion having the DEP configuration. The EW configuration can be an electro-optic wet configuration or a dielectric wetting (EWOD) configuration, both of which are known in the art. In some EW configurations, the support structure 104 has an electrode activation substrate 206 sandwiched between a dielectric layer (not shown) and the bottom electrode 204. The dielectric layer can comprise a hydrophobic material and/or can be coated with a hydrophobic material. For a microfluidic device 200 having an EW configuration, the inner surface 208 of the support structure 104 is the inner surface of the dielectric layer or its hydrophobic coating.

介電層(圖中未展示)可包括一或多個氧化層，且可具有約50奈米至約250奈米(例如，約125奈米至約175奈米)之一厚度。在某些實施例中，介電層可包括一層氧化物，諸如一金屬氧化物(例如氧化鋁或氧化鉿)。在某些實施例中，介電層可包括除一金屬氧化物之外之一介電材料，諸如氧化矽或氮化物。無論準確組合物及厚度如何，介電層可具有約10千歐姆至約50千歐姆之一阻抗。 The dielectric layer (not shown) may include one or more oxide layers and may have a thickness of from about 50 nanometers to about 250 nanometers (eg, from about 125 nanometers to about 175 nanometers). In certain embodiments, the dielectric layer can include a layer of an oxide, such as a metal oxide (eg, aluminum oxide or hafnium oxide). In some embodiments, the dielectric layer can include a dielectric material other than a metal oxide, such as hafnium oxide or nitride. Regardless of the exact composition and thickness, the dielectric layer can have an impedance of from about 10 kilo ohms to about 50 kilo ohms.

在一些實施例中，向內面向區域/室202之介電層之表面塗佈有一疏水材料。該疏水材料可包括(例如)氟化碳分子。氟化碳分子之實例包含全氟聚合物，諸如聚四氟乙烯(例如TEFLON®)或聚(2,3-二氟亞甲基-全氟四氫呋喃)(例如CYTOP^TM)。組成該疏水材料之分子可共價鍵聯至介電層之表面。例如，該疏水材料之分子可藉由一鍵聯子(諸 如矽氧烷基、膦酸基或硫醇基)而共價鍵聯至介電層之表面。因此，在一些實施例中，該疏水材料可包括烷基封端之矽氧烷、烷基封端之膦酸或烷基封端之硫醇。烷基可為長鏈碳氫化合物(例如，具有至少10個碳或至少16個、18個、20個、22個或22個以上碳之一鏈)。替代地，氟化(或全氟化)碳鏈可用於替換烷基。因此，例如，該疏水材料可包括氟烷基封端之矽氧烷、氟烷基封端之膦酸或氟烷基封端之硫醇。在一些實施例中，該疏水塗層具有約10奈米至約50奈米之一厚度。在其他實施例中，該疏水塗層具有小於10奈米(例如，小於5奈米或約1.5奈米至約3.0奈米)之一厚度。 In some embodiments, the surface of the dielectric layer facing the inward facing region/chamber 202 is coated with a hydrophobic material. The hydrophobic material can include, for example, a carbon fluoride molecule. Examples of fluorinated carbon molecules comprising perfluoropolymer, such as polytetrafluoroethylene (e.g., TEFLON ®) or poly (methylene-2,3-difluoro - perfluoro tetrahydrofuran) (e.g., CYTOP ^TM). Molecules constituting the hydrophobic material may be covalently bonded to the surface of the dielectric layer. For example, the molecules of the hydrophobic material can be covalently bonded to the surface of the dielectric layer by a bond (such as a decyloxy group, a phosphonic acid group, or a thiol group). Thus, in some embodiments, the hydrophobic material can include an alkyl terminated alkane, an alkyl terminated phosphonic acid, or an alkyl terminated thiol. The alkyl group can be a long chain hydrocarbon (e.g., having at least 10 carbons or a chain of at least 16, 18, 20, 22 or more than 22 carbons). Alternatively, a fluorinated (or perfluorinated) carbon chain can be used to replace the alkyl group. Thus, for example, the hydrophobic material can include a fluoroalkyl-terminated oxime, a fluoroalkyl-terminated phosphonic acid, or a fluoroalkyl-terminated thiol. In some embodiments, the hydrophobic coating has a thickness from about 10 nanometers to about 50 nanometers. In other embodiments, the hydrophobic coating has a thickness of less than 10 nanometers (eg, less than 5 nanometers or from about 1.5 nanometers to about 3.0 nanometers).

在一些實施例中，具有一電潤濕構形之一微流體器件200之蓋110亦塗佈有一疏水材料(圖中未展示)。該疏水材料可為用以塗佈支撐結構104之介電層之相同疏水材料，且該疏水塗層可具有實質上相同於支撐結構104之介電層上之疏水塗層之厚度的一厚度。而且，蓋110可包括依支撐結構104之方式夾於一介電層與頂部電極210之間的一電極啟動基板206。電極啟動基板206及蓋110之介電層可具有相同於支撐結構104之電極啟動基板206及介電層之組合物及/或尺寸。因此，微流體器件200可具有兩個電潤濕表面。 In some embodiments, the cover 110 of one of the microfluidic devices 200 having an electrowetting configuration is also coated with a hydrophobic material (not shown). The hydrophobic material can be the same hydrophobic material used to coat the dielectric layer of the support structure 104, and the hydrophobic coating can have a thickness that is substantially the same as the thickness of the hydrophobic coating on the dielectric layer of the support structure 104. Moreover, the cover 110 can include an electrode activation substrate 206 sandwiched between a dielectric layer and the top electrode 210 in a manner that supports the structure 104. The dielectric layers of the electrode enable substrate 206 and the cover 110 can have the same composition and/or dimensions as the electrode enable substrate 206 and the dielectric layer of the support structure 104. Thus, the microfluidic device 200 can have two electrowetting surfaces.

在一些實施例中，電極啟動基板206可包括一光導材料，諸如上文所描述。據此，在某些實施例中，電極啟動基板206可包括一層氫化非晶矽(a-Si：H)或由一層氫化非晶矽(a-Si：H)組成。例如，a-Si：H可包括約8%至40%氫(經計算為100*氫原子之數目/氫原子及矽原子之總數目)。a-Si：H層可具有約500奈米至約2.0微米之一厚度。替代地，電極啟動基板206可包括由光電晶體開關控制之電極(例如導電金屬電極)，如上文所描述。此項技術中已知及/或可使用此項技術中已知之電極啟動基板來建構具有一光電潤濕構形之微流體器件。例如，美國專利第6,958,132號(Chiou等人)(該專利之全部內容以引用的方式併入 本文中)揭示具有一光導材料(諸如a-Si：H)之光電潤濕構形，而上文所引用之美國專利公開案第2014/0124370號(Short等人)揭示具有由光電晶體開關控制之電極之電極啟動基板。 In some embodiments, the electrode activation substrate 206 can include a light guiding material such as described above. Accordingly, in some embodiments, the electrode-activated substrate 206 can comprise a layer of hydrogenated amorphous germanium (a-Si:H) or a layer of hydrogenated amorphous germanium (a-Si:H). For example, a-Si:H may comprise from about 8% to 40% hydrogen (calculated as the number of 100* hydrogen atoms / the total number of hydrogen atoms and helium atoms). The a-Si:H layer may have a thickness of from about 500 nm to about 2.0 microns. Alternatively, electrode activation substrate 206 can include an electrode (eg, a conductive metal electrode) controlled by a photodiode switch, as described above. Electrode-starting substrates known in the art are known and/or can be used in the art to construct microfluidic devices having a photo-wetting configuration. For example, U.S. Patent No. 6,958,132 (Chiou et al.), the entire content of which is incorporated by reference. The present invention discloses a photovoltaic wetting configuration having a photoconductive material such as a-Si:H, and the above-cited U.S. Patent Publication No. 2014/0124370 (Short et al.), which is incorporated herein by reference. The electrode of the electrode activates the substrate.

因此，微流體器件200可具有一光電潤濕構形，且光圖案222可用以啟動電極啟動基板206中之光導EW區域或光響應EW電極。電極啟動基板206之此等啟動EW區域或EW電極可在支撐結構104之內表面208(即，上覆介電層或其疏水塗層之內表面)處產生一電潤濕力。可藉由改變入射於電極啟動基板206上之光圖案222(或相對於光源220移動微流體器件200)而使接觸支撐結構104之內表面208之液滴(例如，其含有一水培養基、溶液或溶劑)移動通過存在於區域/室202中之一不溶混流體(例如一油培養基)。 Thus, the microfluidic device 200 can have a photo-wetting configuration, and the light pattern 222 can be used to activate the photoconductive EW region or the photoresponsive EW electrode in the electrode-activated substrate 206. The actuating EW regions or EW electrodes of the electrode actuating substrate 206 can create an electrowetting force at the inner surface 208 of the support structure 104 (i.e., the inner surface of the overlying dielectric layer or its hydrophobic coating). The droplets contacting the inner surface 208 of the support structure 104 can be made by changing the light pattern 222 incident on the electrode activation substrate 206 (or moving the microfluidic device 200 relative to the light source 220) (eg, it contains a water medium, solution) Or solvent) moves through one of the immiscible fluids (eg, an oil medium) present in the zone/chamber 202.

在其他實施例中，微流體器件200可具有一EWOD構形，且電極啟動基板206可包括不依賴於用於啟動之光之可選擇性定址及供能之電極。因此，電極啟動基板206可包含此等電潤濕(EW)電極之一圖案。例如，該圖案可為配置成列及行之實質上呈正方形之EW電極之一陣列，諸如圖2B中所展示。替代地，該圖案可為形成一六方晶格之實質上呈六邊形之EW電極之一陣列。無論圖案如何，可由電開關(例如一半導體基板中之電晶體開關)選擇性地啟動(或不啟動)EW電極。可藉由選擇性地啟動及不啟動電極啟動基板206中之EW電極而使接觸上覆介電層或其疏水塗層之內表面208之液滴(圖中未展示)在區域/室202內移動。圖1中之動力模組162可控制此等開關且因此啟動及不啟動個別EW電極來選擇及移動區域/室202周圍之特定液滴。此項技術中已知且(例如)美國專利第8,685,344號(Sundarsan等人)中已描述具有一EWOD構形(其具有可選擇性定址及供能之電極)之微流體器件，該專利之全部內容以引用的方式併入本文中。 In other embodiments, the microfluidic device 200 can have an EWOD configuration, and the electrode activation substrate 206 can include electrodes that are selectively addressable and energized independent of the light used for activation. Thus, electrode activation substrate 206 can comprise a pattern of one of these electrowetting (EW) electrodes. For example, the pattern can be an array of substantially square EW electrodes configured in columns and rows, such as shown in Figure 2B. Alternatively, the pattern can be an array of substantially hexagonal EW electrodes forming a hexagonal lattice. Regardless of the pattern, the EW electrode can be selectively activated (or not activated) by an electrical switch, such as a transistor switch in a semiconductor substrate. Droplets (not shown) contacting the inner surface 208 of the overlying dielectric layer or its hydrophobic coating can be placed in the region/chamber 202 by selectively activating and deactivating the EW electrode in the substrate 206. mobile. The power module 162 of FIG. 1 can control the switches and thus activate and deactivate individual EW electrodes to select and move specific droplets around the region/chamber 202. Microfluidic devices having an EWOD configuration (which has selectively addressable and energizable electrodes) have been described in the art and are described, for example, in U.S. Patent No. 8,685,344 (Sundarsan et al. The content is incorporated herein by reference.

無論微流體器件200之構形如何，一電源212可用以提供對微流 體器件200之電路供電之一電位(例如一AC電壓電位)。電源212可相同於圖1中所參考之電源192或可為圖1中所參考之電源192之一組件。電源212可經構形以將一AC電壓及/或電流提供至頂部電極210及底部電極204。針對一AC電壓，電源212可提供一頻率範圍及一平均或峰值電力(例如電壓或電流)範圍，其足以產生足夠強之凈DEP力(或電潤濕力)來捕集及移動區域/室202中之微小物體(圖中未展示)(如上文所討論)及/或改變區域/室202中之支撐結構104之內表面208(即，介電層及/或介電層上之疏水塗層)之濕潤性質(亦如上文所討論)。此等頻率範圍及平均或峰值電力範圍在此項技術中係已知的。例如，參閱美國專利第6,958,132號(Chiou等人)、美國專利第RE44,711號(Wu等人)(最初發表為美國專利第7,612,355號)及美國專利申請公開案第US2014/0124370號(Short等人)、第US2015/0306598號(Khandros等人)及第US2015/0306599號(Khandros等人)。 Regardless of the configuration of the microfluidic device 200, a power source 212 can be used to provide a pair of microfluidics The circuit of body device 200 is powered by a potential (eg, an AC voltage potential). The power source 212 can be identical to the power source 192 referenced in FIG. 1 or can be one of the components of the power source 192 referenced in FIG. Power source 212 can be configured to provide an AC voltage and/or current to top electrode 210 and bottom electrode 204. For an AC voltage, the power supply 212 can provide a range of frequencies and an average or peak power (eg, voltage or current) range sufficient to generate a sufficiently strong net DEP force (or electrowetting force) to capture and move the area/room A small object (not shown) in 202 (as discussed above) and/or an inner surface 208 of the support structure 104 in the region/chamber 202 (ie, a hydrophobic coating on the dielectric layer and/or dielectric layer) The wetting properties of the layer) (also as discussed above). These frequency ranges and average or peak power ranges are known in the art. For example, see U.S. Patent No. 6,958,132 (Chiou et al.), U.S. Patent No. 4,711, (Wu et al.), issued to U.S. Patent No. 7,612,355, and U.S. Patent Application Publication No. (US) No. US2015/0306598 (Khandros et al.) and US2015/0306599 (Khandros et al.).

生長室Growth room

圖2C及圖2D中所描繪之微流體器件240內展示通用生長室244、246及248之非限制性實例。各生長室244、246及248可包括一隔離結構250，其界定一隔離區域258及將隔離區域258流體地連接至一通道122之一連接區域254。連接區域254可包括至通道122之一近端開口252及至隔離區域258之一遠端開口256。連接區域254可經構形使得自通道122流動至生長室244、246、248中之一流體培養基(圖中未展示)之一流動之最大滲透深度不延伸至隔離區域258中。因此，歸因於連接區域254，安置於一生長室244、246、248之一隔離區域258中之一微小物體(圖中未展示)或其他材料(圖中未展示)可因此與通道122中之培養基180之一流動隔離且實質上不受通道122中之培養基180之一流動影響。 Non-limiting examples of universal growth chambers 244, 246, and 248 are shown within microfluidic device 240 depicted in Figures 2C and 2D. Each growth chamber 244, 246, and 248 can include an isolation structure 250 that defines an isolation region 258 and fluidly connects the isolation region 258 to a connection region 254 of a channel 122. Connection region 254 can include a proximal opening 252 to one of channel 122 and a distal opening 256 to one of isolation regions 258. The connection region 254 can be configured such that the maximum penetration depth of one of the fluid media (not shown) flowing from the channel 122 to one of the growth chambers 244, 246, 248 does not extend into the isolation region 258. Thus, due to the connection region 254, one of the tiny objects (not shown) or other material (not shown) disposed in one of the isolation regions 258 of a growth chamber 244, 246, 248 can thus be in the channel 122. One of the media 180 is fluidly isolated and substantially unaffected by one of the media 180 in the channel 122.

因此，通道122可為一掃及區域之一實例，且生長室244、246、 248之隔離區域258可為未掃及區域之實例。如本文所提及，通道122及生長室244、246、248可經構形以含有一或多個流體培養基180。在圖2C至圖2D所展示之實例中，端口242連接至通道122且允許將一流體培養基180引入至微流體器件240中或自微流體器件240移除一流體培養基180。在引入流體培養基180之前，微流體器件可預充有諸如二氧化碳氣體之一氣體。一旦微流體器件240含有流體培養基180，則可選擇性地產生且停止通道122中之流體培養基180之流260。例如，如圖中所展示，可將端口242安置於通道122之不同位置(例如相對端)處，且可自用作一入口之一端口242至用作一出口之另一端口242產生培養基之一流260。 Thus, channel 122 can be an instance of a sweep and region, and growth chambers 244, 246, The isolation region 258 of 248 can be an example of an unswept region. As mentioned herein, channel 122 and growth chambers 244, 246, 248 can be configured to contain one or more fluid media 180. In the example shown in FIGS. 2C-2D, port 242 is coupled to channel 122 and allows a fluid medium 180 to be introduced into or removed from microfluidic device 240. Prior to introduction of fluid medium 180, the microfluidic device can be pre-charged with a gas such as carbon dioxide gas. Once the microfluidic device 240 contains the fluid medium 180, the flow 260 of the fluid medium 180 in the channel 122 can be selectively generated and stopped. For example, as shown in the figures, port 242 can be placed at a different location (e.g., opposite end) of channel 122, and can be used as one of the inlet ports 242 to another port 242 that serves as an outlet to generate a flow of media. 260.

圖2E繪示根據本發明之一生長室244之一實例之一詳細圖。圖中亦展示微小物體270之實例。 2E is a detailed view of one of the examples of growth chamber 244 in accordance with the present invention. An example of a tiny object 270 is also shown.

眾所周知，一微流體通道122中之流體培養基180之一流260通過生長室244之一近端開口252可致使培養基180之一副流262進入及/或流出生長室244。為使一生長室244之隔離區域258中之微小物體270與副流262隔離，生長室244之連接區域254之長度L_con(即，自近端開口252至遠端開口256)應大於副流262進入連接區域254之滲透深度D_p。副流262之滲透深度D_p取決於在通道122中流動之流體培養基180之速度及與通道122之構形及至通道122之連接區域254之近端開口252相關之各種參數。針對一給定微流體器件，通道122及開口252之構形將為固定的，而通道122中之流體培養基180之流260之速率將為可變的。據此，針對各生長室244，可識別通道122中之流體培養基180之流260之一最大速度Vmax，其確保副流262之滲透深度D_p不超過連接區域254之長度L_con。只要通道122中之流體培養基180之流260之速率不超過最大速度Vmax，則所得副流262可受限於通道122及連接區域254且保持不進入隔離區域258。因此，通道122中之培養基180之流260不會 自隔離區域258帶出微小物體270。確切而言，無論通道122中之流體培養基180之流260如何，定位於隔離區域258中之微小物體270將留在隔離區域258中。 As is known, one of the streams 260 of fluid medium 180 in a microfluidic channel 122 through one of the proximal openings 252 of the growth chamber 244 can cause a secondary stream 262 of the medium 180 to enter and/or exit the growth chamber 244. In order to isolate the growth chamber 244 of a region 258 of the object 270 and the fine separator sidestream 262, 244 connected to the growth chamber region 254 of length L _con (i.e., the opening 252 from the proximal to the distal end opening 256) should be greater than the secondary flow 262 enters the penetration depth D _{p of the} connection region 254. The penetration depth _Dp of the secondary stream 262 depends on the velocity of the fluid medium 180 flowing in the channel 122 and various parameters associated with the configuration of the channel 122 and the proximal opening 252 to the junction region 254 of the channel 122. For a given microfluidic device, the configuration of channel 122 and opening 252 will be fixed, while the rate of flow 260 of fluid medium 180 in channel 122 will be variable. Accordingly, for each growth chamber 244, one of the maximum velocities Vmax of the stream 260 of the fluid medium 180 in the channel 122 can be identified, which ensures that the penetration depth _{Dp of the} secondary stream 262 does not exceed the length _{Lcon of the} connection region 254. As long as the rate of flow 260 of fluid medium 180 in channel 122 does not exceed the maximum velocity Vmax, the resulting secondary stream 262 can be limited to channel 122 and connection region 254 and remain out of isolation region 258. Thus, stream 260 of medium 180 in channel 122 does not carry tiny objects 270 from isolation region 258. Rather, regardless of the flow 260 of fluid medium 180 in channel 122, the tiny objects 270 positioned in isolation region 258 will remain in isolation region 258.

而且，只要通道122中之培養基180之流260之速率不超過Vmax，則通道122中之流體培養基180之流260不會將混雜粒子(例如微粒及/或奈米粒子)自通道122移動至一生長室244之隔離區域258中。因此，使連接區域254之長度L_con大於副流262之最大滲透深度D_p可防止來自通道122或另一生長室(例如圖2D中之生長室246、248)之混雜粒子污染一生長室244。 Moreover, as long as the rate of stream 260 of medium 180 in channel 122 does not exceed Vmax, stream 260 of fluid medium 180 in channel 122 does not move hybrid particles (e.g., particles and/or nanoparticles) from channel 122 to one. The isolation region 258 of the growth chamber 244. Thus, having the length _{Lcon of} the attachment region 254 greater than the maximum penetration depth _{Dp of the} secondary stream 262 prevents contaminating particles from the channel 122 or another growth chamber (eg, growth chambers 246, 248 in FIG. 2D) from contaminating a growth chamber 244 .

因為通道122及生長室244、246、248之連接區域254會受通道122中之培養基180之流260影響，所以通道122及連接區域254可被視作微流體器件240之掃及(或流動)區域。在另一方面，生長室244、246、248之隔離區域258可被視作未掃及(或非流動)區域。例如，通道122中之一第一流體培養基180中之組分(圖中未展示)可實質上僅藉由第一培養基180之組分自通道122擴散通過連接區域254且進入隔離區域258中之一第二流體培養基280而與隔離區域258中之第二流體培養基280混合。類似地，隔離區域258中之第二培養基280之組分(圖中未展示)可實質上僅藉由第二培養基280之組分自隔離區域258擴散通過連接區域254且進入通道122中之第一培養基180而與通道122中之第一培養基180混合。第一培養基180可為相同於或不同於第二培養基280之一培養基。而且，第一培養基180及第二培養基280可起初相同，接著變為不同(例如，透過由隔離區域258中之一或多個細胞調節第二培養基280，或藉由改變流動通過通道122之培養基180)。 Because the connection region 254 of the channel 122 and the growth chambers 244, 246, 248 are affected by the flow 260 of the medium 180 in the channel 122, the channel 122 and the connection region 254 can be viewed as a sweep and/or flow of the microfluidic device 240. region. In another aspect, the isolation regions 258 of the growth chambers 244, 246, 248 can be considered as unswept and (or non-flowing) regions. For example, a component of one of the channels 122 in the first fluid medium 180 (not shown) can diffuse from the channel 122 through the connection region 254 and into the isolation region 258 substantially only by the components of the first medium 180. A second fluid medium 280 is mixed with the second fluid medium 280 in the isolation region 258. Similarly, the components of the second medium 280 in the isolation region 258 (not shown) may be substantially diffused from the isolation region 258 through the connection region 254 and into the channel 122 by only the components of the second medium 280. A medium 180 is mixed with the first medium 180 in the channel 122. The first medium 180 can be the same medium as or different from the second medium 280. Moreover, the first medium 180 and the second medium 280 can be initially identical and then become different (eg, by modulating the second medium 280 from one or more cells in the isolated region 258, or by changing the medium flowing through the channel 122) 180).

由通道122中之流體培養基180之流260所致之副流262之最大滲透深度D_p可取決於如上文所提及之若干參數。此等參數之實例包含通道122之形狀(例如，通道可將培養基導引至連接區域254中，自連接 區域254轉走培養基，或在實質上垂直於至通道122之連接區域254之近端開口252之一方向上導引培養基)、近端開口252處之通道122之一寬度W_ch(或橫截面積)、近端開口252處之連接區域254之一寬度W_con(或橫截面積)、通道122中之流體培養基180之流260之速度V、第一培養基180及/或第二培養基280之黏度、或其類似者。 The maximum penetration depth _Dp of the secondary stream 262 resulting from stream 260 of fluid medium 180 in channel 122 may depend on several parameters as mentioned above. Examples of such parameters include the shape of the channel 122 (eg, the channel can direct the culture medium into the attachment region 254, the culture medium is removed from the attachment region 254, or the proximal opening is substantially perpendicular to the attachment region 254 to the channel 122. one of the media guide 252 direction), the proximal opening 252 of one channel 122 width W _ch (or cross-sectional area), the proximal opening 252 of one of the connection region 254 width W _con (or cross-sectional area), The velocity V of stream 260 of fluid medium 180 in channel 122, the viscosity of first medium 180 and/or second medium 280, or the like.

在一些實施例中，通道122及生長室244、246、248之尺寸可相對於通道122中之流體培養基180之流260之向量而定向如下：通道寬度W_ch(或通道122之橫截面積)可實質上垂直於培養基180之流260；開口252處之連接區域254之寬度W_con(或橫截面積)可實質上平行於通道122中之培養基180之流260；及/或連接區域之長度L_con可實質上垂直於通道122中之培養基180之流260。上述定向僅為實例，且通道122及生長室244、246、248之相對位置可在相對於彼此之其他定向上。 In some embodiments, the dimensions of channel 122 and growth chambers 244, 246, 248 can be oriented relative to the vector of stream 260 of fluid medium 180 in channel 122 as follows: channel width _Wch (or cross-sectional area of channel 122) The flow 260 may be substantially perpendicular to the flow 180; the width _Wcon (or cross-sectional area) of the attachment region 254 at the opening 252 may be substantially parallel to the flow 260 of the culture medium 180 in the channel 122; and/or the length of the attachment region L _con may be substantially perpendicular to stream 260 of medium 180 in channel 122. The above orientation is merely an example, and the relative positions of the channel 122 and the growth chambers 244, 246, 248 may be in other orientations relative to each other.

如圖2E中所繪示，自近端開口252至遠端開口256之連接區域254之寬度W_con可為均勻的。因此，遠端開口256處之連接區域254之寬度W_con可在本文中針對近端開口252處之連接區域254之寬度W_con所識別之範圍之任何者內。替代地，遠端開口256處之連接區域254之寬度W_con可大於近端開口252處之連接區域254之寬度W_con。 As depicted in Figure 2E, the width _Wcon of the attachment region 254 from the proximal opening 252 to the distal opening 256 can be uniform. Thus, the width W _con of the connection region 254 at the distal opening 256 can be within any of the ranges identified herein for the width W _con of the connection region 254 at the proximal opening 252. Alternatively, the distal opening of the connection region 256 of the width W _con 254 may be greater than the proximal opening of the connection region 252 of width 254 W _con.

如圖2E中所繪示，遠端開口256處之隔離區域258之寬度可實質上相同於近端開口252處之連接區域254之寬度W_con。因此，遠端開口256處之隔離區域258之寬度可在本文中針對近端開口252處之連接區域254之寬度W_con所識別之範圍之任何者內。替代地，遠端開口256處之隔離區域258之寬度可大於或小於近端開口252處之連接區域254之寬度W_con。而且，遠端開口256可小於近端開口252且連接區域254之寬度W_con可在近端開口252與遠端開口256之間變窄。例如，可使用各種不同幾何形狀來使連接區域254在近端開口與遠端開口之間變窄(例如，斜裁連接區域，斜切連接區域)。進一步言之，連接區域254之任 何部分或子部分可變窄(例如相鄰於近端開口252之連接區域之一部分)。 Depicted in FIG. 2E, the distal opening of the isolation region 256 may be substantially the same as the width 258 of the proximal end 252 of the opening width of the connection region 254. W _con. Thus, the width of the isolation region 258 at the distal opening 256 can be within any of the ranges identified herein for the width W _con of the connection region 254 at the proximal opening 252. Alternatively, the width of the isolation region 258 at the distal opening 256 may be greater or less than the width W _con of the connection region 254 at the proximal opening 252. Moreover, the distal opening 256 can be smaller than the proximal opening 252 and the width W _{con of the} connecting region 254 can be narrowed between the proximal opening 252 and the distal opening 256. For example, a variety of different geometries can be used to narrow the attachment region 254 between the proximal opening and the distal opening (eg, obliquely connecting the region, beveling the connecting region). Further, any portion or sub-portion of the connection region 254 can be narrow (eg, a portion of the connection region adjacent to the proximal opening 252).

圖4A至圖4C描繪含有一微流體線路432及流動通道434之一微流體器件400之另一例示性實施例，微流體器件400、微流體線路432及流動通道434係圖1之各自微流體器件100、線路132及通道134之變體。微流體器件400亦具有複數個生長室436，其係上文所描述之生長室124、126、128、130、244、246或248之額外變體。特定言之，應瞭解，圖4A至圖4C中所展示之器件400之生長室436可替換器件100、200、240及290中之上文所描述之生長室124、126、128、130、244、246或248之任何者。同樣地，微流體器件400係微流體器件100之另一變體且亦可具有相同於或不同於上文所描述之微流體器件100、200、240、290之DEP構形以及本文中所描述之其他微流體系統組件之任何者。 4A-4C depict another exemplary embodiment of a microfluidic device 400 comprising a microfluidic circuit 432 and a flow channel 434, each of which is a microfluidic device 400, a microfluidic circuit 432, and a flow channel 434 A variation of device 100, line 132, and channel 134. The microfluidic device 400 also has a plurality of growth chambers 436 that are additional variations of the growth chambers 124, 126, 128, 130, 244, 246 or 248 described above. In particular, it should be appreciated that the growth chamber 436 of the device 400 shown in FIGS. 4A-4C can replace the growth chambers 124, 126, 128, 130, 244 described above in the devices 100, 200, 240, and 290. Any of 246 or 248. Likewise, microfluidic device 400 is another variation of microfluidic device 100 and may also have the same or different DEP configuration of microfluidic devices 100, 200, 240, 290 as described above and as described herein. Any of the other microfluidic system components.

圖4A至圖4C之微流體器件400包括一支撐結構(圖4A至圖4C中不可見，但可相同於或大體上類似於圖1中所描繪之器件100之支撐結構104)、一微流體線路結構412及一蓋(圖4A至圖4C中不可見，但可相同於或大體上類似於圖1中所描繪之器件100之蓋110)。微流體線路結構412包含一框架414及微流體線路材料416，其等可相同於或大體上類似於圖1中所展示之器件100之框架114及微流體線路材料116。如圖4A中所展示，由微流體線路材料416界定之微流體線路432可包括多個生長室436流體地連接至其之多個通道434(圖中展示兩個，但可存在更多個)。 The microfluidic device 400 of Figures 4A-4C includes a support structure (not visible in Figures 4A-4C, but may be the same or substantially similar to the support structure 104 of the device 100 depicted in Figure 1), a microfluidic The line structure 412 and a cover (not visible in Figures 4A-4C, but may be the same or substantially similar to the cover 110 of the device 100 depicted in Figure 1). Microfluidic line structure 412 includes a frame 414 and microfluidic line material 416, which may be the same or substantially similar to frame 114 of device 100 and microfluidic line material 116 as shown in FIG. As shown in FIG. 4A, the microfluidic line 432 defined by the microfluidic line material 416 can include a plurality of channels 434 to which the plurality of growth chambers 436 are fluidly coupled (two are shown, but more can be present) .

各生長室436可包括一隔離結構446、隔離結構446內之一隔離區域444、及一連接區域442。連接區域442自通道434處之一近端開口472至隔離結構436處之一遠端開口474將通道434流體地連接至隔離區域444。一般而言，根據圖2D及圖2E之上述討論，一通道434中之一 第一流體培養基402之一流482可產生自通道434進入生長室436之各自連接區域442及/或離開生長室436之各自連接區域442之第一培養基402之副流484。 Each growth chamber 436 can include an isolation structure 446, an isolation region 444 within the isolation structure 446, and a connection region 442. The connection region 442 fluidly connects the channel 434 to the isolation region 444 from one of the proximal openings 472 at the channel 434 to one of the distal openings 474 at the isolation structure 436. In general, one of the channels 434 is discussed in accordance with the above discussion of Figures 2D and 2E. One stream 482 of first fluid medium 402 can generate a secondary stream 484 from channel 434 into respective junction regions 442 of growth chamber 436 and/or first medium 402 exiting respective junction regions 442 of growth chamber 436.

如圖4B中所繪示，各生長室436之連接區域442大體上包含延伸於至一通道434之近端開口472與至一隔離結構446之遠端開口474之間的區域。連接區域442之長度L_con可大於副流484之最大滲透深度D_p，在該情況中，副流484將延伸至連接區域442中且未被轉向隔離區域444(如圖4A中所展示)。替代地，如圖4C中所繪示，連接區域442可具有小於最大滲透深度D_p之一長度L_con，在該情況中，副流484將延伸通過連接區域442且被轉向隔離區域444。在此後一情況中，連接區域442之長度L_c1及L_c2之總和大於最大滲透深度D_p，使得副流484不會延伸至隔離區域444中。無論連接區域442之長度L_con大於滲透深度D_p或連接區域442之長度L_c1及L_c2之總和大於滲透深度D_p，通道434中之一第一培養基402之一流482(其不超過一最大速度V_max)將產生具有一滲透深度D_p之一副流，且一生長室436之隔離區域444中之微小物體(圖中未展示，但可相同於或大體上類似於圖2E中所展示之微小物體270)不會被通道434中之第一培養基402之一流482帶出隔離區域444。通道434中之流482亦不會將混雜材料(圖中未展示)自通道434帶入至一生長室436之隔離區域444中。因而，擴散係通道434中之一第一培養基402中之組分可自通道434移動至一生長室436之一隔離區域444中之一第二培養基404中之唯一機制。同樣地，擴散係一生長室436之一隔離區域444中之一第二培養基404中之組分可自隔離區域444移動至通道434中之一第一培養基402之唯一機制。第一培養基402可為相同於第二培養基404之培養基，或第一培養基402可為不同於第二培養基404之一培養基。替代地，第一培養基402及第二培養基404可起初相同，接著變為不同，例如，透過由隔離區域444中之一或多個細胞調 節第二培養基，或藉由改變流動通過通道434之培養基。 As shown in FIG. 4B, the connection region 442 of each growth chamber 436 generally includes a region extending between a proximal opening 472 to a channel 434 and a distal opening 474 to an isolation structure 446. The length L _{con of the} connection region 442 may be greater than the maximum penetration depth D _p of the secondary flow 484, in which case the secondary flow 484 will extend into the connection region 442 and not be diverted to the isolation region 444 (as shown in Figure 4A). Alternatively, as depicted in Figure 4C, the connection region 442 may have one less than the maximum length of a penetration depth D _p L _con, in this case, the secondary stream 484 and the extension 444 is diverted through the connecting region isolation region 442. In this latter case, the sum of the lengths _Lc1 and _Lc2 of the connection region 442 is greater than the maximum penetration depth _Dp such that the secondary flow 484 does not extend into the isolation region 444. Regardless of whether the length L _{con of the} attachment region 442 is greater than the penetration depth D _p or the sum of the lengths L _c1 and L _c2 of the attachment region 442 is greater than the penetration depth D _p , one of the first mediums 402 in the channel 434 flows 482 (which does not exceed a maximum The velocity V _max ) will produce a secondary object having a secondary flow of a penetration depth D _p and an isolation region 444 of a growth chamber 436 (not shown, but may be the same or substantially similar to that shown in Figure 2E) The tiny object 270) is not carried out of the isolation region 444 by a stream 482 of the first medium 402 in the channel 434. Stream 482 in channel 434 also does not carry hybrid material (not shown) from channel 434 into isolation region 444 of a growth chamber 436. Thus, the only mechanism in which one of the first medium 402 in the diffusion channel 434 can move from the channel 434 to one of the second media 404 in one of the isolation regions 444 of the growth chamber 436. Likewise, the only mechanism in which the components of the second medium 404 in one of the isolation regions 444 of the diffusion system-growth chamber 436 can move from the isolation region 444 to one of the first media 402 in the channel 434. The first medium 402 may be the same medium as the second medium 404, or the first medium 402 may be one different from the second medium 404. Alternatively, the first medium 402 and the second medium 404 may be initially identical and then become different, for example, by modulating the second medium by one or more cells in the isolated region 444, or by changing the medium flowing through the channel 434. .

如圖4B中所繪示，通道434中之通道434之寬度W_ch(即，橫向於流動通過通道之一流體培養基之方向(由圖4A中之箭頭482指示)而取得)可實質上垂直於近端開口472之一寬度W_con1且因此實質上平行於遠端開口474之一寬度W_con2。然而，近端開口472之寬度W_con1及遠端開口474之寬度W_con2無需實質上彼此垂直。例如，近端開口472之寬度W_con1定向於其上之一軸(圖中未展示)與遠端開口474之寬度W_con2定向於其上之另一軸之間的一角度可不成直角且因此不為90°。替代定向角之實例包含下列範圍之任何者內之角度：自約30°至約90°、自約45°至約90°、自約60°至約90°或其類似者。 Depicted in FIG. 4B, the channel 434 in the channel width of W _ch 434 (i.e., transverse to the flow direction of the fluid through the medium of one of the channels (indicated by the arrow 482 in FIG. 4A) is acquired) may be substantially perpendicular to the One of the proximal openings 472 has a width W _con1 and is therefore substantially parallel to one of the widths W _{con2 of the} distal opening 474. However, the width W _{con1 of the} proximal opening 472 and the width W _{con2 of the} distal opening 474 need not be substantially perpendicular to each other. For example, an angle between the width W _{con1 of the} proximal opening 472 that is oriented on one of the axes (not shown) and the other axis on which the width W _{con2 of the} distal opening 474 is oriented may not be at right angles and therefore is not 90°. Examples of alternative orientation angles include angles within any of the following ranges: from about 30° to about 90°, from about 45° to about 90°, from about 60° to about 90°, or the like.

在生長室(例如124、126、128、130、244、246、248或436)之各種實施例中，隔離區域(例如258或444)經構形以含有複數個微小物體。在其他實施例中，隔離區域可經構形以僅含有1個、2個、3個、4個、5個或類似相對較小數目個微小物體。據此，一隔離區域之體積可為(例如)至少3×10³立方微米、6×10³立方微米、9×10³立方微米、1×10⁴立方微米、2×10⁴立方微米、4×10⁴立方微米、8×10⁴立方微米、1×10⁵立方微米、2×10⁵立方微米、4×10⁵立方微米、8×10⁵立方微米、1×10⁶立方微米、2×10⁶立方微米、4×10⁶立方微米、6×10⁶立方微米、1×10⁷立方微米、2×10⁷立方微米、4×10⁷立方微米、6×10⁷立方微米、1×10⁸立方微米或更大。 In various embodiments of the growth chamber (eg, 124, 126, 128, 130, 244, 246, 248, or 436), the isolation region (eg, 258 or 444) is configured to contain a plurality of minute objects. In other embodiments, the isolation regions can be configured to contain only one, two, three, four, five, or a relatively relatively small number of tiny objects. Accordingly, the volume of an isolation region can be, for example, at least 3 × 10 ³ cubic micrometers, 6 × 10 ³ cubic micrometers, 9 × 10 ³ cubic micrometers, 1 × 10 ⁴ cubic micrometers, 2 × 10 ⁴ cubic micrometers, 4 ×10 ⁴ cubic micrometers, 8 × 10 ⁴ cubic micrometers, 1 × 10 ⁵ cubic micrometers, 2 × 10 ⁵ cubic micrometers, 4 × 10 ⁵ cubic micrometers, 8 × 10 ⁵ cubic micrometers, 1 × 10 ⁶ cubic micrometers, 2 × 10 ⁶ cubic micrometers, 4×10 ⁶ cubic micrometers, 6×10 ⁶ cubic micrometers, 1×10 ⁷ cubic micrometers, 2×10 ⁷ cubic micrometers, 4×10 ⁷ cubic micrometers, 6×10 ⁷ cubic micrometers, 1×10 ⁸ cubic microns or larger.

在生長室之各種實施例中，一近端開口(例如252、472)處之通道122、434之寬度W_ch可在下列範圍之任何者內：50微米至1000微米、50微米至500微米、50微米至400微米、50微米至300微米、50微米至250微米、50微米至200微米、50微米至150微米、50微米至100微米、70微米至500微米、70微米至400微米、70微米至300微米、70微米至250微米、70微米至200微米、70微米至150微米、90微米至400微米、 90微米至300微米、90微米至250微米、90微米至200微米、90微米至150微米、100微米至300微米、100微米至250微米、100微米至200微米、100微米至150微米及100微米至120微米。上述範圍僅為實例，且通道122、434之寬度W_ch可在其他範圍(例如由上文所列之端點之任何者界定之一範圍)內。而且，通道122、434之W_ch可經選定為在除一生長室之一近端開口處之外之通道之區域中之此等區域之任何者內。 In various embodiments of the growth chamber, a proximal end opening of the channel (e.g., 252,472) of the width W _ch 122,434 may be any of those within the following ranges: 50 to 1000 micrometers, 50 micrometers to 500 micrometers, 50 microns to 400 microns, 50 microns to 300 microns, 50 microns to 250 microns, 50 microns to 200 microns, 50 microns to 150 microns, 50 microns to 100 microns, 70 microns to 500 microns, 70 microns to 400 microns, 70 microns Up to 300 microns, 70 microns to 250 microns, 70 microns to 200 microns, 70 microns to 150 microns, 90 microns to 400 microns, 90 microns to 300 microns, 90 microns to 250 microns, 90 microns to 200 microns, 90 microns to 150 Micron, 100 micrometers to 300 micrometers, 100 micrometers to 250 micrometers, 100 micrometers to 200 micrometers, 100 micrometers to 150 micrometers, and 100 micrometers to 120 micrometers. The above ranges are merely examples, and the channel width W _ch 122,434 may be in other ranges (e.g. defined by any endpoint of one range listed above) within. Further, the channel W _ch 122,434 may be selected to be within any region of the channel of either of these area outside the proximal end of one of a growth chamber in addition to opening.

在一些實施例中，一生長室具有約30微米至約200微米或約50微米至約150微米之一橫截面高度。在一些實施例中，生長室具有約100,000平方微米至約2,500,000平方微米或約200,000平方微米至約2,000,000平方微米之一橫截面積。在一些實施例中，一連接區域具有匹配對應生長室之橫截面高度之一橫截面高度。在一些實施例中，連接區域具有約50微米至約500微米或約100微米至約300微米之一橫截面寬度。 In some embodiments, a growth chamber has a cross-sectional height of from about 30 microns to about 200 microns or from about 50 microns to about 150 microns. In some embodiments, the growth chamber has a cross-sectional area of from about 100,000 square microns to about 2,500,000 square microns or from about 200,000 square microns to about 2,000,000 square microns. In some embodiments, a joint region has a cross-sectional height that matches one of the cross-sectional heights of the corresponding growth chamber. In some embodiments, the attachment region has a cross-sectional width of from about 50 microns to about 500 microns or from about 100 microns to about 300 microns.

在生長室之各種實施例中，一近端開口252、472處之通道122、434之高度H_ch可在下列範圍之任何者內：20微米至100微米、20微米至90微米、20微米至80微米、20微米至70微米、20微米至60微米、20微米至50微米、30微米至100微米、30微米至90微米、30微米至80微米、30微米至70微米、30微米至60微米、30微米至50微米、40微米至100微米、40微米至90微米、40微米至80微米、40微米至70微米、40微米至60微米或40微米至50微米。上述範圍僅為實例，且通道122、434之高度H_ch可在其他範圍(例如由上文所列之端點之任何者界定之一範圍)內。通道122、434之高度H_ch可經選擇為在除一生長室之一近端開口處之外之通道之區域中之此等範圍之任何者內。 In various embodiments of the growth chamber, a proximal opening of the channel height H _ch 122,434 252,472 may be any of those of the following ranges: 20 to 100 micrometers, 20 micrometers to 90 micrometers, 20 micrometers to 80 microns, 20 microns to 70 microns, 20 microns to 60 microns, 20 microns to 50 microns, 30 microns to 100 microns, 30 microns to 90 microns, 30 microns to 80 microns, 30 microns to 70 microns, 30 microns to 60 microns 30 microns to 50 microns, 40 microns to 100 microns, 40 microns to 90 microns, 40 microns to 80 microns, 40 microns to 70 microns, 40 microns to 60 microns or 40 microns to 50 microns. The above ranges are merely examples, and the channel height H _ch 122,434 may be in other ranges (e.g. defined by any endpoint of one range listed above) within. The channel height H _ch 122,434 may be selected to be within any of these ranges are outside of the channel region of one opening in a growth chamber at the proximal end addition.

在生長室之各種實施例中，一近端開口252、472處之通道122、434之一橫截面積可在下列範圍之任何者內：500平方微米至50,000平方微米、500平方微米至40,000平方微米、500平方微米至30,000平方 微米、500平方微米至25,000平方微米、500平方微米至20,000平方微米、500平方微米至15,000平方微米、500平方微米至10,000平方微米、500平方微米至7,500平方微米、500平方微米至5,000平方微米、1,000平方微米至25,000平方微米、1,000平方微米至20,000平方微米、1,000平方微米至15,000平方微米、1,000平方微米至10,000平方微米、1,000平方微米至7,500平方微米、1,000平方微米至5,000平方微米、2,000平方微米至20,000平方微米、2,000平方微米至15,000平方微米、2,000平方微米至10,000平方微米、2,000平方微米至7,500平方微米、2,000平方微米至6,000平方微米、3,000平方微米至20,000平方微米、3,000平方微米至15,000平方微米、3,000平方微米至10,000平方微米、3,000平方微米至7,500平方微米或3,000平方微米至6,000平方微米。上述範圍僅為實例，且一近端開口252、472處之通道122之橫截面積可在其他範圍(例如由上文所列之端點之任何者界定之一範圍)內。 In various embodiments of the growth chamber, one of the channels 122, 434 at a proximal opening 252, 472 may have a cross-sectional area in any of the following ranges: 500 square microns to 50,000 square microns, 500 square microns to 40,000 square meters. Micron, 500 square microns to 30,000 square Micron, 500 square microns to 25,000 square microns, 500 square microns to 20,000 square microns, 500 square microns to 15,000 square microns, 500 square microns to 10,000 square microns, 500 square microns to 7,500 square microns, 500 square microns to 5,000 square microns, 1,000 square microns to 25,000 square microns, 1,000 square microns to 20,000 square microns, 1,000 square microns to 15,000 square microns, 1,000 square microns to 10,000 square microns, 1,000 square microns to 7,500 square microns, 1,000 square microns to 5,000 square microns, 2,000 squares Micron to 20,000 square micrometers, 2,000 square micrometers to 15,000 square micrometers, 2,000 square micrometers to 10,000 square micrometers, 2,000 square micrometers to 7,500 square micrometers, 2,000 square micrometers to 6,000 square micrometers, 3,000 square micrometers to 20,000 square micrometers, 3,000 square micrometers to 15,000 square micrometers, 3,000 square micrometers to 10,000 square micrometers, 3,000 square micrometers to 7,500 square micrometers, or 3,000 square micrometers to 6,000 square micrometers. The above ranges are merely examples, and the cross-sectional area of the channel 122 at a proximal opening 252, 472 can be within other ranges (eg, by one of the endpoints listed above).

在生長室之各種實施例中，連接區域254、442之長度L_con可在下列範圍之任何者內：1微米至200微米、5微米至150微米、10微米至100微米、15微米至80微米、20微米至60微米、20微米至500微米、40微米至400微米、60微米至300微米、80微米至200微米及100微米至150微米。上述範圍僅為實例，且一連接區域254、442之長度L_con可在不同於上述實例之一範圍(例如由上文所列之端點之任何者界定之一範圍)內。 In various embodiments of the growth chamber, the length L _{con of the} attachment regions 254, 442 can be within any of the following ranges: 1 micrometer to 200 micrometers, 5 micrometers to 150 micrometers, 10 micrometers to 100 micrometers, 15 micrometers to 80 micrometers. 20 microns to 60 microns, 20 microns to 500 microns, 40 microns to 400 microns, 60 microns to 300 microns, 80 microns to 200 microns, and 100 microns to 150 microns. The above ranges are merely examples, and the length L _{con of} a connection region 254, 442 may be within a range different from one of the above examples (eg, a range defined by any of the endpoints listed above).

在生長室之各種實施例中，一近端開口252處之一連接區域254、442之寬度W_con可在下列範圍之任何者內：20微米至500微米、20微米至400微米、20微米至300微米、20微米至200微米、20微米至150微米、20微米至100微米、20微米至80微米、20微米至60微米、30微米至400微米、30微米至300微米、30微米至200微米、30微米至150 微米、30微米至100微米、30微米至80微米、30微米至60微米、40微米至300微米、40微米至200微米、40微米至150微米、40微米至100微米、40微米至80微米、40微米至60微米、50微米至250微米、50微米至200微米、50微米至150微米、50微米至100微米、50微米至80微米、60微米至200微米、60微米至150微米、60微米至100微米、60微米至80微米、70微米至150微米、70微米至100微米及80微米至100微米。上述範圍僅為實例，且一近端開口252處之一連接區域254、442之寬度W_con可不同於上述實例(例如由上文所列之端點之任何者界定之一範圍)。 In various embodiments of the growth chamber, the width W _con of one of the connection regions 254, 442 at a proximal opening 252 can be within any of the following ranges: 20 microns to 500 microns, 20 microns to 400 microns, 20 microns to 300 microns, 20 microns to 200 microns, 20 microns to 150 microns, 20 microns to 100 microns, 20 microns to 80 microns, 20 microns to 60 microns, 30 microns to 400 microns, 30 microns to 300 microns, 30 microns to 200 microns 30 microns to 150 microns, 30 microns to 100 microns, 30 microns to 80 microns, 30 microns to 60 microns, 40 microns to 300 microns, 40 microns to 200 microns, 40 microns to 150 microns, 40 microns to 100 microns, 40 Micron to 80, 40 to 60, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 60 to 200, 60 to 150 microns, 60 microns to 100 microns, 60 microns to 80 microns, 70 microns to 150 microns, 70 microns to 100 microns, and 80 microns to 100 microns. The above ranges are merely examples, and the width W _con of one of the connection regions 254, 442 at a proximal opening 252 may differ from the above examples (eg, a range defined by any of the endpoints listed above).

在生長室之各種實施例中，一近端開口252、472處之一連接區域254、442之寬度W_con可在下列範圍之任何者內：2微米至35微米、2微米至25微米、2微米至20微米、2微米至15微米、2微米至10微米、2微米至7微米、2微米至5微米、2微米至3微米、3微米至25微米、3微米至20微米、3微米至15微米、3微米至10微米、3微米至7微米、3微米至5微米、3微米至4微米、4微米至20微米、4微米至15微米、4微米至10微米、4微米至7微米、4微米至5微米、5微米至15微米、5微米至10微米、5微米至7微米、6微米至15微米、6微米至10微米、6微米至7微米、7微米至15微米、7微米至10微米、8微米至15微米及8微米至10微米。上述範圍僅為實例，且一近端開口252、472處之一連接區域254、442之寬度W_con可不同於上述實例(例如由上文所列之端點之任何者界定之一範圍)。 In various embodiments of the growth chamber, a proximal end connected to one of the opening area 254,442 252,472 The width W _con may be any of those within the following ranges: from 2 microns to 35 microns, 2 to 25 microns, 2 Micron to 20 microns, 2 microns to 15 microns, 2 microns to 10 microns, 2 microns to 7 microns, 2 microns to 5 microns, 2 microns to 3 microns, 3 microns to 25 microns, 3 microns to 20 microns, 3 microns to 15 microns, 3 microns to 10 microns, 3 microns to 7 microns, 3 microns to 5 microns, 3 microns to 4 microns, 4 microns to 20 microns, 4 microns to 15 microns, 4 microns to 10 microns, 4 microns to 7 microns 4 microns to 5 microns, 5 microns to 15 microns, 5 microns to 10 microns, 5 microns to 7 microns, 6 microns to 15 microns, 6 microns to 10 microns, 6 microns to 7 microns, 7 microns to 15 microns, 7 Micron to 10 microns, 8 microns to 15 microns, and 8 microns to 10 microns. The above ranges are merely examples, and the width W _con of one of the connection regions 254, 442 at a proximal opening 252, 472 may differ from the above examples (eg, a range defined by any of the endpoints listed above).

在生長室之各種實施例中，近端開口252、472處之一連接區域254、442之長度L_con與連接區域254、442之一寬度W_con之一比率可大於或等於下列比率之任何者：0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0、6.0、7.0、8.0、9.0、10.0或更大。上述比率僅為實例，且近端開口252、472處之一連接區域254之長度L_con與連接區域 254、442之一寬度W_con之比率可不同於上述實例。 In various embodiments of the growth chamber, the ratio of the length L _con of one of the connection regions 254, 442 at one of the proximal openings 252, 472 to the width W _con of one of the connection regions 254, 442 may be greater than or equal to any of the following ratios : 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or more. The above ratios are merely examples, and the ratio of the length L _con of one of the connection regions 254 at the proximal openings 252, 472 to the width W _con of one of the connection regions 254, 442 may be different from the above examples.

在微流體器件100、200、240、290、400之各種實施例中，V_max可經設定為約0.2微升/秒、約0.3微升/秒、約0.4微升/秒、約0.5微升/秒、約0.6微升/秒、約0.7微升/秒、約0.8微升/秒、約0.9微升/秒、約1.0微升/秒、約1.1微升/秒、約1.2微升/秒、約1.3微升/秒、約1.4微升/秒或約1.5微升/秒。在一些其他實施例中，替代地，V_max可經設定為約0.2微升/秒、約0.3微升/秒、約0.4微升/秒、約0.5微升/秒、約0.6微升/秒、約0.7微升/秒、約0.8微升/秒、約0.9微升/秒、約1.0微升/秒、約1.1微升/秒、約1.2微升/秒、約1.3微升/秒、約1.4微升/秒、約1.5微升/秒、約1.6微升/秒、約1.7微升/秒、約1.8微升/秒、約1.9微升/秒、約2.0微升/秒、約2.1微升/秒、約2.2微升/秒、約2.3微升/秒、約2.4微升/秒或約2.5微升/秒。在其他實施例中，V_max可經設定為或大致設定為2.0微升/秒、2.2微升/秒、2.4微升/秒、2.6微升/秒、2.8微升/秒、3.0微升/秒、3.2微升/秒、3.4微升/秒、3.6微升/秒、3.8微升/秒、4.0微升/秒、4.2微升/秒、4.4微升/秒、4.6微升/秒、4.8微升/秒、5.0微升/秒、6.0微升/秒、7.0微升/秒、8.0微升/秒或9.0微升/秒。 In various embodiments of microfluidic devices 100, 200, 240, 290, 400, _Vmax can be set to about 0.2 microliters/second, about 0.3 microliters/second, about 0.4 microliters/second, about 0.5 microliters. /sec, about 0.6 microliters/second, about 0.7 microliters/second, about 0.8 microliters/second, about 0.9 microliters/second, about 1.0 microliters/second, about 1.1 microliters/second, about 1.2 microliters/ Seconds, about 1.3 microliters per second, about 1.4 microliters per second or about 1.5 microliters per second. In some other embodiments, alternatively, _Vmax can be set to about 0.2 microliters/second, about 0.3 microliters/second, about 0.4 microliters/second, about 0.5 microliters/second, about 0.6 microliters/second. , about 0.7 microliters per second, about 0.8 microliters per second, about 0.9 microliters per second, about 1.0 microliters per second, about 1.1 microliters per second, about 1.2 microliters per second, about 1.3 microliters per second, About 1.4 microliters per second, about 1.5 microliters per second, about 1.6 microliters per second, about 1.7 microliters per second, about 1.8 microliters per second, about 1.9 microliters per second, about 2.0 microliters per second, about 2.1 microliters per second, about 2.2 microliters per second, about 2.3 microliters per second, about 2.4 microliters per second or about 2.5 microliters per second. In other embodiments, _Vmax can be set to or substantially set to 2.0 microliters/second, 2.2 microliters/second, 2.4 microliters/second, 2.6 microliters/second, 2.8 microliters/second, 3.0 microliters/ Seconds, 3.2 microliters/second, 3.4 microliters/second, 3.6 microliters/second, 3.8 microliters/second, 4.0 microliters/second, 4.2 microliters/second, 4.4 microliters/second, 4.6 microliters/second, 4.8 microliters per second, 5.0 microliters per second, 6.0 microliters per second, 7.0 microliters per second, 8.0 microliters per second or 9.0 microliters per second.

在具有生長室之微流體器件之各種實施例中，一生長室之一隔離區域258、444之體積可為(例如)至少3×10³立方微米、6×10³立方微米、9×10³立方微米、1×10⁴立方微米、2×10⁴立方微米、4×10⁴立方微米、8×10⁴立方微米、1×10⁵立方微米、2×10⁵立方微米、4×10⁵立方微米、8×10⁵立方微米、1×10⁶立方微米、2×10⁶立方微米、4×10⁶立方微米、6×10⁶立方微米或更大。在具有生長室之微流體器件之各種實施例中，一生長室之體積可為約5×10³立方微米、約7×10³立方微米、約1×10⁴立方微米、約3×10⁴立方微米、約5×10⁴立方微米、約8×10⁴立方微米、約1×10⁵立方微米、約2×10⁵立方微米、約4×10⁵立方微米、約6×10⁵立方微米、約8×10⁵立方微米、約1×10⁶立方微米、約2×10⁶立方 微米、約4×10⁶立方微米、約8×10⁶立方微米、約1×10⁷立方微米、約3×10⁷立方微米、約5×10⁷立方微米或約8×10⁷立方微米或更大。在一些實施例中，微流體器件具有其中可維持不超過1×10²個生物細胞之生長室，且一生長室之體積可不超過2×10⁶立方微米。在一些實施例中，微流體器件具有其中可維持不超過1×10²個生物細胞之生長室，且一生長室可不超過4×10⁵立方微米。在其他實施例中，微流體器件具有其中可維持不超過50個生物細胞之生長室，一生長室可不超過4×10⁵立方微米。 In various embodiments of the microfluidic device having a growth chamber, the volume of one of the isolation regions 258, 444 of a growth chamber can be, for example, at least 3 x 10 ³ cubic micrometers, 6 x 10 ³ cubic micrometers, 9 x 10 ³ Cubic micron, 1 x 10 ⁴ cubic micron, 2 x 10 ⁴ cubic micron, 4 x 10 ⁴ cubic micron, 8 x 10 ⁴ cubic micron, 1 x 10 ⁵ cubic micron, 2 x 10 ⁵ cubic micron, 4 x 10 ⁵ cubic Micron, 8 x 10 ⁵ cubic micrometers, 1 x 10 ⁶ cubic micrometers, 2 x 10 ⁶ cubic micrometers, 4 x 10 ⁶ cubic micrometers, 6 x 10 ⁶ cubic micrometers or more. In various embodiments of a microfluidic device having a growth chamber, a growth chamber may have a volume of about 5 x 10 ³ cubic microns, about 7 x 10 ³ cubic microns, about 1 x 10 ⁴ cubic microns, and about 3 x 10 ^{4 .} Cubic microns, about 5 x 10 ⁴ cubic microns, about 8 x 10 ⁴ cubic microns, about 1 x 10 ⁵ cubic microns, about 2 x 10 ⁵ cubic microns, about 4 x 10 ⁵ cubic microns, about 6 x 10 ⁵ cubic microns , about 8×10 ⁵ cubic micrometers, about 1×10 ⁶ cubic micrometers, about 2×10 ⁶ cubic micrometers, about 4×10 ⁶ cubic micrometers, about 8×10 ⁶ cubic micrometers, about 1×10 ⁷ cubic micrometers, about 3 x 10 ⁷ cubic microns, about 5 x 10 ⁷ cubic microns or about 8 x 10 ⁷ cubic microns or more. In some embodiments, wherein the microfluidic device may be maintained with no more than 1 × 10 ² The Growth chambers of a biological cell, and a volume of the growth chamber may not exceed 2 × 10 ⁶ cubic microns. In some embodiments, wherein the microfluidic device may be maintained with no more than 1 × 10 ² The Growth chambers of a biological cell, and a growth chamber be not more than 4 × 10 ⁵ cubic microns. In other embodiments, wherein the microfluidic device having no more than 50 can be maintained in a growth chamber of a biological cell, a growth chamber be not more than 4 × 10 ⁵ cubic microns.

在各種實施例中，微流體器件具有如同本文中所討論之實施例之任何者般構形之生長室，其中微流體器件具有約100個至約500個生長室、約200個至約1000個生長室、約500個至約1500個生長室、約1000個至約2000個生長室或約1000個至約3500個生長室。 In various embodiments, the microfluidic device has a growth chamber configured as in any of the embodiments discussed herein, wherein the microfluidic device has from about 100 to about 500 growth chambers, from about 200 to about 1000 Growth chambers, from about 500 to about 1500 growth chambers, from about 1000 to about 2000 growth chambers or from about 1000 to about 3500 growth chambers.

在一些其他實施例中，微流體器件具有如同本文中所討論之實施例之任何者般構形之生長室，其中微流體器件具有約1500個至約3000個生長室、約2000個至約3500個生長室、約2500個至約4000個生長室、約3000個至約4500個生長室、約3500個至約5000個生長室、約4000個至約5500個生長室、約4500個至約6000個生長室、約5000個至約6500個生長室、約5500個至約7000個生長室、約6000個至約7500個生長室、約6500個至約8000個生長室、約7000個至約8500個生長室、約7500個至約9000個生長室、約8000個至約9500個生長室、約8500個至約10,000個生長室、約9000個至約10,500個生長室、約9500個至約11,000個生長室、約10,000個至約11,500個生長室、約10,500個至約12,000個生長室、約11,000個至約12,500個生長室、約11,500個至約13,000個生長室、約12,000個至約13,500個生長室、約12,500個至約14,000個生長室、約13,000個至約14,500個生長室、約13,500個至約15,000個生長室、約14,000個至約15,500個生長室、約14,500個至約 16,000個生長室、約15,000個至約16,500個生長室、約15,500個至約17,000個生長室、約16,000個至約17,500個生長室、約16,500個至約18,000個生長室、約17,000個至約18,500個生長室、約17,500個至約19,000個生長室、約18,000個至約19,500個生長室、約18,500個至約20,000個生長室、約19,000個至約20,500個生長室、約19,500個至約21,000個生長室或約20,000個至約21,500個生長室。 In some other embodiments, the microfluidic device has a growth chamber configured as in any of the embodiments discussed herein, wherein the microfluidic device has from about 1500 to about 3000 growth chambers, from about 2000 to about 3500 Growth chambers, from about 2,500 to about 4,000 growth chambers, from about 3,000 to about 4,500 growth chambers, from about 3,500 to about 5,000 growth chambers, from about 4,000 to about 5,500 growth chambers, from about 4,500 to about 6,000 Growth chambers, from about 5,000 to about 6,500 growth chambers, from about 5,500 to about 7,000 growth chambers, from about 6,000 to about 7,500 growth chambers, from about 6,500 to about 8,000 growth chambers, from about 7,000 to about 8500 Growth chambers, from about 7,500 to about 9,000 growth chambers, from about 8,000 to about 9,500 growth chambers, from about 8,500 to about 10,000 growth chambers, from about 9,000 to about 10,500 growth chambers, from about 9,500 to about 11,000 Growth chambers, from about 10,000 to about 11,500 growth chambers, from about 10,500 to about 12,000 growth chambers, from about 11,000 to about 12,500 growth chambers, from about 11,500 to about 13,000 growth chambers, from about 12,000 to about 13,500 Growth chambers, from about 12,500 to about 14,000 growth chambers, about 13,000 to Approximately 14,500 growth chambers, from about 13,500 to about 15,000 growth chambers, from about 14,000 to about 15,500 growth chambers, from about 14,500 to about 16,000 growth chambers, from about 15,000 to about 16,500 growth chambers, from about 15,500 to about 17,000 growth chambers, from about 16,000 to about 17,500 growth chambers, from about 16,500 to about 18,000 growth chambers, from about 17,000 to about 18,500 growth chambers, from about 17,500 to about 19,000 growth chambers, from about 18,000 to about 19,500 growth chambers, from about 18,500 to about 20,000 growth chambers, from about 19,000 to about 20,500 growth chambers, from about 19,500 to about 21,000 growth chambers or from about 20,000 to about 21,500 growth chambers.

圖2F繪示根據一實施例之一微流體器件290。圖2F中所繪示之微流體器件290係一微流體器件100之一典型圖。實際上，微流體器件290及其組成線路元件(例如通道122及生長室128)將具有本文中所討論之尺寸。圖2F中所繪示之微流體線路120具有兩個端口107、四個不同通道122及四個不同流動路徑106。微流體器件290進一步包括朝向各通道122敞開之複數個生長室。在圖2F所繪示之微流體器件中，生長室具有類似於圖2E中所繪示之圍欄之一幾何形狀，且因此具有連接區域及隔離區域兩者。據此，微流體線路120包含掃及區域(例如通道122及副流262之最大滲透深度D_p內之連接區域254之部分)及未掃及區域(例如隔離區域258及不在副流262之最大滲透深度D_p內之連接區域254之部分)兩者。 2F illustrates a microfluidic device 290 in accordance with an embodiment. The microfluidic device 290 illustrated in FIG. 2F is a typical diagram of a microfluidic device 100. In effect, the microfluidic device 290 and its constituent line elements (e.g., channel 122 and growth chamber 128) will have the dimensions discussed herein. The microfluidic line 120 depicted in FIG. 2F has two ports 107, four different channels 122, and four different flow paths 106. The microfluidic device 290 further includes a plurality of growth chambers that are open toward each of the channels 122. In the microfluidic device depicted in Figure 2F, the growth chamber has a geometry similar to that of the fence depicted in Figure 2E, and thus has both a junction region and an isolation region. Accordingly, the microfluidic line 120 includes sweeps and regions (e.g., portions of the connection regions 254 within the maximum penetration depth _Dp of the channels 122 and the secondary streams 262) and unswept regions (e.g., the isolation regions 258 and the maximum of the secondary streams 262). Both of the penetration regions 254 within the depth _Dp are penetrated.

圖3A及圖3B展示根據本發明之可用以操作及觀測微流體器件(例如100、200、240、290)之系統150之各種實施例。如圖3A中所繪示，系統150可包含經構形以保持一微流體器件100(圖中未展示)或本文中所描述之任何其他微流體器件之一結構(「巢套」)300。巢套300可包含能夠與微流體器件360(例如一光學致動之電動器件100)界接且提供自電源192至微流體器件360之電連接之一插座302。巢套300可進一步包含一整合電信號產生子系統304。電信號產生子系統304可經構形以將一偏壓電壓供應至插座302，使得當由插座302保持微流體器件360時，橫跨微流體器件360中之一對電極而施加該偏壓電壓。因此，電 信號產生子系統304可為電源192之部分。將一偏壓電壓施加於微流體器件360之能力不意謂將在由插座302保持微流體器件360時始終施加一偏壓電壓。確切而言，在大多數情況中，將間歇地施加偏壓電壓，例如，僅在需要促進在微流體器件360中產生電動力(諸如介電泳或電潤濕)時施加偏壓電壓。 3A and 3B show various embodiments of a system 150 that can be used to operate and observe microfluidic devices (e.g., 100, 200, 240, 290) in accordance with the present invention. As shown in FIG. 3A, system 150 can include a structure ("socket") 300 configured to hold a microfluidic device 100 (not shown) or any other microfluidic device described herein. The nest 300 can include a socket 302 that can interface with the microfluidic device 360 (e.g., an optically actuated motor device 100) and provide electrical connection from the power source 192 to the microfluidic device 360. The nest 300 can further include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 can be configured to supply a bias voltage to the receptacle 302 such that when the microfluidic device 360 is held by the receptacle 302, the bias voltage is applied across one of the pair of microfluidic devices 360 . Therefore, electricity Signal generation subsystem 304 can be part of power supply 192. The ability to apply a bias voltage to the microfluidic device 360 does not mean that a bias voltage will always be applied while the microfluidic device 360 is being held by the receptacle 302. Specifically, in most cases, the bias voltage will be applied intermittently, for example, only when it is desired to promote the generation of electrical power (such as dielectrophoresis or electrowetting) in the microfluidic device 360.

如圖3A中所繪示，巢套300可包含一印刷電路板總成(PCBA)320。電信號產生子系統304可安裝於PCBA 320上且電整合至PCBA 320中。例示性巢套300亦包含安裝於PCBA 320上之插座302。 As shown in FIG. 3A, the nest 300 can include a printed circuit board assembly (PCBA) 320. Electrical signal generation subsystem 304 can be mounted on PCBA 320 and electrically integrated into PCBA 320. The exemplary nest 300 also includes a receptacle 302 that is mounted to the PCBA 320.

通常，電信號產生子系統304將包含一波形產生器(圖中未展示)。電信號產生子系統304可進一步包含經構形以放大自該波形產生器接收之一波形之一示波器(圖中未展示)及/或一波形放大電路(圖中未展示)。該示波器(若存在)可經構形以量測供應至由插座302保持之微流體器件360之波形。在某些實施例中，該示波器量測接近於微流體器件360(且遠離於該波形產生器)之一位置處之波形以因此確保量測實際上施加於器件之波形時之較大精確度。例如，可將自該示波器量測獲得之資料作為回饋提供至該波形產生器，且該波形產生器可經構形以基於此回饋而調整其輸出。一適當組合之波形產生器及示波器之一實例係Red Pitaya^TM。 Typically, electrical signal generation subsystem 304 will include a waveform generator (not shown). The electrical signal generation subsystem 304 can further include an oscilloscope (not shown) and/or a waveform amplification circuit (not shown) configured to amplify one of the waveforms received from the waveform generator. The oscilloscope, if present, can be configured to measure the waveform supplied to the microfluidic device 360 held by the receptacle 302. In some embodiments, the oscilloscope measures a waveform at a location near one of the microfluidic device 360 (and away from the waveform generator) to thereby ensure greater accuracy in measuring the waveform actually applied to the device. . For example, data obtained from the oscilloscope measurements can be provided as feedback to the waveform generator, and the waveform generator can be configured to adjust its output based on this feedback. A suitable combination of the waveform generator and oscilloscope example one based Red Pitaya ^TM.

在某些實施例中，巢套300進一步包括一控制器308，諸如用以感測及/或控制電信號產生子系統304之一微處理器。適合微處理器之實例包含Arduino^TM微處理器，諸如Arduino Nano^TM。控制器308可用以執行功能及分析或可與一外部主控制器154(如圖1中所展示)通信以執行功能及分析。在圖3A所繪示之實施例中，控制器308透過一介面310(例如一插頭或連接器)而與一主控制器154通信。 In some embodiments, the nest 300 further includes a controller 308, such as a microprocessor for sensing and/or controlling one of the electrical signal generation subsystems 304. Suitable examples include a microprocessor Arduino ^TM microprocessor, such as Arduino Nano ^TM. Controller 308 can be used to perform functions and analysis or can communicate with an external host controller 154 (as shown in Figure 1) to perform functions and analysis. In the embodiment illustrated in FIG. 3A, controller 308 is in communication with a host controller 154 via an interface 310 (eg, a plug or connector).

在一些實施例中，巢套300可包括一電信號產生子系統304，其包括一Red Pitaya^TM波形產生器/示波器單元(「Red Pitaya單元」)及一 波形放大電路，該波形放大電路放大由該Red Pitaya單元產生之波形且將放大電壓傳至微流體器件100。在一些實施例中，該Red Pitaya單元經構形以量測微流體器件360處之放大電壓且接著根據需要而調整其自身輸出電壓，使得微流體器件360處之量測電壓係所要值。在一些實施例中，該波形放大線路可具有由安裝於PCBA 320上之一對DC-DC轉換器產生之一+6.5伏特至-6.5伏特電源供應器以導致微流體器件100處之高達13Vpp之一信號。 In some embodiments, the nest 300 may comprise a subsystem 304 generates an electrical signal, comprising a waveform generator Red Pitaya ^TM / oscilloscope unit ( "Red Pitaya unit") and a waveform amplifying circuit, the amplifying circuit is amplified by the waveform The Red Pitaya unit generates a waveform and transmits the amplified voltage to the microfluidic device 100. In some embodiments, the Red Pitaya unit is configured to measure the amplified voltage at the microfluidic device 360 and then adjust its own output voltage as needed such that the measured voltage at the microfluidic device 360 is a desired value. In some embodiments, the waveform amplifying line can have a +6.5 volt to -6.5 volt power supply generated by one of the pair of DC-DC converters mounted on the PCBA 320 to cause up to 13 Vpp at the microfluidic device 100. a signal.

如圖3A中所繪示，巢套300可進一步包含一熱控制子系統306。熱控制子系統306可經構形以調節由巢套300保持之微流體器件360之溫度。例如，熱控制子系統306可包含一帕耳帖(Peltier)熱電器件(圖中未展示)及一冷卻單元(圖中未展示)。該帕耳帖熱電器件可具有經構形以與微流體器件360之至少一表面界接之一第一表面。該冷卻單元可為(例如)一冷卻塊(圖中未展示)，諸如一液冷鋁塊。該帕耳帖熱電器件之一第二表面(例如相對於該第一表面之一表面)可經構形以與此一冷卻塊之一表面界接。該冷卻塊可連接至經構形以使冷卻流體循環通過該冷卻塊之一流體路徑330。在圖3A所繪示之實施例中，巢套300包括一入口332及一出口334來自一外部儲液器(圖中未展示)接收冷卻流體，將該冷卻流體引入至流體路徑330中且通過該冷卻塊，且接著使該冷卻流體返回至該外部儲液器。在一些實施例中，該帕耳帖熱電器件、該冷卻單元及/或流體路徑330可安裝於巢套300之一罩殼340上。在一些實施例中，熱控制子系統306經構形以調節該帕耳帖熱電器件之溫度以便達到微流體器件360之一目標溫度。例如，可藉由一熱電電源供應器(諸如一Pololu^TM熱電電源供應器(Pololu Robotics and Electronics公司))而達成該帕耳帖熱電器件之溫度調節。熱控制子系統306可包含一回饋電路，諸如由一類比電路提供之一溫度值。替代地，該回饋電路可由一數位電路提供。 As shown in FIG. 3A, the nest 300 can further include a thermal control subsystem 306. Thermal control subsystem 306 can be configured to adjust the temperature of microfluidic device 360 held by nest 300. For example, thermal control subsystem 306 can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device can have a first surface that is configured to interface with at least one surface of the microfluidic device 360. The cooling unit can be, for example, a cooling block (not shown), such as a liquid cold aluminum block. A second surface of the Peltier thermoelectric device (e.g., relative to a surface of the first surface) can be configured to interface with one of the surfaces of the cooling block. The cooling block can be coupled to a fluid path 330 configured to circulate a cooling fluid through the cooling block. In the embodiment illustrated in FIG. 3A, the nest 300 includes an inlet 332 and an outlet 334 from an external reservoir (not shown) for receiving cooling fluid, introducing the cooling fluid into the fluid path 330 and passing The cooling block is then returned to the external reservoir. In some embodiments, the Peltier thermoelectric device, the cooling unit, and/or the fluid path 330 can be mounted to a housing 340 of the nest 300. In some embodiments, the thermal control subsystem 306 is configured to adjust the temperature of the Peltier thermoelectric device to achieve a target temperature of the microfluidic device 360. For example, by a thermoelectric power supply (such as a thermoelectric power supply Pololu ^TM (Pololu Robotics and Electronics Corporation)) and reached the temperature of the Peltier thermoelectric device of regulation. Thermal control subsystem 306 can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit.

在一些實施例中，巢套300可包含具有一回饋電路(其係一類比分壓器電路(圖中未展示))之一熱控制子系統306，其包含一電阻器(例如，具有1千歐姆+/-0.1%之電阻、+/-0.02ppm/C0之溫度係數)及一NTC熱阻器(例如，具有1千歐姆+/-0.01%之標稱電阻)。在一些例項中，熱控制子系統306自回饋電路量測電壓且接著將計算溫度值用作為至一板上PID控制迴路演算法之輸入。來自該PID控制迴路演算法之輸出可驅動(例如)一Pololu^TM馬達驅動器(圖中未展示)上之一方向接針及一脈寬調變信號接針兩者來致動熱電電源供應器，藉此控制帕耳帖熱電器件。 In some embodiments, the nest 300 can include a thermal control subsystem 306 having a feedback circuit (which is an analog voltage divider circuit (not shown)) that includes a resistor (eg, having one thousand Ohms +/- 0.1% resistance, +/- 0.02 ppm/C0 temperature coefficient) and an NTC thermistor (for example, with a nominal resistance of 1 k ohm +/- 0.01%). In some examples, thermal control subsystem 306 measures the voltage from the feedback circuit and then uses the calculated temperature value as an input to an on-board PID control loop algorithm. The output from the PID control loop of the algorithm can be driven (e.g.) a Pololu ^TM motor drive (not shown in the drawing) connected to both the one direction, and a needle contact pins PWM signal to actuate the thermoelectric power supply, Thereby controlling the Peltier thermoelectric device.

巢套300可包含一串列埠350，其允許控制器308之微處理器經由介面310而與一外部主控制器154通信。另外，控制器308之微處理器可與電信號產生子系統304及熱控制子系統306通信(例如，經由一Plink工具(圖中未展示))。因此，經由控制器308、介面310及串列埠350之組合，電信號產生子系統304及熱控制子系統306可與外部主控制器154通信。依此方式，主控制器154可尤其藉由對輸出電壓調整執行縮放計算而輔助電信號產生子系統304。經由耦合至外部主控制器154之一顯示器件170而提供之一圖形使用者介面(GUI)(圖中未展示)可經構形以繪製分別自熱控制子系統306及電信號產生子系統304獲得之溫度及波形資料。替代地或另外，該GUI可允許更新控制器308、熱控制子系統306及電信號產生子系統304。 The nest 300 can include a serial port 350 that allows the microprocessor of the controller 308 to communicate with an external host controller 154 via the interface 310. Additionally, the microprocessor of controller 308 can be in communication with electrical signal generation subsystem 304 and thermal control subsystem 306 (e.g., via a Plink tool (not shown)). Thus, the electrical signal generation subsystem 304 and the thermal control subsystem 306 can communicate with the external host controller 154 via a combination of the controller 308, the interface 310, and the serial port 350. In this manner, main controller 154 can assist electrical signal generation subsystem 304, inter alia, by performing scaling calculations on output voltage adjustments. A graphical user interface (GUI) (not shown) may be configured via one of the display devices 170 coupled to the external host controller 154 to be configured to draw the respective self-heating control subsystem 306 and the electrical signal generation subsystem 304. Obtain temperature and waveform data. Alternatively or additionally, the GUI may allow for updating controller 308, thermal control subsystem 306, and electrical signal generation subsystem 304.

如上文所討論，系統150可包含一成像器件194。在一些實施例中，成像器件194包括一光調變子系統422。光調變子系統422可包含一數位鏡面器件(DMD)或一微快門陣列系統(MSA)，其等之任一者可經構形以自一光源420接收光且將該接收光之一子集傳輸至顯微鏡450之一光學元件串中。替代地，光調變子系統422可包含自身產生光(且因此無需一光源420)之一器件，諸如一有機發光二極體顯示器 (OLED)、一矽上液晶(LCOS)器件、一矽上鐵電液晶器件(FLCOS)或一透射式液晶顯示器(LCD)。光調變子系統422可為(例如)一投射器。因此，光調變子系統422能夠發射結構光及非結構光兩者。一適合光調變子系統422之一實例係來自Andor Technologies^TM之Mosaic^TM系統。在某些實施例中，系統150之成像模組164及/或動力模組162可控制光調變子系統422。 As discussed above, system 150 can include an imaging device 194. In some embodiments, imaging device 194 includes a light modulation subsystem 422. The light modulation subsystem 422 can include a digital mirror device (DMD) or a micro shutter array system (MSA), any of which can be configured to receive light from a light source 420 and to receive the light. The set is transmitted to a string of optical elements in one of the microscopes 450. Alternatively, the light modulation subsystem 422 can include a device that generates light by itself (and thus does not require a light source 420), such as an organic light emitting diode display (OLED), a liquid crystal on a liquid crystal (LCOS) device, a stack of Ferroelectric liquid crystal device (FLCOS) or a transmissive liquid crystal display (LCD). Light modulation subsystem 422 can be, for example, a projector. Thus, the light modulation subsystem 422 is capable of emitting both structured and unstructured light. A light modulator suitable subsystem 422, one instance of the system from Andor Technologies ^TM Mosaic ^TM system. In some embodiments, imaging module 164 and/or power module 162 of system 150 can control light modulation subsystem 422.

在某些實施例中，成像器件194進一步包括一顯微鏡450。在此等實施例中，巢套300及光調變子系統422可個別地經構形以安裝於顯微鏡450上。顯微鏡450可為(例如)一標準研究級光學顯微鏡或螢光顯微鏡。因此，巢套300可經構形以安裝於顯微鏡450之置物台426上及/或光調變子系統422可經構形以安裝於顯微鏡450之一端口上。在其他實施例中，本文中所描述之巢套300及光調變子系統422可為顯微鏡450之整合組件。 In some embodiments, imaging device 194 further includes a microscope 450. In such embodiments, the nest 300 and the light modulation subsystem 422 can be individually configured to be mounted on the microscope 450. The microscope 450 can be, for example, a standard research grade optical microscope or a fluorescent microscope. Thus, the nest 300 can be configured to be mounted to the stage 426 of the microscope 450 and/or the light modulation subsystem 422 can be configured to be mounted to one of the ports of the microscope 450. In other embodiments, the nest 300 and light modulation subsystem 422 described herein can be an integrated component of the microscope 450.

在某些實施例中，顯微鏡450可進一步包含一或多個偵測器440。在一些實施例中，偵測器440由成像模組164控制。偵測器440可包含一目鏡、一電荷耦合器件(CCD)、一攝影機(例如一數位攝影機)或其等之任何組合。若存在至少兩個偵測器440，則一偵測器可為(例如)一快圖框速率攝影機且另一偵測器可為一高敏感度攝影機。此外，顯微鏡450可包含一光學元件串，其經構形以自微流體器件360接收反射光及/或發射光且將該反射光及/或發射光之至少一部分聚焦於一或多個偵測器440上。顯微鏡之該光學元件串亦可包含用於不同偵測器之不同鏡筒透鏡(圖中未展示)，使得各偵測器上之最終放大倍數可不同。 In some embodiments, microscope 450 can further include one or more detectors 440. In some embodiments, the detector 440 is controlled by the imaging module 164. Detector 440 can include an eyepiece, a charge coupled device (CCD), a camera (e.g., a digital camera), or any combination thereof. If there are at least two detectors 440, one detector can be, for example, a fast frame rate camera and the other detector can be a high sensitivity camera. Additionally, microscope 450 can include a string of optical elements configured to receive reflected light and/or emitted light from microfluidic device 360 and focus at least a portion of the reflected and/or emitted light to one or more of the detected On the 440. The string of optical elements of the microscope may also include different barrel lenses (not shown) for different detectors such that the final magnification on each detector may vary.

在某些實施例中，成像器件194經構形以使用至少兩個光源。例如，一第一光源420可用以產生結構光(例如，經由光調變子系統422)且一第二光源430可用以提供非結構光。第一光源420可產生用於光學 致動電動及/或螢光激發之結構光，且第二光源430可用以提供亮場照明。在此等實施例中，動力模組164可用以控制第一光源420且成像模組164可用以控制第二光源430。顯微鏡450之光學元件串可經構形以：(1)自光調變子系統422接收結構光且當一微流體器件(諸如一光學致動之電動器件)由巢套300保持時將該結構光聚焦於該器件中之至少一第一區域上；及(2)自該微流體器件接收反射光及/或發射光且將此反射光及/或發射光之至少一部分聚焦至偵測器440上。光學元件串可進一步經構形以自一第二光源接收非結構光且當該微流體器件由巢套300保持時將該非結構光聚焦於該器件之至少一第二區域上。在某些實施例中，該微流體器件之該第一區域及該第二區域可為重疊區域。例如，該第一區域可為該第二區域之一子集。 In some embodiments, imaging device 194 is configured to use at least two light sources. For example, a first light source 420 can be used to generate structured light (eg, via light modulation subsystem 422) and a second light source 430 can be used to provide unstructured light. The first light source 420 can be generated for optics The electrically and/or fluorescently excited structured light is actuated and the second light source 430 can be used to provide bright field illumination. In such embodiments, the power module 164 can be used to control the first light source 420 and the imaging module 164 can be used to control the second light source 430. The optical element string of microscope 450 can be configured to: (1) receive structured light from optical modulation subsystem 422 and when the microfluidic device (such as an optically actuated motor device) is held by nest 304 Light is focused on at least a first region of the device; and (2) receiving reflected light and/or emitted light from the microfluidic device and focusing at least a portion of the reflected and/or emitted light to detector 440 on. The string of optical elements can be further configured to receive unstructured light from a second source and to focus the unstructured light onto at least a second region of the device when the microfluidic device is held by the nest 300. In some embodiments, the first region and the second region of the microfluidic device can be overlapping regions. For example, the first region can be a subset of the second region.

圖3B中展示將光供應至一光調變子系統422之第一光源420，光調變子系統422將結構光提供至系統450之顯微鏡450之光學元件串。圖中展示經由一分光器424而將非結構提供至光學元件串之第二光源430。來自光調變子系統422之結構光及來自第二光源430之非結構光一起自分光器424行進穿過光學元件串而到達一第二分光器424(或二向色濾光器448，取決於由光調變子系統422提供之光)，其中該光經反射以向下穿過物鏡454而到達樣本平面428。接著，來自樣本平面428之反射光及/或發射光向上反向行進穿過物鏡454，穿過分光器及/或二向色濾光器448而到達二向色濾光器452。到達二向色濾光器452之光之僅一部分穿過且到達偵測器440。 A first light source 420 that supplies light to a light modulation subsystem 422 that provides structured light to a string of optical elements of a microscope 450 of system 450 is shown in FIG. 3B. A second light source 430 that provides a non-structural structure to a string of optical elements via a beam splitter 424 is shown. The structured light from the optical modulation subsystem 422 and the unstructured light from the second source 430 travel from the optical splitter 424 through the optical element string to a second optical splitter 424 (or dichroic filter 448, depending on The light provided by the light modulation subsystem 422, wherein the light is reflected to pass down through the objective 454 to the sample plane 428. The reflected and/or emitted light from the sample plane 428 then travels up through the objective lens 454, through the beam splitter and/or dichroic filter 448, to the dichroic filter 452. Only a portion of the light that reaches the dichroic filter 452 passes through and reaches the detector 440.

在一些實施例中，第二光源430發射藍光。使用一適當二向色濾光器452，自樣本平面428反射之藍光能夠穿過二向色濾光器452而到達偵測器440。相比而言，來自光調變子系統422之結構光自樣本平面428反射，但不穿過二向色濾光器452。在此實例中，二向色濾光器452濾除具有長於495奈米之一波長之可見光。自光調變子系統422濾 除該光將僅在自光調變子系統發射之光不包含短於495奈米之任何波長時完成(如圖中所展示)。實際上，若來自光調變子系統422之光包含短於495奈米之波長(例如藍光波長)，則來自光調變子系統之一些光將穿過濾光器452而到達偵測器440。在此一實施例中，濾光器452用以改變自第一光源420及第二光源430到達偵測器440之光量之間的平衡。若第一光源420顯著地強於第二光源430，此可為有益的。在其他實施例中，第二光源430可發射紅光，且二向色濾光器452可濾除除紅光之外之可見光(例如具有短於650奈米之一波長之可見光)。 In some embodiments, the second light source 430 emits blue light. Using a suitable dichroic filter 452, blue light reflected from the sample plane 428 can pass through the dichroic filter 452 to the detector 440. In contrast, structured light from the light modulation subsystem 422 reflects from the sample plane 428 but does not pass through the dichroic filter 452. In this example, dichroic filter 452 filters visible light having a wavelength longer than 495 nm. Self-light modulation subsystem 422 filter Except that the light will only be completed when the light emitted from the photomodulation subsystem does not contain any wavelength shorter than 495 nm (as shown in the figure). In fact, if the light from the light modulation subsystem 422 contains wavelengths shorter than 495 nanometers (e.g., blue light wavelengths), some of the light from the light modulation subsystem will pass through the filter 452 to the detector 440. In this embodiment, the filter 452 is used to change the balance between the amount of light from the first light source 420 and the second light source 430 to the detector 440. This may be beneficial if the first source 420 is significantly stronger than the second source 430. In other embodiments, the second light source 430 can emit red light, and the dichroic filter 452 can filter visible light other than red light (eg, visible light having a wavelength shorter than 650 nm).

用於維持微流體器件之生長室內之細胞之存活性之額外系統組件Additional system components for maintaining the viability of cells in the growth chamber of a microfluidic device

為促進細胞群體之生長及/或擴增，有利於維持功能細胞之環境條件可由系統之額外組件提供。例如，此等額外組件可提供營養素、細胞生長傳信物種、pH調變、氣體交換、溫度控制及自細胞移除廢棄物。 To promote the growth and/or expansion of a population of cells, environmental conditions that facilitate the maintenance of functional cells can be provided by additional components of the system. For example, such additional components can provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste from cells.

微流體器件之調節表面Adjustment surface of microfluidic device

在一些實施例中，微流體器件之至少一表面經調節以支援細胞生長、存活性、可移植性或其等之任何組合。在一些實施例中，調節實質上全部內表面。一調節表面可為促進微流體器件內之成功細胞培育之元件之一者。識別一適當調節表面需要平衡若干操作要求。第一，調節表面可提供用以使細胞免遭可用於製造此類微流體器件之材料類型影響之一接觸表面。在不受理論限制之情況下，調節表面可由水合水包圍，該水合水提供與細胞之一水接觸層而非一金屬接觸層。第二，調節表面可提供可在培育期間使用其來適當支撐至少一生物細胞之一接觸表面，其不會實質上抑制在培育完成之後自生長室移除細胞之能力。例如，諸多細胞需要具有一定程度之親水性之一接觸表面來充分貼附以可存活及/或生長。替代地，一些細胞需要具有一定程 度之疏水性之一接觸表面來生長且呈現存活性之所要位準。另外，一些細胞需要選定蛋白質或肽基元存在於接觸表面內以啟始存活性/生長回應。第三，調節至少一表面可容許用於微流體器件中之動力實質上在正常作用電力範圍內起作用。例如，若採用光致動之動力，則調節表面可實質上容許光穿過調節表面，使得光致動之動力實質上不受抑制。 In some embodiments, at least one surface of the microfluidic device is conditioned to support cell growth, viability, portability, or any combination thereof. In some embodiments, substantially all of the inner surface is adjusted. A conditioning surface can be one of the components that facilitate successful cell culture within the microfluidic device. Identifying an appropriate adjustment surface requires balancing several operational requirements. First, the conditioning surface can provide a contact surface to protect the cells from the type of material that can be used to make such microfluidic devices. Without being bound by theory, the conditioning surface may be surrounded by hydrated water that provides a water contact layer with one of the cells rather than a metal contact layer. Second, the conditioning surface can provide for the ability to properly support one of the at least one biological cell contact surface during incubation, which does not substantially inhibit the ability to remove cells from the growth chamber after completion of the incubation. For example, many cells require a contact surface with a degree of hydrophilicity to adhere sufficiently to survive and/or grow. Alternatively, some cells need to have a certain course One of the hydrophobicities of the degree contacts the surface to grow and exhibits the desired level of viability. In addition, some cells require selected proteins or peptide motifs to be present in the contact surface to initiate a viability/growth response. Third, adjusting at least one surface may allow the power used in the microfluidic device to function substantially within the normal operating power range. For example, if light actuated power is employed, the conditioning surface can substantially allow light to pass through the conditioning surface such that the power of the light actuation is substantially uninhibited.

至少一調節表面可包含生長室之一表面或流動區域之一表面或其等之一組合。在一些實施例中，複數個生長室之各者具有至少一調節表面。在其他實施例中，複數個流動區域之各者具有至少一調節表面。在一些實施例中，複數個生長室之各者及複數個流動區域之各者之至少一表面係調節表面。 The at least one conditioning surface can comprise a surface of one of the growth chambers or a surface of one of the flow regions or a combination thereof. In some embodiments, each of the plurality of growth chambers has at least one conditioning surface. In other embodiments, each of the plurality of flow regions has at least one conditioning surface. In some embodiments, at least one surface of each of the plurality of growth chambers and each of the plurality of flow regions is an adjustment surface.

包含一聚合物之調節表面a conditioning surface comprising a polymer

至少一調節表面可包含一聚合物。該聚合物可共價鍵聯或非共價鍵聯至至少一表面。聚合物可具有各種結構基元，其包含嵌段聚合物(及共聚物)、星狀聚合物(星狀共聚物)、及接枝或梳形聚合物(接枝共聚物)，其等之全部可適合用於本文中。 The at least one conditioning surface can comprise a polymer. The polymer can be covalently bonded or non-covalently bonded to at least one surface. The polymer may have various structural units including block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), etc. All are suitable for use herein.

聚合物可包含具有伸烷基醚部分之一聚合物。各種含有伸烷基醚之聚合物可適合用於本文中所描述之微流體器件中。含有伸烷基醚之聚合物之一非限制性例示性類別係兩親性非離子嵌段共聚物，其包含在聚合物鏈內具有不同比率且位於不同位置中之聚環氧乙烷(PEO)亞單元及聚環氧丙烷(PPO)亞單元之嵌段。Pluronic®聚合物(BASF)係此類型之嵌段共聚物且在此項技術中被視為適合用在與活細胞接觸時。聚合物之平均分子量M_w在自約2000Da至約20KDa之範圍內。在一些實施例中，PEO-PPO嵌段共聚物可具有大於約10之一親水親油平衡值(HLB)(例如12至18)。用於產生一調節表面之特定Pluronic®聚合物包含Pluronic® L44、L64、P85及F127(其包含F127NF)。含有伸烷 基醚之聚合物之另一類別係聚乙二醇(PEG,Mw<100,000Da)或替代地聚環氧乙烷(PEO,M_w>100,000)。在一些實施例中，一PEG可具有約1000Da、約5000Da、約10,000Da或約20,000Da之一M_w。 The polymer may comprise a polymer having one of the alkyl ether moieties. A wide variety of alkyl ether-containing polymers are suitable for use in the microfluidic devices described herein. One non-limiting exemplary class of polymers containing alkylene ethers are amphiphilic nonionic block copolymers comprising polyethylene oxide having different ratios in the polymer chain and located in different positions (PEO) a block of subunits and polypropylene oxide (PPO) subunits. Pluronic® polymers (BASF) are block copolymers of this type and are considered suitable for use in contact with living cells in the art. The average molecular weight M _{w of the} polymer ranges from about 2000 Da to about 20 KDa. In some embodiments, the PEO-PPO block copolymer can have a hydrophilic-lipophilic balance (HLB) of greater than about 10 (eg, 12 to 18). The specific Pluronic® polymer used to create a conditioning surface comprises Pluronic® L44, L64, P85 and F127 (which comprises F127NF). Another class of polymers containing alkylene ethers is polyethylene glycol (PEG, Mw < 100,000 Da) or alternatively polyethylene oxide (PEO, _Mw > 100,000). In some embodiments, a PEG may have about 1000 Da, about 5000 Da, or from about one to about 10,000Da 20,000Da M _w.

在其他實施例中，聚合物調節表面可包含含有羧酸部分之一聚合物。羧酸亞單元可為含有烷基部分、烯基部分或芳香族部分之一亞單元。一非限制性實例係聚乳酸(PLA)。 In other embodiments, the polymeric conditioning surface can comprise a polymer comprising one of the carboxylic acid moieties. The carboxylic acid subunit may be a subunit containing one of an alkyl moiety, an alkenyl moiety or an aromatic moiety. A non-limiting example is polylactic acid (PLA).

在一些其他實施例中，聚合物調節表面可包含含有胺基甲酸酯部分之一聚合物，諸如(但不限於)聚胺基甲酸酯。 In some other embodiments, the polymeric conditioning surface can comprise a polymer comprising one of the urethane moieties, such as, but not limited to, a polyurethane.

在其他實施例中，聚合物調節表面可包含含有磺酸部分之一聚合物。磺酸亞單元可為含有烷基部分、烯基部分或芳香族部分之一亞單元。一非限制性實例係聚苯乙烯磺酸(PSSA)或聚茴香腦磺酸。此等後一例示性聚合物係聚電解質且可更改表面之特性以輔助/阻止黏著。 In other embodiments, the polymeric conditioning surface can comprise a polymer comprising one of the sulfonic acid moieties. The sulfonic acid subunit may be a subunit containing one of an alkyl moiety, an alkenyl moiety or an aromatic moiety. A non-limiting example is polystyrene sulfonic acid (PSSA) or polyceane sulfonic acid. These latter exemplary polymers are polyelectrolytes and can modify the properties of the surface to aid/block adhesion.

在其他實施例中，聚合物調節表面可包含一聚合物，其在聚合物主鏈之一末端或來自該聚合物之主鏈之側鏈處含有磷酸部分。 In other embodiments, the polymeric conditioning surface can comprise a polymer comprising a phosphate moiety at one end of the polymer backbone or from a side chain of the backbone of the polymer.

在其他實施例中，聚合物調節表面可包含含有糖類部分之一聚合物。在一非限制性實例中，多糖(諸如衍生自藻類或真菌多糖(諸如黃原膠或聚葡萄糖))可適合用於形成可輔助或防止細胞黏著之一聚合物調節表面。例如，具有約3Kda之一大小之聚葡萄糖聚合物可用以提供一微流體器件內之一調節表面。 In other embodiments, the polymeric conditioning surface can comprise a polymer comprising one of the saccharide moieties. In one non-limiting example, a polysaccharide, such as derived from an algal or fungal polysaccharide such as xanthan gum or polydextrose, can be suitably used to form a polymeric conditioning surface that can aid or prevent cell adhesion. For example, a polydextrose polymer having a size of about 3 Kda can be used to provide a conditioning surface within a microfluidic device.

在其他實施例中，聚合物調節表面可包含含有核苷酸部分(即，核酸)之一聚合物，其可具有核糖核苷酸部分或脫氧核糖核苷酸部分。核酸可僅含有天然核苷酸部分或可含有非天然核苷酸部分，其包括核鹼基、核糖或磷酸部分類似物，諸如(但不限於)7-去氮腺嘌呤、戊醣、甲基膦酸酯或硫代磷酸酯部分。含有核酸之一聚合物可包含可輔助或防止黏著之聚電解質。 In other embodiments, the polymeric conditioning surface can comprise a polymer comprising a nucleotide moiety (ie, a nucleic acid), which can have a ribonucleotide moiety or a deoxyribonucleotide moiety. The nucleic acid may contain only a portion of the natural nucleotide or may contain a portion of the non-natural nucleotide, including nucleobases, ribose or phosphate moiety analogs such as, but not limited to, 7-deaza adenine, pentose, methyl Phosphonate or phosphorothioate moiety. The polymer containing one of the nucleic acids may comprise a polyelectrolyte that can assist or prevent adhesion.

在其他實施例中，聚合物調節表面可包含含有胺基酸部分之一聚合物。含有胺基酸部分之聚合物可包含含有天然胺基酸之一聚合物或含有非天然胺基酸之一聚合物，其等之任一者可包含肽、多肽或蛋白質。在一非限制性實例中，蛋白質可為牛血清白蛋白(BSA)。在一些實施例中，一細胞外間質(ECM)蛋白質可提供於調節表面內以用於促進細胞生長之最佳化細胞黏著。可包含於一調節表面中之一細胞基質蛋白質可包含(但不限於)一膠原蛋白、一彈性蛋白、一含RGD之鈦(例如一纖連蛋白)或一層黏連蛋白。在其他實施例中，生長因子、細胞激素、內分泌素或其他細胞傳信物種可提供於微流體器件之至少一調節表面內。 In other embodiments, the polymeric conditioning surface can comprise a polymer comprising one of the amino acid moieties. The polymer containing an amino acid moiety may comprise a polymer comprising one of the natural amino acids or a polymer containing one of the non-natural amino acids, any of which may comprise a peptide, polypeptide or protein. In a non-limiting example, the protein can be bovine serum albumin (BSA). In some embodiments, an extracellular matrix (ECM) protein can be provided to optimize cell adhesion within the regulatory surface for promoting cell growth. One of the cell matrix proteins that may be included in a conditioning surface may include, but is not limited to, a collagen, an elastin, an RGD-containing titanium (eg, a fibronectin), or a layer of adhesion protein. In other embodiments, a growth factor, cytokine, endocrine, or other cell signaling species can be provided within at least one of the conditioning surfaces of the microfluidic device.

在進一步實施例中，聚合物調節表面可包含具有胺基部分之一聚合物。多胺聚合物可包含一天然多胺聚合物或一合成多胺聚合物。天然多胺之實例包含精胺、亞精胺及腐胺。 In a further embodiment, the polymeric conditioning surface can comprise a polymer having one of the amine moiety. The polyamine polymer may comprise a natural polyamine polymer or a synthetic polyamine polymer. Examples of natural polyamines include spermine, spermidine, and putrescine.

在一些實施例中，聚合物調節表面可包含含有下列之一者以上之一聚合物：伸烷基氧化物部分、羧酸部分、磺酸部分、磷酸部分、糖類部分、核苷酸部分或胺基酸部分。在其他實施例中，聚合物調節表面可包含各具有下列各者之一個以上聚合物之一混合物：伸烷基氧化物部分、羧酸部分、磺酸部分、磷酸部分、糖類部分、核苷酸部分及/或胺基酸部分，其等可獨立地或同時併入至調節表面中。 In some embodiments, the polymeric conditioning surface can comprise a polymer comprising one or more of the following: an alkylene oxide moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphate moiety, a carbohydrate moiety, a nucleotide moiety, or an amine. Base acid part. In other embodiments, the polymeric conditioning surface can comprise a mixture of one or more polymers each having the following: an alkyl oxide moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphate moiety, a carbohydrate moiety, a nucleotide Partial and/or amino acid moieties, which may be incorporated into the conditioning surface, either independently or simultaneously.

共價鍵聯之調節表面Covalently bonded adjustment surface

在一些實施例中，至少一調節表面包含經構形以支援微流體器件內之一或多個生物細胞之細胞生長、存活性、可移植性或其等之任何組合之一共價鍵聯部分。該共價鍵聯部分可包含一鍵聯基團，其中該鍵聯基團共價鍵聯至微流體器件之一表面。該鍵聯基團亦鍵聯至經構形以支援微流體器件內之一或多個生物細胞之細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯基團鍵聯至其之該表面 可包含微流體器件之基板之一表面，針對其中微流體器件包含一DEP構形之實施例，該表面可包含矽及/或二氧化矽。在一些實施例中，該共價鍵聯調節表面包含微流體器件之全部內表面。 In some embodiments, the at least one conditioning surface comprises a covalently bonded moiety configured to support one of cell growth, viability, portability, or the like of any one or more biological cells within the microfluidic device. The covalently bonded moiety can comprise a linking group wherein the linking group is covalently bonded to one surface of the microfluidic device. The linking group is also linked to the portion configured to support cell growth, viability, portability, or the like, of any one or more of the biological cells within the microfluidic device. The bonding group is bonded to the surface thereof One of the surfaces of the substrate of the microfluidic device can be included, and for embodiments in which the microfluidic device comprises a DEP configuration, the surface can comprise tantalum and/or hafnium oxide. In some embodiments, the covalently bonded conditioning surface comprises all of the inner surface of the microfluidic device.

圖9中展示具有一調節表面之一微流體器件之一示意圖。如圖9中所見，一微流體器件900具有面向該微流體器件之一封閉區域902之一第一DEP基板904及一第二DEP基板906，微流體器件900可包含至少一生長室及/或流動區域。器件900可依其他方式構形，如同微流體器件100、200、240、290、400、500A至500E或600之任何者。封閉區域902可為其中維持生物細胞或將生物細胞輸入至其中或自其輸出生物細胞之區域。(第二DEP基板906之)內表面910及(第一DEP基板904之)內表面912經一調節表面916改質，調節表面916可為支援細胞生長、存活性、可移植性或其等之任何組合之任何部分。在此實施例中，調節表面經由矽烷氧基鍵聯基團914而共價鍵聯至內表面之氧化官能基。 A schematic of one of the microfluidic devices having an adjustment surface is shown in FIG. As seen in FIG. 9, a microfluidic device 900 has a first DEP substrate 904 facing a closed region 902 of the microfluidic device and a second DEP substrate 906, which may include at least one growth chamber and/or Flow area. Device 900 can be configured in other ways, like any of microfluidic devices 100, 200, 240, 290, 400, 500A through 500E, or 600. The enclosed region 902 can be a region in which biological cells are maintained or biological cells are input thereto or from which biological cells are exported. The inner surface 910 (of the second DEP substrate 906) and the inner surface 912 (of the first DEP substrate 904) are modified by a conditioning surface 916 that can support cell growth, viability, portability, or the like. Any part of any combination. In this embodiment, the conditioning surface is covalently bonded to the oxidative functional group of the inner surface via a decyloxy linkage group 914.

在一些實施例中，經構形以支援細胞生長、存活性、可移植性或其等之任何組合之共價鍵聯部分可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷基化胺、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。 In some embodiments, the covalently bonded moiety configured to support cell growth, viability, portability, or any combination thereof, etc., can comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group) moiety Monosaccharide or polysaccharide (which may include, but is not limited to, polydextrose); alcohols (including but not limited to, propargyl alcohol); polyols including, but not limited to, polyvinyl alcohol; alkyl An ether comprising, but not limited to, polyethylene glycol; a polyelectrolyte comprising (but not limited to, polyacrylic acid or polyvinylphosphonic acid); an amine group comprising a derivative thereof such as, but not limited to, an alkyl group An amine, a hydroxyalkylated amine group, a phosphonium salt, and a heterocyclic group containing an unaromatized nitrogen ring atom, such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid, including but not limited to a propiolic acid (which can provide a carboxylate anion surface); a phosphonic acid comprising, but not limited to, an ethynylphosphonic acid (which can provide a phosphonate anion surface); a sulfonic acid anion; a carboxybetaine; a sulfobetaine Amine sulfonic acid; or an amino acid.

經構形以支援微流體器件內之一或多個生物細胞之細胞生長、 存活性、可移植性或其等之任何組合之共價鍵聯部分可為如本文中所描述之任何聚合物，且可包含一或多個聚合物，其含有伸烷基氧化物部分、羧酸部分、糖類部分、磺酸部分、磷酸部分、胺基酸部分、核酸部分或胺基部分。 Configuring to support cell growth of one or more biological cells within a microfluidic device, The covalently bonded moiety of any combination of viability, portability, or the like, can be any polymer as described herein, and can comprise one or more polymers containing an alkylene oxide moiety, a carboxy group An acid moiety, a saccharide moiety, a sulfonic acid moiety, a phosphoric acid moiety, an amino acid moiety, a nucleic acid moiety or an amine moiety.

在其他實施例中，經構形以支援一或多個生物細胞之細胞生長、存活性、可移植性或其等之任何組合之共價鍵聯部分可包含非聚合部分，諸如烷基部分、氟烷基部分(其包含(但不限於)全氟烷基)、胺基酸部分、醇部分、胺基部分、羧酸部分、膦酸部分、磺酸部分、胺磺酸部分或糖類部分。 In other embodiments, the covalently bonded moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells can comprise a non-polymeric moiety, such as an alkyl moiety, A fluoroalkyl moiety (which includes, but is not limited to, a perfluoroalkyl group), an amino acid moiety, an alcohol moiety, an amine moiety, a carboxylic acid moiety, a phosphonic acid moiety, a sulfonic acid moiety, an aminesulfonic acid moiety, or a carbohydrate moiety.

在一些實施例中，共價鍵聯部分可為烷基。該烷基可包括形成一直鏈之碳原子(例如至少10個碳或至少14個、16個、18個、20個、22個或22個以上碳之一直鏈)。因此，該烷基可為一無支鏈烷基。在一些實施例中，該烷基可包含一經取代之烷基(例如，該烷基中之一些碳可經氟化或經全氟化)。該烷基可包括接合至未經取代碳之一直鏈之經取代(例如，經氟化或經全氟化)碳之一直鏈。例如，該烷基可包含接合至一第二鏈段(其可包含一未經取代烷基)之一第一鏈段(其可包含全氟烷基)。該第一鏈段及該第二鏈段可直接或間接接合(例如，藉由一醚鍵)。該烷基之該第一鏈段可定位於鍵聯基團之遠端處，且該烷基之該第二鏈段可定位於鍵聯基團之近端處。在其他實施例中，該烷基可包含一支鏈烷基且可進一步具有中斷該烷基之烷基主鏈之一或多個伸芳基。在一些實施例中，該烷基或氟化烷基之一支鏈部分或經伸芳基中斷部分定位於至表面之共價鍵之遠端處之一點處。 In some embodiments, the covalently bonded moiety can be an alkyl group. The alkyl group can include a carbon atom that forms a straight chain (eg, at least 10 carbons or a straight chain of at least 14, 16, 18, 20, 22, or 22 carbons). Thus, the alkyl group can be an unbranched alkyl group. In some embodiments, the alkyl group can comprise a substituted alkyl group (eg, some of the carbons in the alkyl group can be fluorinated or perfluorinated). The alkyl group can include a straight chain of substituted (e.g., fluorinated or perfluorinated) carbon bonded to the straight chain of unsubstituted carbon. For example, the alkyl group can comprise a first segment (which can comprise a perfluoroalkyl group) joined to a second segment (which can comprise an unsubstituted alkyl group). The first segment and the second segment can be joined directly or indirectly (eg, by an ether linkage). The first segment of the alkyl group can be positioned at the distal end of the linking group and the second segment of the alkyl group can be positioned at the proximal end of the linking group. In other embodiments, the alkyl group can comprise a single chain alkyl group and can further have one or more extended aryl groups that interrupt the alkyl backbone of the alkyl group. In some embodiments, one of the branched or extended aryl interrupting moieties of the alkyl or fluorinated alkyl group is positioned at a point to the distal end of the covalent bond to the surface.

在其他實施例中，共價鍵聯部分可包含至少一個胺基酸，其可包含一個以上胺基酸。共價鍵聯部分可包含肽或蛋白質。在一些實施例中，共價鍵聯部分可包含胺基酸，其可提供用於支援細胞生長、存活性、可移植性或其等之任何組合之一兩性離子表面。 In other embodiments, the covalently bonded moiety can comprise at least one amino acid, which can comprise more than one amino acid. The covalently linked moiety can comprise a peptide or protein. In some embodiments, the covalently linked moiety can comprise an amino acid that can provide a zwitterionic surface for supporting cell growth, viability, portability, or any combination thereof.

共價鍵聯部分可包含一或多個糖。該等共價鍵聯糖可為單糖、雙糖或多糖。該等共價鍵聯糖可經改質以引入容許偶合至或(詳細言之)附接至表面之一反應配對部分。例示性反應配對部分可包含醛基、炔基或鹵基部分。可使多糖依一隨機方式改質，其中糖類單體之各者可經改質或該多糖內之糖類單體之僅一部分經改質以提供可直接或間接偶合至一表面之一反應配對部分。一範例可包含可經由一無支鏈鍵聯子而間接偶合至一表面之聚葡萄糖多糖。 The covalently linked moiety can comprise one or more sugars. The covalently linked sugars can be monosaccharides, disaccharides or polysaccharides. The covalently linked sugars can be modified to introduce a reaction pairing moiety that allows for coupling to or (in detail) attachment to one of the surfaces. An exemplary reaction pairing moiety can comprise an aldehyde, alkynyl or halo moiety. The polysaccharide may be modified in a random manner, wherein each of the saccharide monomers may be modified or only a portion of the saccharide monomer in the polysaccharide may be modified to provide a direct or indirect coupling to a surface of a reaction pairing moiety . An example can include a polydextrose polysaccharide that can be indirectly coupled to a surface via an unbranched linkage.

共價鍵聯部分可包含一或多個胺基。該胺基可為一經取代之胺部分、胍部分、含氮雜環部分或雜芳基部分。含胺基部分可具有容許微流體器件內及(視情況)生長室內之環境之pH改質之結構。 The covalently bonded moiety can comprise one or more amine groups. The amine group can be a substituted amine moiety, a hydrazine moiety, a nitrogen-containing heterocyclic moiety or a heteroaryl moiety. The amine-containing moiety can have a structure that permits pH upgrading of the environment within the microfluidic device and, where appropriate, the growth chamber.

共價鍵聯部分可包含一或多個羧酸、膦酸、胺磺酸或磺酸部分。在一些實施例中，共價鍵聯部分可包含一或多個核酸部分，其可具有經設計以自微流體器件內之生物細胞捕獲核酸之個別核苷酸之一序列。該等捕獲核酸可具有與來自生物細胞之核酸互補且可藉由雜交而捕獲核酸之一核苷酸序列。 The covalently bonded moiety can comprise one or more carboxylic acid, phosphonic acid, amine sulfonic acid or sulfonic acid moieties. In some embodiments, the covalently linked moiety can comprise one or more nucleic acid moieties, which can have a sequence of individual nucleotides designed to capture nucleic acids from biological cells within the microfluidic device. The capture nucleic acids can have a nucleotide sequence that is complementary to the nucleic acid from the biological cell and can capture one of the nucleic acids by hybridization.

調節表面可由僅一種部分組成或可包含一種以上不同部分。例如，氟烷基調節表面(其包含全氟烷基)可具有全部相同之複數個共價鍵聯部分，例如，具有至表面之相同共價附接且具有相同數目個氟亞甲基單元(其包括支援生長及/或存活性及/或可移植性之氟烷基部分)。替代地，調節表面可具有附接至表面之一種以上部分。例如，調節表面可包含具有指定數目個亞甲基或氟亞甲基單元之烷基或氟烷基且可進一步包含附接至具有一帶電部分之表面之另一組部分，該帶電部分附接至具有更大數目個亞甲基或氟亞甲基單元之烷基或氟烷基鏈。在一些實施例中，具有一種以上附接部分之調節表面可經設計使得具有較大數目個主鏈原子且因此具有自共價附接至表面之一較大長度之一第一組附接配位基可提供容量來使體積較大部分呈現於調節表 面處，而具有空間需求較小之不同末端且具有較少主鏈原子之一第二組附接配位基可有助於使整個基板表面官能化以防止與一矽或氧化鋁基板本身之非所要黏著或接觸。在另一實例中，附接至表面之部分可提供一兩性離子表面以依一隨機方式將交變電荷呈現於該表面上。 The conditioning surface may consist of only one portion or may comprise more than one different portion. For example, a fluoroalkyl modulating surface (which comprises a perfluoroalkyl group) can have all of the same plurality of covalently bonded moieties, for example, having the same covalent attachment to the surface and having the same number of fluoromethylene units ( It includes a fluoroalkyl moiety that supports growth and/or viability and/or portability. Alternatively, the conditioning surface can have more than one portion attached to the surface. For example, the conditioning surface can comprise an alkyl or fluoroalkyl group having a specified number of methylene or fluoromethylene units and can further comprise another set of moieties attached to a surface having a charged portion attached to the charged portion To an alkyl or fluoroalkyl chain having a greater number of methylene or fluoromethylene units. In some embodiments, the conditioning surface having more than one attachment portion can be designed such that it has a greater number of backbone atoms and thus has one of the larger lengths from one of the covalent attachments to the surface. The bit base can provide capacity to make the larger part of the volume appear in the adjustment table At the face, a second set of attachment ligands having a smaller space requirement and having fewer of the backbone atoms can help to functionalize the entire substrate surface to prevent interaction with the tantalum or alumina substrate itself. Do not want to stick or touch. In another example, a portion attached to the surface can provide a zwitterionic surface to present an alternating charge on the surface in a random manner.

調節表面性質Adjusting surface properties

在一些實施例中，共價鍵聯部分可在共價鍵聯至微流體器件之表面(例如一經DEP構形之基板表面)時形成一單層。在一些實施例中，由共價鍵聯部分形成之調節表面可具有小於10奈米(例如，小於5奈米或約1.5奈米至約3.0奈米)之一厚度。在其他實施例中，由共價鍵聯部分形成之調節表面可具有約10奈米至約50奈米之一厚度。在一些實施例中，調節表面無需一完美形成單層適當地運用於一DEP構形內之操作。 In some embodiments, the covalently bonded moiety can form a single layer upon covalent bonding to the surface of the microfluidic device (eg, a DEP-configured substrate surface). In some embodiments, the conditioning surface formed from the covalently bonded moiety can have a thickness of less than 10 nanometers (eg, less than 5 nanometers or from about 1.5 nanometers to about 3.0 nanometers). In other embodiments, the conditioning surface formed from the covalently bonded moiety can have a thickness from about 10 nanometers to about 50 nanometers. In some embodiments, the conditioning surface does not require a perfect formation of a single layer for proper operation within a DEP configuration.

在各種實施例中，微流體器件之(若干)調節表面可提供所要電性質。在不受理論限制之情況下，影響一調節表面之穩健性之一因數係本徵電荷捕集。不同表面調節材料可捕集會導致材料被擊穿之電子。調節表面中之缺陷會導致電荷捕集且進一步導致調節表面被擊穿。 In various embodiments, the conditioning surface(s) of the microfluidic device can provide the desired electrical properties. Without being bound by theory, one factor that affects the robustness of an conditioned surface is intrinsic charge trapping. Different surface conditioning materials can trap electrons that can cause the material to break down. Adjusting defects in the surface can result in charge trapping and further cause the conditioning surface to be broken down.

除調節表面之組合物之外，其他因數(諸如疏水性材料之實體厚度)亦可影響DEP力。各種因數可更改調節表面之實體厚度，諸如調節表面形成於基板上之方式(例如氣相沈積、液相沈積、旋轉塗佈、溢流及靜電塗佈)。可使用一橢偏計來量測調節表面之實體厚度及均勻性。 In addition to adjusting the composition of the surface, other factors, such as the physical thickness of the hydrophobic material, can also affect the DEP force. Various factors can alter the physical thickness of the conditioning surface, such as the manner in which the conditioning surface is formed on the substrate (eg, vapor deposition, liquid deposition, spin coating, flooding, and electrostatic coating). An ellipsometer can be used to measure the physical thickness and uniformity of the conditioning surface.

除調節表面之電性質之外，調節表面亦可具有有益於與生物分子一起使用之性質。例如，含有氟化(或全氟化)碳鏈之一調節表面可提供比烷基封端之鏈減少表面污損量之一益處。如本文中所使用，表面污損係指無差別材料沈積於微流體器件之表面上之量，其可包含諸如蛋白質及其降解物、核酸及各自降解物及其類似者之生物材料之永 久性或半永久性沈積。 In addition to adjusting the electrical properties of the surface, the conditioning surface can also have properties that are beneficial for use with biomolecules. For example, adjusting the surface with one of the fluorinated (or perfluorinated) carbon chains can provide one benefit in reducing the amount of surface fouling compared to the alkyl terminated chain. As used herein, surface fouling refers to the amount by which an indiscriminate material is deposited on the surface of a microfluidic device, which may comprise a permanent material such as a protein and its degradants, nucleic acids, and respective degradants and the like. Permanent or semi-permanent deposition.

可用於DEP構形中之調節表面之各種性質包含於下表中。如表中可見，針對條目1至7(其等全部為如本文中所描述之共價鍵聯調節表面)，藉由橢圓偏振術而量測之厚度始終薄於條目8(藉由非共價旋轉塗佈而形成之一CYTOP表面)之厚度(N/A表示整個表中無資料可用)。吾人發現，污損更多取決於表面之化學性質而非形成之模式，此係因為氟化表面通常比烷(烴)調節表面更少污損。 The various properties of the conditioning surface that can be used in the DEP configuration are included in the table below. As can be seen in the table, for items 1 to 7 (which are all covalently bonded to the surface as described herein), the thickness measured by ellipsometry is always thinner than item 8 (by non-covalent The thickness of one of the CYTOP surfaces formed by spin coating (N/A means no data is available in the entire table). We have found that fouling is more dependent on the chemical nature of the surface rather than the pattern of formation, since fluorinated surfaces are generally less fouling than alkane (hydrocarbon) conditioning surfaces.

1. CYTOP結構： 1. CYTOP structure:

2. 旋轉塗佈，非共價。 2. Spin coating, non-covalent.

至表面之鍵聯基團Bonding group to the surface

形成調節表面之共價鍵聯部分經由一鍵聯基團而附接至表面。該鍵聯基團可為藉由使一含矽氧烷之試劑與基板表面(其可由矽或氧化鋁形成)之氧化物反應而形成之矽烷氧基鍵聯基團。在一些其他實施例中，該鍵聯基團可為藉由使一含膦酸之試劑與矽或鋁基板表面之氧化物反應而形成之膦酸酯。 The covalently bonded moiety forming the conditioning surface is attached to the surface via a linking group. The linking group may be a decyloxy linking group formed by reacting a hydrazine-containing reagent with an oxide of a substrate surface which may be formed of ruthenium or alumina. In some other embodiments, the linking group can be a phosphonate formed by reacting a phosphonic acid-containing reagent with an oxide of the surface of a tantalum or aluminum substrate.

多部分調節表面Multi-part adjustment surface

可藉由一表面調節試劑(其經構形以已含有提供調節表面之部分)(例如烷基矽氧烷試劑或一經氟取代之烷基矽氧烷試劑，其可包含全氟矽氧烷試劑)之反應而形成共價鍵聯調節表面，如下文將描述。替代地，可藉由將支援細胞生長、存活性、可移植性或其等之任何組合之部分偶合至一表面改質配位基(其本身共價鍵聯至表面)而形成調節表面。 By a surface conditioning agent (which is configured to already contain a portion that provides a conditioning surface) (eg, an alkyl oxane reagent or a fluorine substituted alkyl decane reagent, which may comprise a perfluorodecane reagent The reaction forms a covalently bonded conditioning surface as will be described below. Alternatively, the conditioning surface can be formed by coupling a portion of any combination that supports cell growth, viability, portability, or the like to a surface modifying ligand that is itself covalently bonded to the surface.

一調節表面之結構及製備方法Structure of a regulating surface and preparation method thereof

在一些實施例中，共價鍵聯至介電泳基板之表面之氧化物的一調節表面具有式1之一結構： In some embodiments, a conditioning surface covalently bonded to the oxide of the surface of the dielectrophoretic substrate has a structure of Formula 1:

調節表面可共價鍵聯至介電泳基板之表面之氧化物。介電泳基板可為矽或氧化鋁，且氧化物可作為基板之天然化學結構之部分而存在或可如下文將討論般被引入。調節表面可經由一鍵聯基團LG而附接至氧化物，鍵聯基團LG可為藉由使矽氧烷或膦酸基與氧化物反應 而形成之矽烷氧基或膦酸酯基。 The conditioning surface can be covalently bonded to the oxide of the surface of the dielectrophoretic substrate. The dielectrophoretic substrate can be germanium or aluminum oxide, and the oxide can exist as part of the natural chemical structure of the substrate or can be introduced as discussed below. The conditioning surface can be attached to the oxide via a linking group LG, which can be reacted with the oxide by a oxoxane or phosphonic acid group The decyloxy or phosphonate group formed.

經構形以支援細胞生長、存活性、可移植性或其等之任何組合之部分可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。烷基或氟烷基部分可具有等於或大於10個碳之一主鏈長度。在一些實施例中，該烷基或氟烷基部分可具有約10個、約12個、約14個、約16個、約18個、約20個或約22個碳之一主鏈長度。 Portions configured to support cell growth, viability, portability, or any combination thereof, etc., may comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or polysaccharide (which may comprise ( But not limited to) polydextrose); alcohols (including but not limited to, propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkyl ethers, including but not limited to poly Ethylene glycol; polyelectrolyte (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amine group (which includes derivatives thereof such as, but not limited to, alkylated amine groups, hydroxyalkylated amine groups, a phosphonium salt and a heterocyclic group containing an unaromatized nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid comprising, but not limited to, a propiolic acid (which provides a carboxylic acid) Anionic surface); phosphonic acid comprising, but not limited to, ethynylphosphonic acid (which provides a phosphonate anionic surface); sulfonate anion; carboxybetaine; sulfobetaine; amine sulfonic acid; or amino acid. The alkyl or fluoroalkyl moiety can have a backbone length equal to or greater than 10 carbons. In some embodiments, the alkyl or fluoroalkyl moiety can have a backbone length of about 10, about 12, about 14, about 16, about 18, about 20, or about 22 carbons.

鍵聯基團LG可直接或間接連接至提供支援微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之部分。當鍵聯基團LG直接連接至該部分時，選用鍵聯子L不存在且n係0。當鍵聯基團LG間接連接至該部分時，鍵聯子L存在且n係1。鍵聯子L可具有一直鏈部分，其中該直鏈部分之一主鏈可包含選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子(經受此項技術中已知之化學接合限制)之任何組合之1個至200個非氫原子。在一些非限制性實例中，可使用選自由醚、胺基、羰基、醯胺基或膦酸酯基組成之群組之一或多個部分之任何組合來中斷鍵聯子L。另外，鍵聯子L可具有中斷鍵聯子之主鏈之一或多個伸芳基、伸雜芳基或雜環基。在一些實施例中，鍵聯子L之主鏈可包含10個至20個原子。在其他實施例中，鍵聯子L之主鏈可包含約5個原子至約200個原子、約10個原子至約80個原子、約10個原 子至約50個原子或約10個原子至約40個原子。在一些實施例中，主鏈原子全部為碳原子。在其他實施例中，主鏈原子並非全部為碳，而是可包含經受此項技術中已知之化學接合限制之矽原子、碳原子、氮原子、氧原子、硫原子或磷原子之任何可行組合。 The linking group LG can be directly or indirectly attached to a moiety that provides support for any combination of cell growth, viability, portability, or the like within the microfluidic device. When the linking group LG is directly attached to the moiety, the linkage L is not present and n is 0. When the linking group LG is indirectly attached to the moiety, the linkage L is present and n is 1. The bond L may have a straight chain moiety, wherein one of the backbone portions may comprise a halogen atom selected from the group consisting of a halogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom (which is subjected to chemical bonding known in the art). Limit any one to 200 non-hydrogen atoms in any combination. In some non-limiting examples, the linkage L can be interrupted using any combination of one or more selected from the group consisting of ether, amine, carbonyl, guanamine or phosphonate groups. Further, the linker L may have one or more of an extended aryl group, a heteroaryl group or a heterocyclic group of the main chain of the interrupted bond. In some embodiments, the backbone of the linkage L can comprise from 10 to 20 atoms. In other embodiments, the backbone of the linkage L may comprise from about 5 atoms to about 200 atoms, from about 10 atoms to about 80 atoms, and about 10 originals. From about 50 atoms or from about 10 atoms to about 40 atoms. In some embodiments, the backbone atoms are all carbon atoms. In other embodiments, the backbone atoms are not all carbon, but may comprise any feasible combination of deuterium atoms, carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms or phosphorus atoms that are subject to chemical bonding limitations known in the art. .

表面調節試劑Surface conditioning reagent

當在一步驟程序中將經構形以支援細胞生長、存活性、可移植性或其等之任何組合且藉此提供調節表面之部分添加至基板之表面時，式6之一表面調節試劑可用以引入調節表面。 One of the surface conditioning agents of Formula 6 may be used when a portion of the procedure is configured to support cell growth, viability, portability, or the like, and thereby providing a portion of the conditioning surface to be added to the surface of the substrate. To introduce an adjustment surface.

表面調節試劑可具有式6之一結構： The surface conditioning reagent can have a structure of Formula 6:

在式6之表面調節試劑中，表面調節試劑可包含一鍵聯基團LG，其可為矽氧烷或膦酸基。鍵聯基團LG可直接或間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之部分。LG可經由連接至一鍵聯子L之一第一端而直接(n=0)或間接(n=1)鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之部分。鍵聯子L可進一步包含一直鏈部分，其中該直鏈部分之一主鏈可具有選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子(經受此項技術中已知之化學接合限制)之任何組合之1個至200個非氫原子。該直鏈部分之主鏈可進一步包含一或多個伸芳基部分。經構形以支援細胞生長、存活性、可移植性或其等之任何組合之部分(「部分」)可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基、胍鹽及含有一未芳 香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。經構形以支援細胞生長、存活性、可移植性或其等之任何組合之部分可包含烷基或全氟烷基部分。該等烷基或全氟烷基部分可具有大於10個碳之一主鏈長度。經構形以支援細胞生長、存活性、可移植性或其等之任何組合之表面調節試劑之部分可包含糖類部分，且可為聚葡萄糖。在其他實施例中，經構形以支援細胞生長、存活性、可移植性或其等之任何組合之表面調節試劑之部分可包含伸烷基醚部分。該等伸烷基醚部分可為聚乙二醇。表面調節試劑可進一步包含一可裂解部分，其可定位於鍵聯子L內或可為經構形以支援細胞生長、存活性、可移植性或其等之任何組合之表面調節試劑之部分之部分。該可裂解部分可經構形以容許破壞調節表面，藉此促進一或多個生物細胞之可移植性。 In the surface conditioning agent of Formula 6, the surface conditioning agent may comprise a linking group LG which may be a decane or a phosphonic acid group. The linking group LG can be directly or indirectly linked to a portion configured to support cell growth, viability, portability, or any combination thereof. LG can be directly (n=0) or indirectly (n=1) linked to the conformation to support cell growth, viability, portability, etc. via a first end connected to a link L Part of any combination. The bond L may further comprise a straight chain moiety, wherein one of the backbone portions may have a halogen atom selected from the group consisting of a halogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom (subject to the chemistry known in the art) One to 200 non-hydrogen atoms of any combination of bonding limits). The backbone of the linear portion may further comprise one or more extended aryl moieties. Portions ("portions") that are configured to support cell growth, viability, portability, or any combination thereof, etc., may comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or polysaccharide (which may include, but is not limited to, polydextrose); alcohols (including but not limited to, propargyl alcohol); polyols including, but not limited to, polyvinyl alcohol; alkyl ethers, which comprise ( But not limited to) polyethylene glycol; polyelectrolyte (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amine group (which includes derivatives thereof such as, but not limited to, alkylated amine groups, hydroxyalkanes) Alkylamine, sulfonium salt and a non-aromatic a heterocyclic group of an aroma nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid comprising, but not limited to, a propiolic acid (which can provide a carboxylate anion surface); a phosphonic acid , which includes, but is not limited to, ethynylphosphonic acid (which provides a phosphonate anion surface); a sulfonic acid anion; a carboxybetaine; a sulfobetaine; an amine sulfonic acid; or an amino acid. Portions that are configured to support cell growth, viability, portability, or any combination thereof, etc., can comprise an alkyl or perfluoroalkyl moiety. The alkyl or perfluoroalkyl moiety can have a backbone length of greater than 10 carbons. Portions of the surface conditioning agent configured to support cell growth, viability, portability, or any combination thereof, etc., may comprise a carbohydrate moiety and may be polydextrose. In other embodiments, a portion of a surface conditioning agent configured to support cell growth, viability, portability, or any combination thereof, etc., can comprise an alkyl ether moiety. The alkylene ether moiety can be polyethylene glycol. The surface conditioning reagent can further comprise a cleavable moiety that can be positioned within the linkage L or can be part of a surface conditioning reagent configured to support cell growth, viability, portability, or any combination thereof, etc. section. The cleavable moiety can be configured to allow disruption of the conditioning surface, thereby promoting portability of one or more biological cells.

在一些實施例中，可在一多步驟程序中將支援細胞生長、存活性、可移植性或其等之任何組合之部分添加至基板之表面。當部分依一逐步方式偶合至表面時，鍵聯子L可進一步包含一偶合基團CG，如式2中所展示。 In some embodiments, portions of any combination that support cell growth, viability, portability, or the like can be added to the surface of the substrate in a multi-step procedure. When a moiety is coupled to the surface in a stepwise manner, the linkage L can further comprise a coupling group CG, as shown in Formula 2.

在一些實施例中，偶合基團CG表示來自一反應部分R_x與經構形以與一反應配對部分R_px反應之一部分之反應之所得部分。例如，一 典型CG可包含甲醯胺基，其係胺基與羧酸之一衍生物(諸如活性酯、醯氯或其類似者)之反應結果。CG可包含可在一反應部分與其各自反應配對部分反應之後形成之伸***基、甲醯胺基、硫代醯胺基、肟、烷硫基、二硫化物、醚或烯基或任何其他適合基團。偶合基團CG可定位於其中附接部分之鍵聯子L之第二端處。在一些其他實施例中，偶合基團CG可中斷鍵聯子L之主鏈。在一些實施例中，偶合基團CG係伸***基，其係炔基與疊氮基之間的一反應之結果，炔基及疊氮基之任一者可為此項技術中已知用於點擊偶合反應中之反應部分或反應配對部分。亦可進一步取代伸***基。例如，二苯并環辛烯基稠合伸***基部分可源於具有二苯并環辛炔基反應配對部分R_px之一調節改質劑與表面改質配位基之疊氮基反應部分R_x之反應，如下列段落中將更詳細描述。各種二苯并環辛炔基改質分子在此項技術中係已知的或可經合成以併入經構形以支援細胞生長、存活性、可移植性或其等之任何組合之一部分。 In some embodiments, the coupling group represented by CG from a portion of the reaction by R _x and configured to react with a mating portion of the resulting part of the reaction portion of the reaction R _px. For example, a typical CG may comprise a methionine group as a result of the reaction of an amine group with a derivative of one of the carboxylic acids, such as an active ester, hydrazine or the like. The CG may comprise a triazole group, a formamidine group, a thioguanamine group, a hydrazine, an alkylthio group, a disulfide, an ether or an alkenyl group which may be formed after the reaction of a reaction moiety with its respective reaction partner moiety or any other suitable Group. The coupling group CG can be positioned at the second end of the linkage L of the attachment portion. In some other embodiments, the coupling group CG can interrupt the backbone of the linkage L. In some embodiments, the coupling group CG is a triazolyl group which is a result of a reaction between an alkynyl group and an azide group, and any of an alkynyl group and an azide group can be used in the art. Click on the reaction moiety or the reaction partner in the coupling reaction. The triazole group can be further substituted. For example, the dibenzocyclooctenyl fused triazole moiety can be derived from an azide reaction moiety having a dibenzocyclooctynyl reaction pairing moiety R _px that modulates the modifier and the surface modifying ligand. The reaction of R _x is described in more detail in the following paragraphs. Various dibenzocyclooctynyl modified molecules are known in the art or can be synthesized to incorporate portions of any combination that are configured to support cell growth, viability, portability, or the like.

當在一多步驟程序中形成調節表面時，可藉由一調節改質劑(式5)與一基板(其具有共價鍵聯至其之一表面改質配位基)之反應而引入支援細胞生長、存活性、可移植性或其等之任何組合之部分，該表面改質配位基具有式3之一結構。 When a conditioning surface is formed in a multi-step procedure, support can be introduced by reacting a conditioning modifier (Formula 5) with a substrate having a covalent bond to one of its surface modifying ligands. The surface modifying ligand has a structure of Formula 3, which is part of any combination of cell growth, viability, portability, or the like.

式3之中間改質表面具有附接至其之一表面改質配位基，其具有-LG-(L")_j-R_x之一式，該式鍵聯至基板之氧化物，且如上文針對式1之 調節表面所描述般類似形成。DEP基板之表面係如上文所描述，且包含基板固有或引入至其內之氧化物。鍵聯基團LG係如上文所描述。一鍵聯子L"可存在(j=1)或不存在(j=0)。鍵聯子L"可具有一直鏈部分，其中該直鏈部分之一主鏈可包含選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子(經受此項技術中已知之化學接合限制)之任何組合之1個至100個非氫原子。在一些非限制性實例中，可使用醚、胺基、羰基、醯胺基或膦酸酯基之任何組合來中斷鍵聯子L"。另外，鍵聯子L"可具有中斷鍵聯子之主鏈之一或多個伸芳基、伸雜芳基或雜環基。在一些實施例中，鍵聯子L"之主鏈可包含10個至20個原子。在其他實施例中，鍵聯子L"之主鏈可包含約5個原子至約100個原子、約10個原子至約80個原子、約10個原子至約50個原子或約10個原子至約40個原子。在一些實施例中，主鏈原子全部為碳原子。在其他實施例中，主鏈原子並非全部為碳，而是可包含經受此項技術中已知之化學接合限制之矽原子、碳原子、氮原子、氧原子、硫原子或磷原子之任何可行組合。 The intermediate modified surface of Formula 3 has a surface modifying ligand attached thereto having a formula of -LG-(L") _j -R _x bonded to the oxide of the substrate, and as above The surface of the DEP substrate is similarly described as described for the conditioning surface of Formula 1. The surface of the DEP substrate is as described above and contains the oxide inherent to or incorporated into the substrate. The linking group LG is as described above. L" may exist (j = 1) or absent (j = 0). The bond L" may have a straight chain moiety, wherein one of the backbone portions may comprise a halogen atom selected from the group consisting of a halogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom (which is subjected to chemistry known in the art). 1 to 100 non-hydrogen atoms of any combination of bonding limits. In some non-limiting examples, any combination of ether, amine, carbonyl, decyl or phosphonate groups can be used to interrupt the linkage L ". In addition, the linkage L" may have one or more of an extended aryl, heteroaryl or heterocyclic group of the backbone of the interrupted linkage. In some embodiments, the backbone of the linkage L" may comprise 10 to 20 atoms. In other embodiments, the backbone of the linkage L" may comprise from about 5 atoms to about 100 atoms, from about 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms, or about 10 atoms. Up to about 40 atoms. In some embodiments, the backbone atoms are all carbon atoms. In other embodiments, the backbone atoms are not all carbon, but may be subjected to chemical bonding limitations known in the art. Any feasible combination of atoms, carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms or phosphorus atoms.

一反應部分R_x存在於表面改質配位基與表面之共價鍵遠端處之表面改質配位基之末端處。反應部分R_x係用於偶合反應以引入支援細胞生長、存活性、可移植性或其等之任何組合之部分之任何適合反應部分。在一些實施例中，反應部分R_x可為疊氮基、胺基、溴基、硫醇基、活性酯基、琥珀醯亞胺基或炔基部分。 A reactive moiety R _{x is} present at the end of the surface modifying ligand at the distal end of the covalent bond of the surface modifying ligand to the surface. The reaction part R _x based coupling reactions to introduce support for cell growth, and any combination of any part of viability, portability, etc., or suitable reactive moieties. In some embodiments, the reactive moiety R _x can be an azide, an amine, a bromo, a thiol, an active ester, an amber imino or an alkynyl moiety.

調節改質劑Adjustment modifier

調節改質劑(式5)經構形以供應支援細胞生長、存活性、可移植性或其等之任何組合之部分。 The modulating modifier (Formula 5) is configured to supply portions that support cell growth, viability, portability, or any combination thereof.

部分-(L')_m-R_px 式5 Part-(L') _m -R _px formula 5

經構形以支援細胞生長、存活性、可移植性或其等之任何組合 之調節改質劑之部分藉由一反應配對部分R_px與反應部分R_x之反應而鍵聯至表面改質配位基。反應配對部分R_px係經構形以與各自反應部分R_x反應之任何適合反應部分。在一非限制性實例中，一適合反應配對部分R_px可為炔基且反應部分R_x可為疊氮基。替代地，反應配對部分R_px可為疊氮基部分且各自反應部分R_x可為炔基。在其他實施例中，反應配對部分R_px可為活性酯官能基且反應部分R_x可為胺基。在其他實施例中，反應配對部分R_px可為醛基且反應部分R_x可為胺基。其他反應部分-反應配對部分組合係可行的，且此等實例絕不具限制性。 Was configured to support cell growth, part of the adjusting any combination of viability, or the like of the portability of the modifier portion by a reaction partner R _px reaction with R _x bonded to the portions of the surface modification with Bit base. The reaction partner moiety R _px is any suitable reactive moiety that is configured to react with the respective reaction moiety R _x . In a non-limiting example, a suitable reactive pairing moiety R _px can be an alkynyl group and the reactive moiety R _x can be an azide group. Alternatively, the reaction partner moiety R _px may be an azide moiety and the respective reaction moiety R _x may be an alkynyl group. In other embodiments, the reaction may be a mating portion R _px active ester functional groups and reactive moieties R _x may be a group. In other embodiments, the reaction partner moiety R _px can be an aldehyde group and the reaction moiety R _x can be an amine group. Other reaction moiety-reaction pairing moiety combinations are possible, and such examples are by no means limiting.

經構形以支援細胞生長、存活性、可移植性或其等之任何組合之式5之調節改質劑之部分可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。 The portion of the conditioning modifier of Formula 5 that is configured to support cell growth, viability, portability, or the like, can comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a sugar or polysaccharide (which may include, but is not limited to, polydextrose); an alcohol (including but not limited to, propargyl alcohol); a polyol comprising, but not limited to, a polyvinyl alcohol; an alkyl ether, It includes, but is not limited to, polyethylene glycol; a polyelectrolyte comprising, but not limited to, polyacrylic acid or polyvinylphosphonic acid; an amine group comprising a derivative thereof such as, but not limited to, an alkylated amine group a hydroxyalkylated amine group, a phosphonium salt, and a heterocyclic group containing an unaromatized nitrogen ring atom, such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid, including but not limited to An acetylene acid (which can provide a carboxylic acid anion surface); a phosphonic acid comprising, but not limited to, an ethynylphosphonic acid (which can provide a phosphonate anion surface); a sulfonic acid anion; a carboxybetaine; a sulfobetaine; an amine a sulfonic acid; or an amino acid.

提供增強細胞生長、存活性、可移植性或其等之任何組合之式5之調節改質劑之部分可直接(L'，其中m=0)或間接連接至反應配對部分R_px。當反應配對部分R_px間接連接至提供增強細胞生長、存活性、可移植性或其等之任何組合之部分時，反應配對部分R_px可連接至一鍵聯子L'(m=1)。反應配對部分R_px可連接至鍵聯子L'之一第一端，且提供增強細胞生長、存活性、可移植性或其等之任何組合之部分可連 接至鍵聯子L'之一第二端。鍵聯子L'可具有一直鏈部分，其中該直鏈部分之一主鏈可包含選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子(經受此項技術中已知之化學接合限制)之任何組合之1個至100個非氫原子。在一些非限制性實例中，可使用醚、胺基、羰基、醯胺基或膦酸酯基之任何組合來中斷鍵聯子L'。另外，鍵聯子L'可具有中斷鍵聯子L'之主鏈之一或多個伸芳基、伸雜芳基或雜環基。在一些實施例中，鍵聯子L'之主鏈可包含10個至20個原子。在其他實施例中，鍵聯子L'之主鏈可包含約5個原子至約100個原子、約10個原子至約80個原子、約10個原子至約50個原子或約10個原子至約40個原子。在一些實施例中，主鏈原子全部為碳原子。在其他實施例中，主鏈原子並非全部為碳，而是可包含經受此項技術中已知之化學接合限制之矽原子、碳原子、氮原子、氧原子、硫原子或磷原子之任何可行組合。 The portion of the modulating modifier of Formula 5 that provides enhanced cell growth, viability, portability, or the like, can be directly (L', where m = 0) or indirectly linked to the reaction partner portion _Rpx . When the reaction partner portion R _{px is} indirectly linked to a moiety providing any combination that enhances cell growth, viability, portability, or the like, the reaction partner portion R _px can be linked to a one-linker L' (m = 1). The reaction pairing moiety R _px can be attached to the first end of one of the linkages L' and can provide a portion that enhances cell growth, viability, portability, or any combination thereof, etc., can be linked to one of the linkages L' Two ends. The bond L' may have a straight chain moiety, wherein one of the backbone portions may comprise a halogen atom selected from the group consisting of a halogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom (which is subjected to chemistry known in the art). One to 100 non-hydrogen atoms of any combination of bonding restrictions). In some non-limiting examples, any combination of ether, amine, carbonyl, guanamine or phosphonate groups can be used to interrupt the linkage L'. Further, the bond L' may have one or more of an extended aryl group, a heteroaryl group or a heterocyclic group interrupting the bond L'. In some embodiments, the backbone of the linkage L' can comprise from 10 to 20 atoms. In other embodiments, the backbone of the linkage L' can comprise from about 5 atoms to about 100 atoms, from about 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms, or about 10 atoms. Up to about 40 atoms. In some embodiments, the backbone atoms are all carbon atoms. In other embodiments, the backbone atoms are not all carbon, but may comprise any feasible combination of deuterium atoms, carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms or phosphorus atoms that are subject to chemical bonding limitations known in the art. .

當調節改質劑(式5)與具有一表面改質配位基(式3)之表面反應時，形成具有式2之一調節表面之一基板。接著，鍵聯子L'及鍵聯子L"形式上係鍵聯子L之部分，且反應配對部分R_px與反應部分R_x之反應產生式2之偶合基團CG。 When the conditioning modifier (Formula 5) is reacted with a surface having a surface modifying ligand (Formula 3), a substrate having one of the conditioning surfaces of Formula 2 is formed. Next, the linkage sub L 'and linkages sub L "form part of line L of the sub-linking, and the reaction partner R _px reaction portion of the reaction part R _x of the production of the coupling group CG 2.

表面改質劑Surface modifier

表面改質劑係具有LG-(L")_j-R_x(式4)之一結構之一化合物。鍵聯基團LG共價鍵聯至介電泳基板之表面之氧化物。介電泳基板可為矽或氧化鋁，且氧化物可作為基板之天然化學結構之部分而存在或可如本文中所討論般被引入。鍵聯基團LG可為由矽氧烷或膦酸基與基板之表面上之氧化物之反應形成之矽烷氧基或膦酸酯基。反應部分R_x係如上文所描述。反應部分R_x可直接(L"，j=0)或經由一鍵聯子L"(j=1)而間接連接至鍵聯基團LG。鍵聯基團LG可附接至鍵聯子L"之一第一端且反應部分R_x可連接至鍵聯子L"之一第二端，在表面改質配位基已 如式3般附接至表面之後，該第二端將位於基板之表面之遠端處。 The surface modifying agent is a compound having one of the structures of LG-(L") _j -R _x (Formula 4). The linking group LG is covalently bonded to the oxide of the surface of the dielectrophoretic substrate. Is ruthenium or aluminum oxide, and the oxide may be present as part of the natural chemical structure of the substrate or may be introduced as discussed herein. The linkage group LG may be a surface of a substrate consisting of a siloxane or a phosphonic acid group a decyloxy or phosphonate group formed by the reaction of the oxide thereon. The reaction moiety R _x is as described above. The reaction moiety R _x may be directly (L", j = 0) or via a bond L" ( j=1) and indirectly connected to the linking group LG. The linking group LG may be attached to one of the first ends of the linkage L" and the reaction portion R _x may be connected to one of the linkages L" End, after the surface modifying ligand has been attached to the surface as in Equation 3, the second end will be located at the distal end of the surface of the substrate.

鍵聯子L"可具有一直鏈部分，其中該直鏈部分之一主鏈包含選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之任何組合之1個至100個非氫原子。在一些非限制性實例中，可使用醚、胺基、羰基、醯胺基或膦酸酯基之任何組合來中斷鍵聯子L"。另外，鍵聯子L"可具有中斷鍵聯子L"之主鏈之一或多個伸芳基、伸雜芳基或雜環基。在一些實施例中，鍵聯子L"之主鏈可包含10個至20個原子。在其他實施例中，鍵聯子L"之主鏈可包含約5個原子至約100個原子、約10個原子至約80個原子、約10個原子至約50個原子或約10個原子至約40個原子。在一些實施例中，主鏈原子全部為碳原子。在其他實施例中，主鏈原子並非全部為碳，而是可包含經受此項技術中已知之化學接合限制之矽原子、碳原子、氮原子、氧原子、硫原子或磷原子之任何可行組合。 The bonder L" may have a straight chain moiety, wherein one of the backbone portions comprises from 1 to 100 non-selective groups selected from the group consisting of a halogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. Hydrogen Atoms. In some non-limiting examples, any combination of ether, amine, carbonyl, guanamine or phosphonate groups can be used to interrupt the linkage L". Further, the linker L" may have one or more of an extended aryl group, a heteroaryl group or a heterocyclic group of the main chain of the interrupt bond L". In some embodiments, the backbone of the linkage L" may comprise from 10 to 20 atoms. In other embodiments, the backbone of the linkage L" may comprise from about 5 atoms to about 100 atoms, about From 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms or from about 10 atoms to about 40 atoms. In some embodiments, the backbone atoms are all carbon atoms. In other embodiments, the backbone atoms are not all carbon, but may comprise any feasible combination of deuterium atoms, carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms or phosphorus atoms that are subject to chemical bonding limitations known in the art. .

可裂解部分Cleavable part

在各種實施例中，下列之任何者可進一步包含一可裂解部分(如下文將討論)：支援細胞生長、存活性、可移植性或其等之任何組合之部分、鍵聯子L、鍵聯子L'、鍵聯子L"或偶合基團CG。該可裂解部分可經構形以容許破壞一微流體器件之一調節表面，其促進一或多個生物細胞之可移植性。在一些實施例中，可期望一或多個生物細胞之可移植性能夠在培養細胞一時間週期之後移動細胞且尤其能夠自微流體器件輸出細胞。 In various embodiments, any of the following may further comprise a cleavable moiety (as discussed below): a portion that supports cell growth, viability, portability, or any combination thereof, linkage L, linkage a sub-L', a linker L" or a coupling group CG. The cleavable moiety can be configured to permit destruction of one of the microfluidic devices to modulate the surface, which promotes portability of one or more biological cells. In embodiments, it may be desirable for the portability of one or more biological cells to be able to move cells after a period of time in which the cells are cultured and in particular to be capable of exporting cells from the microfluidic device.

基板之組合物Substrate composition

據此，提供一組合物，其包含：一基板，其具有一介電泳(DEP)構形及一表面；及一調節表面，其共價鍵聯至該基板之該表面之氧化物部分。該基板上之該調節表面可具有式1或式2之一結構： Accordingly, a kit is provided comprising: a substrate having a dielectrophoresis (DEP) configuration and a surface; and an conditioning surface covalently bonded to the oxide portion of the surface of the substrate. The adjustment surface on the substrate may have a structure of Equation 1 or Formula 2:

其中：LG係一鍵聯基團；L係一鍵聯子，其可存在(n=1)或不存在(n=0)；部分係支援微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之部分；及CG係一偶合基團，如本文中所界定。 Among them: LG is a one-linking group; L is a one-linker, which may exist (n=1) or not (n=0); part of it supports cell growth, viability, and portability in microfluidic devices a moiety of any combination of properties or the like; and a coupling group of CG, as defined herein.

調節表面可包含共價鍵聯至表面之氧化物部分之一鍵聯基團LG。該鍵聯基團可進一步鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之一部分。該鍵聯基團可為矽烷氧基鍵聯基團。在其他實施例中，該鍵聯基團可為膦酸酯基。該鍵聯基團可直接或間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯基團可經由連接至一鍵聯子之一第一端而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯子可進一步包含一直鏈部分，其中該直鏈部分之一主鏈可具有選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之任何組合之1個至200個非氫原子，如上文所討論。該直鏈部分之主鏈可進一步包含一或多個伸芳基部分。 The conditioning surface can comprise a linking group LG that is covalently bonded to one of the oxide moieties of the surface. The linking group can be further linked to a portion configured to support cell growth, viability, portability, or the like. The linking group can be a decyloxy linking group. In other embodiments, the linking group can be a phosphonate group. The linking group can be directly or indirectly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly linked to the portion configured to support cell growth, viability, portability, or the like, via attachment to one of the first ends of a linkage. The linker may further comprise a straight chain moiety, wherein one of the backbone portions may have from 1 to 200 selected from the group consisting of a ruthenium atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. Non-hydrogen atoms, as discussed above. The backbone of the linear portion may further comprise one or more extended aryl moieties.

鍵聯子可具有如上文所界定之一偶合基團CG。偶合基團CG可包 含伸***基部分。該伸***基部分可中斷鍵聯子之直鏈部分或可連接至鍵聯子之直鏈部分之一第二端。鍵聯子之第二端可位於基板之表面之遠端處。經構形以支援細胞生長、存活性、可移植性或其等之任何組合之部分可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。在一些實施例中，將不同部分之一混合物(諸如(但不限於)提供一兩性離子調節表面之陰離子官能基及陽離子官能基之一混合物)併入於調節表面中。調節表面可包含烷基或全氟烷基部分。該等烷基或全氟烷基部分可具有大於10個碳之一主鏈長度。調節表面可包含糖類部分，且可為聚葡萄糖。在其他實施例中，調節表面可包含伸烷基醚部分。該等伸烷基醚部分可為聚乙二醇。調節表面可進一步包含一可裂解部分。該可裂解部分可經構形以容許破壞調節表面，藉此促進一或多個生物細胞之可移植性。 The bonder can have one of the coupling groups CG as defined above. Coupling group CG can be packaged Containing a triazole-based moiety. The triazole moiety can interrupt the linear portion of the bond or can be attached to the second end of one of the linear portions of the bond. The second end of the bonder can be located at the distal end of the surface of the substrate. Portions configured to support cell growth, viability, portability, or any combination thereof, etc., may comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or polysaccharide (which may comprise ( But not limited to) polydextrose); alcohols (including but not limited to, propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkyl ethers, including but not limited to poly Ethylene glycol; polyelectrolyte (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amine group (which includes derivatives thereof such as, but not limited to, alkylated amine groups, hydroxyalkylated amine groups, a phosphonium salt and a heterocyclic group containing an unaromatized nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid comprising, but not limited to, a propiolic acid (which provides a carboxylic acid) Anionic surface); phosphonic acid comprising, but not limited to, ethynylphosphonic acid (which provides a phosphonate anionic surface); sulfonate anion; carboxybetaine; sulfobetaine; amine sulfonic acid; or amino acid. In some embodiments, a mixture of different portions, such as, but not limited to, a mixture of anionic functional groups and one of cationic functional groups that provide a zwitterionic conditioning surface, is incorporated into the conditioning surface. The conditioning surface can comprise an alkyl or perfluoroalkyl moiety. The alkyl or perfluoroalkyl moiety can have a backbone length of greater than 10 carbons. The conditioning surface can comprise a carbohydrate moiety and can be polydextrose. In other embodiments, the conditioning surface can comprise an alkyl ether moiety. The alkylene ether moiety can be polyethylene glycol. The conditioning surface can further comprise a cleavable portion. The cleavable moiety can be configured to allow disruption of the conditioning surface, thereby promoting portability of one or more biological cells.

提供另一組合物，其包含：一基板，其包含一介電泳(DEP)構形及一表面；及一表面改質配位基，其共價鍵聯至該基板之該表面之氧化物部分。具有一表面改質配位基之該基板可具有式3之一結構： Another composition is provided comprising: a substrate comprising a dielectrophoresis (DEP) configuration and a surface; and a surface modifying ligand covalently bonded to the oxide portion of the surface of the substrate. The substrate having a surface modifying ligand may have a structure of Formula 3:

其中：LG係鍵聯基團；L"係一選用鍵聯子，j係0或1。鍵聯子L"當j=1時，鍵聯子L"存在，且當j=0時，鍵聯子L"不存在；及R_x係如本文中所描述之一反應部分。 Wherein: LG is a linkage group; L" is a linkage, j is 0 or 1. The linkage L" is when the j = 1, the linkage L" exists, and when j = 0, the key sub-linked L "absent; and R _x represent one of the line portions as the reaction described herein.

表面改質配位基之反應部分可為疊氮基、胺基、溴基、硫醇基、活性酯基、琥珀醯亞胺基或炔基部分。表面改質配位基可經由一鍵聯基團而共價鍵聯至氧化物部分。該鍵聯基團可為矽烷氧基部分。在其他實施例中，該鍵聯基團可為膦酸酯基。該鍵聯基團可經由一鍵聯子而間接連接至表面改質配位基之反應部分。該鍵聯基團可附接至該鍵聯子之一第一端且該反應部分可附接至該鍵聯子之一第二端。鍵聯子L"可包含一直鏈部分，其中該直鏈部分之一主鏈包含選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之任何組合之1個至100個非氫原子。鍵聯子L"之主鏈可包含10個至20個原子。在其他實施例中，鍵聯子L"之主鏈可包含約5個原子至約50個原子。在一些實施例中，鍵聯子L"之主鏈可全部為碳原子。該直鏈部分之主鏈可包含一或多個伸芳基部分。鍵聯子L"可包含伸***基部分。該伸***基部分可中斷鍵聯子L"或可附接於鍵聯子L"之一末端處。表面改質配位基可包含一可裂解部分。該可裂解部分可經構形以容許破壞一微流體器件之一調節表面，藉此促進一或多個生物細胞之可移植性。 The reaction portion of the surface modifying ligand may be an azide group, an amine group, a bromo group, a thiol group, an active ester group, an amber quinone group or an alkynyl moiety. The surface modifying ligand can be covalently bonded to the oxide moiety via a linking group. The linking group can be a decyloxy moiety. In other embodiments, the linking group can be a phosphonate group. The linking group can be indirectly attached to the reactive portion of the surface modifying ligand via a bond. The linking group can be attached to one of the first ends of the linkage and the reactive portion can be attached to one of the second ends of the linkage. The linkage L" may comprise a straight chain moiety, wherein one of the backbone portions comprises from 1 to 100 non-selective groups selected from the group consisting of a ruthenium atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom and a phosphorus atom. The hydrogen atom. The backbone of the linkage L" may contain from 10 to 20 atoms. In other embodiments, the backbone of the linkage L" can comprise from about 5 atoms to about 50 atoms. In some embodiments, the backbone of the linkage L" can be all carbon atoms. The backbone of the linear moiety can comprise one or more extended aryl moieties. The linkage L" may comprise a triazolyl moiety. The triazole moiety may interrupt the linkage L" or may be attached to one end of the linkage L". The surface modifying ligand may comprise a cleavable moiety The cleavable portion can be configured to allow destruction of one of the microfluidic devices to modulate the surface, thereby facilitating portability of one or more biological cells.

製備之方法Method of preparation

在一些實施例中，使用化學氣相沈積來將調節表面或表面改質 配位基沈積於微流體器件之內表面上。調節表面/表面改質配位基可透過分子之氣相沈積而達成緊密堆積單層，其中包括調節表面/表面改質配位基之分子共價鍵聯至微流體器件(100、200、240、290、400、500A至500E、600)之任何者之內表面之分子。為達成一所要堆積密度，可在至少110℃之一溫度(例如至少120℃、130℃、140℃、150℃、160℃等等)處氣相沈積包括(例如)烷基封端之矽氧烷之分子達至少15小時之一週期(例如至少20、25、30、35、40、45或45以上小時)。通常在真空下且在存在一水源時執行此氣相沈積，諸如一含水硫酸鹽(例如MgSO₄‧7H₂0)。通常，氣相沈積之溫度升高及持續時間增加產生調節表面/表面改質配位基之改良特性。在一些實施例中，可藉由一液相中之反應而引入調節表面或表面改質配位基。 In some embodiments, chemical vapor deposition is used to deposit a conditioning surface or surface modifying ligand on the inner surface of the microfluidic device. Adjusting surface/surface modifying ligands can achieve a tightly packed monolayer through vapor deposition of molecules, including molecules that modulate surface/surface modifying ligands to covalently bond to microfluidic devices (100, 200, 240) Molecules of the inner surface of any of 290, 400, 500A to 500E, 600). To achieve a desired bulk density, vapor deposition can be carried out at a temperature of at least one of 110 ° C (eg, at least 120 ° C, 130 ° C, 140 ° C, 150 ° C, 160 ° C, etc.) including, for example, alkyl terminated oxygen The molecules of the alkane are for a period of at least 15 hours (eg, at least 20, 25, 30, 35, 40, 45 or 45 hours). And this is usually performed in the presence of a vapor deposition source of water under vacuum, such as an aqueous sulfates (e.g. MgSO ₄ ‧7H ₂ 0). Generally, the increase in temperature and duration of vapor deposition results in improved properties of the conditioning surface/surface modifying ligand. In some embodiments, the conditioning surface or surface modifying ligand can be introduced by a reaction in a liquid phase.

為製備微流體表面，可藉由一氧氣電漿處理而處理蓋、微流體線路材料及電極活性基板，該氧氣電漿處理可移除各種雜質，同時引入氧化表面(例如可如本文中所描述般共價改質之表面處之氧化物)。可(例如)在真空條件下依100W操作氧氣電漿清洗器達60秒。替代地，可使用液相處理，其包含用以氧化表面之氧化劑，諸如過氧化氫。例如，鹽酸及過氧化氫之一混合物或硫酸及過氧化氫之一混合物(例如食人魚溶液，其可具有自約3：1至約7：1範圍內之硫酸與過氧化氫之一比率)。 To prepare a microfluidic surface, the cap, microfluidic line material, and electrode active substrate can be treated by an oxygen plasma treatment that removes various impurities while introducing an oxidized surface (eg, as described herein) The oxide at the surface of the covalently modified surface). The oxygen plasma cleaner can be operated, for example, at 100 W under vacuum for 60 seconds. Alternatively, a liquid phase treatment comprising an oxidant to oxidize the surface, such as hydrogen peroxide, can be used. For example, a mixture of hydrochloric acid and hydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide (eg, a piranha solution, which may have a ratio of sulfuric acid to hydrogen peroxide ranging from about 3:1 to about 7:1) .

例如，可藉由預清洗蓋、微流體線路材料及電極活性基板而視情況改良氣相沈積程序。例如，此預清洗可包含一溶劑浴，諸如丙酮浴、乙醇浴或其等之一組合。該溶劑浴可包含音波處理。 For example, the vapor deposition process can be modified as appropriate by pre-cleaning the lid, the microfluidic line material, and the electrode active substrate. For example, this pre-cleaning can comprise a solvent bath, such as a combination of an acetone bath, an ethanol bath, or the like. The solvent bath can include sonication.

在一些實施例中，在已組裝微流體器件以形成界定一微流體線路之一封閉體之後使用氣相沈積來塗佈微流體器件之(若干)內表面。 In some embodiments, the inner surface(s) of the microfluidic device are coated using vapor deposition after the microfluidic device has been assembled to form an enclosure defining one of the microfluidic lines.

當具有一表面改質配位基之一基板進一步與調節改質劑反應以製備具有一調節表面之基板時，可就地使用任何適合溶劑來執行反 應，該任何適合溶劑將溶解試劑且不會破壞微流體線路材料或具有一表面改質配位基之表面。在一些實施例中，溶劑係一水溶液。 When a substrate having a surface modifying ligand is further reacted with a conditioning modifier to prepare a substrate having a conditioning surface, any suitable solvent may be used in situ to perform the reaction. Any suitable solvent will dissolve the reagent without destroying the microfluidic line material or the surface having a surface modifying ligand. In some embodiments, the solvent is an aqueous solution.

製備一調節表面或包含一表面改質配位基之一表面之方法A method of preparing a conditioning surface or a surface comprising a surface modifying ligand

據此，提供製備具有一介電泳(DEP)構形之一微流體器件之一改質表面之一方法，其包含下列步驟：提供一微流體器件之一基板之一表面，其中該基板包含一DEP構形；使該表面之氧化物與一改質劑反應，藉此將該基板之該表面轉換成一改質表面。在一些實施例中，該基板之該表面可經電漿清洗以將氧化物提供於該表面上。在一些實施例中，可在組裝該微流體器件之前電漿清洗該表面。在其他實施例中，可在組裝該微流體器件之後電漿清洗該表面。 Accordingly, a method of preparing a modified surface of one of the microfluidic devices having a dielectrophoretic (DEP) configuration is provided, comprising the steps of: providing a surface of one of the substrates of a microfluidic device, wherein the substrate comprises a DEP Forming; reacting the oxide of the surface with a modifying agent to convert the surface of the substrate into a modified surface. In some embodiments, the surface of the substrate can be plasma cleaned to provide an oxide on the surface. In some embodiments, the surface can be plasma cleaned prior to assembly of the microfluidic device. In other embodiments, the surface can be plasma cleaned after assembly of the microfluidic device.

藉由使表面暴露於包括改質劑之一液體而執行方法中之使表面之氧化物與改質劑反應之步驟。在一些實施例中，可藉由在減小壓力處使表面暴露於含有改質劑之一蒸汽而執行使表面之氧化物反應之步驟。 The step of reacting the oxide of the surface with the modifying agent in the method is performed by exposing the surface to a liquid comprising one of the modifying agents. In some embodiments, the step of reacting the oxide of the surface can be performed by exposing the surface to vapor containing one of the modifiers at a reduced pressure.

在一些實施例中，改質劑可包含一表面調節試劑，其具有經構形以與表面共價反應之一第一部分及經構形以支援細胞生長、存活性、可移植性或其等之任何組合之一第二部分，藉此使表面改質為經調節以支援細胞生長、存活性、可移植性或其等之任何組合之一表面。 In some embodiments, the modifying agent can comprise a surface conditioning agent having a first portion configured to covalently react with the surface and configured to support cell growth, viability, portability, or the like. A second portion of any combination whereby the surface is modified to a surface that is adjusted to support cell growth, viability, portability, or any combination thereof.

表面調節試劑可具有式6之一結構：部分-(L)_n-LG 式6 The surface conditioning reagent may have a structure of the formula 6: a moiety - (L) _n - LG

第一部分可包含一鍵聯基團LG，其可為矽氧烷或膦酸基。鍵聯基團LG可直接或間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之部分。第一部分可直接(n=0)或經由連接至一鍵聯子L之一第一端而間接(n=1)鍵聯至第二部分，該第二部分係經 構形以支援細胞生長、存活性、可移植性或其等之任何組合之部分。鍵聯子L可進一步包含一直鏈部分，其中該直鏈部分之一主鏈可具有選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之任何組合之1個至200個非氫原子。該直鏈部分之主鏈可進一步包含一或多個伸芳基部分。表面調節試劑之第二部分(「部分」)可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。表面調節試劑之第二部分可包含烷基或全氟烷基部分。該等烷基或全氟烷基部分可具有大於10個碳之一主鏈長度。表面調節試劑之第二部分可包含糖類部分，且可為聚葡萄糖。在其他實施例中，表面調節試劑之第二部分可包含伸烷基醚部分。該等伸烷基醚部分可為聚乙二醇。表面調節試劑之第二部分可進一步包含一可裂解部分，其可定位於鍵聯子L內或可為表面調節試劑之第二部分之部分。該可裂解部分可經構形以容許破壞調節表面，藉此促進一或多個生物細胞之可移植性。 The first portion may comprise a linking group LG which may be a decane or a phosphonic acid group. The linking group LG can be directly or indirectly linked to a portion configured to support cell growth, viability, portability, or any combination thereof. The first portion may be directly (n = 0) or indirectly (n = 1) linked to the second portion via a first end connected to a bond L, the second portion being The configuration is to support portions of cell growth, viability, portability, or any combination thereof. The bond L may further comprise a straight chain moiety, wherein one of the backbone portions may have from 1 to 200 selected from the group consisting of a ruthenium atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. Non-hydrogen atoms. The backbone of the linear portion may further comprise one or more extended aryl moieties. The second portion of the surface conditioning reagent ("portion") may comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or polysaccharide (which may include, but is not limited to, polydextrose); an alcohol (which includes, but is not limited to, propargyl alcohol); a polyol comprising, but not limited to, a polyvinyl alcohol; an alkyl ether comprising, but not limited to, polyethylene glycol; a polyelectrolyte (which comprises ( But not limited to) polyacrylic acid or polyvinylphosphonic acid); amine groups (including derivatives thereof such as, but not limited to, alkylated amine groups, hydroxyalkylated amine groups, phosphonium salts, and containing an unaromatic nitrogen ring) a heterocyclic group of an atom such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid comprising, but not limited to, a propiolic acid (which can provide a carboxylate anion surface); a phosphonic acid comprising But not limited to) ethynylphosphonic acid (which provides a phosphonate anion surface); sulfonate anion; carboxybetaine; sulfobetaine; amine sulfonic acid; or amino acid. The second portion of the surface conditioning agent can comprise an alkyl or perfluoroalkyl moiety. The alkyl or perfluoroalkyl moiety can have a backbone length of greater than 10 carbons. The second portion of the surface conditioning agent can comprise a carbohydrate moiety and can be polydextrose. In other embodiments, the second portion of the surface conditioning agent can comprise an alkyl ether moiety. The alkylene ether moiety can be polyethylene glycol. The second portion of the surface conditioning reagent can further comprise a cleavable moiety that can be positioned within the linkage L or can be part of a second portion of the surface conditioning reagent. The cleavable moiety can be configured to allow disruption of the conditioning surface, thereby promoting portability of one or more biological cells.

在各種實施例中，改質劑可包含一表面改質劑，其具有如上文所界定之式4之一結構，其中該表面改質劑包含：一第一部分LG，其經構形以與表面反應；及一第二部分R_x，其可為一反應部分或可經改質以包含一反應部分(該反應部分包含疊氮基、胺基、溴基、硫醇基、活性酯基、琥珀醯亞胺基或炔基部分)，藉此將表面轉換成包括 一表面改質配位基之一表面，該表面改質配位基具有式3之一結構，如上文所描述。在一些實施例中，該表面改質劑之該第一部分(其經構形以與表面之氧化物反應)可為矽氧烷或膦酸。 In various embodiments, the modifier may comprise a surface modifying agent having a structure of Formula 4 as defined above, wherein the surface modifying agent comprises: a first portion LG configured to conform to the surface And a second portion R _x which may be a reactive moiety or may be modified to comprise a reactive moiety comprising azide, an amine group, a bromo group, a thiol group, an active ester group, amber The quinone imine or alkynyl moiety, thereby converting the surface to include a surface of a surface modifying ligand having a structure of Formula 3, as described above. In some embodiments, the first portion of the surface modifying agent (which is configured to react with the oxide of the surface) can be a oxoxane or a phosphonic acid.

在一些實施例中，該方法包含下列步驟：使包括一表面改質配位基(式3)之表面與一調節改質劑反應，該調節改質劑包含經構形以支援細胞生長、存活性、可移植性或其等之任何組合之一第一部分及經構形以與該表面改質配位基之反應部分反應之一第二部分R_px；藉此提供經構形以支援一生物細胞之細胞生長、存活性、可移植性或其等之任何組合之一表面，該表面具有式2之一結構，如上文所描述。該調節改質劑可具有式5之一結構。在一些實施例中，該調節改質劑之該第一部分包括下列之至少一者：環氧烷部分、胺基酸部分、糖類部分、陰離子部分、陽離子部分及兩性離子部分。 In some embodiments, the method comprises the steps of reacting a surface comprising a surface modifying ligand (Formula 3) with an modulating modifier comprising a conformation to support cell growth, storage activity, portability, etc., or any combination of one of the first portion and configured to react via ligand modification of the surface of one portion of the second portion of the reaction R _px; thereby provide a configuration to support a biological The surface of one of any combination of cell growth, viability, portability, or the like of the cell having a structure of Formula 2, as described above. The conditioning modifier may have a structure of Formula 5. In some embodiments, the first portion of the conditioning modifier comprises at least one of an alkylene oxide moiety, an amino acid moiety, a carbohydrate moiety, an anion moiety, a cationic moiety, and a zwitterionic moiety.

在各種實施例中，表面調節試劑、表面改質劑或調節改質劑之任何者可進一步包含如本文中所描述之一可裂解部分。 In various embodiments, any of the surface conditioning agents, surface modifying agents, or conditioning modifying agents can further comprise a cleavable moiety as described herein.

含有其他組分之調節表面Adjustment surface containing other components

調節表面可包含除由一共價鍵聯部分形成之一聚合物或一調節表面之外之其他組分，或調節表面除包含由一共價鍵聯部分形成之一聚合物或一調節表面之外，亦可另外包含其他組分，該共價鍵聯部分包含生物相容金屬離子(例如鈣、鈉、鉀或鎂)、抗氧化劑、表面活性劑及/或必需營養素。一非限制性例示性列表包含：維生素，諸如B7、α-生育酚、α-生育酚乙酸酯、維生素A及其乙酸酯；蛋白質，諸如BSA、過氧化氫酶、胰島素、轉鐵蛋白、超氧化物歧化酶；小分子，諸如皮質固酮、D-半乳糖、乙醇胺鹽酸鹽、還原谷胱甘肽、L-肉鹼鹽酸鹽、亞油酸、亞麻酸、黃體固酮、腐胺二鹽酸鹽及三碘甲腺原氨酸；及鹽，其包含(但不限於)亞硒酸鈉、磷酸鈉、磷酸鉀、磷酸鈣及/或磷酸鎂。抗氧化劑可包含(但不限於)類胡蘿蔔素、肉桂酸及其衍 生物、阿魏酸、多酚(諸如類黃酮、醌及其衍生物)(其包含米托蒽醌-Q)、N-乙醯半胱胺酸及抗氧化維生素(諸如抗壞血酸、維生素E及其類似者)。調節表面可包含一培養基補充劑(諸如B-27®補充劑)，其含有抗氧化劑及上文所列之其他組分之諸多者。B-27®補充劑係來自ThermoFisher Scientific(Cat# 17504044)之市售(50倍)無血清培養基。 The conditioning surface may comprise a component other than a polymer or a conditioning surface formed by a covalently bonded moiety, or the conditioning surface comprises a polymer or a conditioning surface formed by a covalently bonded moiety, Other components may additionally be included, the covalently bonded moiety comprising a biocompatible metal ion (eg, calcium, sodium, potassium or magnesium), an antioxidant, a surfactant, and/or an essential nutrient. A non-limiting, illustrative list includes: vitamins such as B7, alpha-tocopherol, alpha-tocopherol acetate, vitamin A and its acetate; proteins such as BSA, catalase, insulin, transferrin , superoxide dismutase; small molecules, such as corticosterone, D-galactose, ethanolamine hydrochloride, reduced glutathione, L-carnitine hydrochloride, linoleic acid, linolenic acid, luteinone, Putrescine dihydrochloride and triiodothyronine; and salts including, but not limited to, sodium selenite, sodium phosphate, potassium phosphate, calcium phosphate and/or magnesium phosphate. Antioxidants may include, but are not limited to, carotenoids, cinnamic acid, and derivatives thereof. Biological, ferulic acid, polyphenols (such as flavonoids, guanidine and its derivatives) (which contain mitoxantrone-Q), N-acetyl cysteine and antioxidant vitamins (such as ascorbic acid, vitamin E and its Similar). The conditioning surface can comprise a media supplement (such as a B-27® supplement) containing antioxidants and many of the other components listed above. The B-27® supplement is a commercially available (50X) serum-free medium from ThermoFisher Scientific (Cat# 17504044).

在一些實施例中，至少一調節表面可包含哺乳動物血清之一或多個組分。在一些實施例中，哺乳動物血清係胎牛血清(FBS)或小牛血清(FCS)。調節表面可包含哺乳動物血清之特定組分(諸如常見於血清中之特定數量及類型之蛋白質)，其之界定數量或類型可自無血清培養基或合成培養基提供。 In some embodiments, the at least one conditioning surface can comprise one or more components of mammalian serum. In some embodiments, the mammalian serum is fetal bovine serum (FBS) or calf serum (FCS). The modulating surface can comprise a particular component of a mammalian serum (such as a particular amount and type of protein commonly found in serum), the defined amount or type of which can be provided from serum-free or synthetic media.

在其他實施例中，至少一調節表面不包含一哺乳動物血清。在各種實施例中，至少一調節表面可不包含任何鈦、鎳或鐵金屬離子。在其他實施例中，至少一調節表面可不包含任何顯著濃度之鈦、鎳或鐵金屬離子。在其他實施例中，至少一調節表面可不包含任何金、鋁或鎢金屬離子。 In other embodiments, the at least one conditioning surface does not comprise a mammalian serum. In various embodiments, the at least one conditioning surface may not comprise any titanium, nickel or iron metal ions. In other embodiments, the at least one conditioning surface may not comprise any significant concentration of titanium, nickel or iron metal ions. In other embodiments, the at least one conditioning surface may not comprise any gold, aluminum or tungsten metal ions.

用以減弱黏著之試劑處理/試劑之混合物a mixture of reagent treatments/reagents for weakening adhesion

當在一微流體器件內培養細胞時，細胞主動分泌蛋白質及其他生物分子且被動地流出可黏著至該微流體器件內之表面之類似生物分子。培養細胞可彼此黏著或黏著至調節表面，且變得難以自生長室移除以自該微流體器件輸出。另外，在一些情況下，可期望將相同於或不同於培養細胞之額外細胞類型帶入至該微流體件中。此等新傳遞細胞亦可變得黏著至累積於微流體環境內之表面污損，且難以在一隨後時間點處自該器件移除。 When cells are cultured in a microfluidic device, the cells actively secrete proteins and other biomolecules and passively flow out similar biomolecules that can adhere to the surface within the microfluidic device. The cultured cells can adhere or adhere to each other to the conditioning surface and become difficult to remove from the growth chamber for output from the microfluidic device. Additionally, in some cases it may be desirable to bring additional cell types that are the same or different from the cultured cells into the microfluidic member. These new delivery cells can also become adherent to surface contamination accumulated in the microfluidic environment and are difficult to remove from the device at a subsequent point in time.

使用蛋白酶(諸如胰蛋白酶或Accutase®(具有蛋白質水解活性及溶膠原活性之一酶促混合物，Innovative Cell Technologies))之處理無法提供充分功效足以(針對一非限制性實例)容許黏著細胞自微流體器 件輸出之效能。提供抗黏著性質之一或多個蛋白質及/或肽可用作用以減弱此兩種情形之黏著之一混合物。可使用具有抵抗各種細胞黏著機制之一者之活性之生物分子或小分子。可受抑制之一些細胞黏著機制可為活性肌動蛋白纖維形成及相關程序，其可藉由使用化合物(諸如細胞鬆弛素B(New England Biosciences Cat No：M0303S)、微纖維延伸之一小分子抑制劑)而達成。可藉由使用含RGD之肽而達成特定受體驅動黏著程序，諸如(但不限於)抑制整合素受體介導黏著至纖連蛋白(其可見於一污損表面上)。另一類型之污損材料(即，自死細胞釋放之核酸)可吸引細胞結合，其可藉由使用一核酸內切酶(其將裂解污損核酸)而達成。一特定核酸內切酶(去氧核糖核酸酶1(DNase 1，Sigma Aldrich，Catalog No.AMPD1-1KT))亦結合至肌動蛋白以因此提供黏著之一雙活性阻斷。在一些實施例中，全部三個阻斷劑之一混合物可用以防止/減弱細胞黏著。 Treatment with a protease such as trypsin or Accutase® (one enzymatically active mixture with proteolytic activity and procollagen activity, Innovative Cell Technologies) does not provide sufficient efficacy (for a non-limiting example) to allow adherent cells to self-microfluid Device The performance of the output. One or more proteins and/or peptides that provide anti-adhesive properties can be used as a mixture to adhere to both of these conditions. Biomolecules or small molecules having activity against one of various cell adhesion mechanisms can be used. Some of the cell adhesion mechanisms that can be inhibited can be active actin fiber formation and related procedures, which can be inhibited by the use of a compound such as cytochalasin B (New England Biosciences Cat No: M0303S), a microfiber extension And achieved). Specific receptor-driven adhesion procedures can be achieved by using RGD-containing peptides, such as, but not limited to, inhibiting integrin receptor-mediated adhesion to fibronectin (which can be found on a stained surface). Another type of fouling material (i.e., nucleic acid released from dead cells) can attract cell binding, which can be achieved by using an endonuclease that will lyse the contaminating nucleic acid. A specific endonuclease (DNase 1, Sigma Aldrich, Catalog No. AMPD-KT) also binds to actin to thereby provide one of the double-activity blockade of adhesion. In some embodiments, a mixture of all three blockers can be used to prevent/reduce cell adhesion.

一般處理協定/在培養之後：General treatment agreement / after training:

針對已在一微流體器件內生長2天、3天、4天或4天以上之細胞，三個抗黏著劑之混合物或單一抗黏著劑(如下文將描述)可流動至該微流體器件中且被允許在輸出細胞之前之約20分鐘、約30分鐘、約40分鐘、約50分鐘或約60分鐘之一時間週期內擴散至生長室中。 For cells that have been grown in a microfluidic device for 2, 3, 4 or more days, a mixture of three anti-adhesive agents or a single anti-adhesive (as described below) can flow into the microfluidic device. And it is allowed to diffuse into the growth chamber for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes before the cells are output.

預處理Pretreatment

針對待輸入至一微流體器件中之細胞，可在含有混合物或單一抗黏著劑之一培養基中預培育細胞達約30分鐘，接著，將細胞輸入至微流體晶片。抑制在無需進一步添加試劑之情況下持續1小時、2小時、3小時或3小時以上之時間週期。 For cells to be input into a microfluidic device, the cells may be pre-incubated in a medium containing the mixture or a single anti-adhesive for about 30 minutes, and then the cells are input to the microfluidic wafer. The time period of 1 hour, 2 hours, 3 hours, or more than 1 hour is inhibited without further addition of the reagent.

RGD三肽(mw.614.6，Santa Cruz Biotechnology Cat No：sc-201176)可以約0.1毫莫耳至約20毫莫耳之一濃度存在於培養基或預輸入培育基中。在一些實施例中，RGD三肽可以約0.1毫莫耳、約0.5毫 莫耳、約0.7毫莫耳、約1.0毫莫耳、約3.0毫莫耳、約5.0毫莫耳、約8.0毫莫耳、約10.0毫莫耳或該範圍內之任何值之一濃度存在。細胞鬆弛素B可以約0.01毫莫耳或約50毫莫耳或約0.01毫莫耳、約0.05毫莫耳、約0.1毫莫耳、約2毫莫耳、約4毫莫耳、約6毫莫耳、約8毫莫耳、約10毫莫耳、約20毫莫耳、約30毫莫耳、約50毫莫耳或該範圍內之任何值之一濃度存在於預輸入培育基中。DNase 1可以約0.001U/微升至約10U/微升或約0.001U/微升、約0.005U/微升、約0.01U/微升、約0.05U/微升、約1.0U/微升、約5.0U/微升、約10U/微升或該範圍內之任何值之一濃度存在。 The RGD tripeptide (mw. 614.6, Santa Cruz Biotechnology Cat No: sc-201176) can be present in the medium or pre-input incubation medium at a concentration of from about 0.1 millimolar to about 20 millimolar. In some embodiments, the RGD tripeptide can be about 0.1 millimolar, about 0.5 milliliter Mole, about 0.7 millimolar, about 1.0 millimolar, about 3.0 millimolar, about 5.0 millimolar, about 8.0 millimolar, about 10.0 millimolar or one of any value in the range is present. Cytochalasin B can be about 0.01 millimolar or about 50 millimolar or about 0.01 millimolar, about 0.05 millimolar, about 0.1 millimolar, about 2 millimolar, about 4 millimolar, about 6 milli. Mole, about 8 millimolar, about 10 millimolar, about 20 millimolar, about 30 millimolar, about 50 millimolar or any concentration in the range is present in the pre-input incubation base. DNase 1 can range from about 0.001 U/microliter to about 10 U/microliter or about 0.001 U/microliter, about 0.005 U/microliter, about 0.01 U/microliter, about 0.05 U/microliter, about 1.0 U/microliter. A concentration of about 5.0 U/μl, about 10 U/μl, or any value within the range is present.

在一些實施例中，一單一試劑可用以減弱已在一微流體器件中培養細胞之前或已在一微流體器件中培養細胞之後之黏著。例如，RGD三肽可以5毫克/毫升之一濃度而用於預培育或可在輸出之前作為一處理劑流入該微流體器件內。 In some embodiments, a single agent can be used to attenuate adhesion after cells have been cultured in a microfluidic device or after cells have been cultured in a microfluidic device. For example, the RGD tripeptide can be used for pre-incubation at a concentration of 5 mg/ml or can be flowed into the microfluidic device as a treating agent prior to export.

可使用之另一抑制劑係四肽纖連蛋白抑制劑(Arg-Gly-Asp-Ser-OH，mw.433.4，Santa Cruz Biotechnology Cat No：sc-202156)。該纖連蛋白抑制劑可以約1.75微克/毫升(4微莫耳)之一濃度使用。 Another inhibitor that can be used is the tetrapeptide fibronectin inhibitor (Arg-Gly-Asp-Ser-OH, mw. 433.4, Santa Cruz Biotechnology Cat No: sc-202156). The fibronectin inhibitor can be used at a concentration of about 1.75 micrograms per milliliter (4 micromoles).

類似於將蛋白質或小分子試劑用以減弱或防止黏著，細胞外黏著相關蛋白質之抗體可用以影響輸出及微流體器件內之可移植性。一非限制性實例係抗B1整合素：選殖M-106(Santa Cruz Biotechnology Cat No：sc-8978)。 Similar to the use of proteins or small molecule reagents to attenuate or prevent adhesion, antibodies to extracellular adhesion-related proteins can be used to affect the portability of the output and microfluidic devices. A non-limiting example is an anti-B1 integrin: M-106 (Santa Cruz Biotechnology Cat No: sc-8978).

含有一可裂解部分之調節表面Adjustment surface containing a cleavable portion

在一些實施例中，調節表面可具有併入於調節表面之共價或非共價鍵聯分子內之可裂解部分。調節表面可包含具有上述之一功能之肽基元(諸如RGD)，或其可具有促進細胞生長或提供細胞增殖之接觸線索之另一肽基元。在其他實施例中，調節表面對細胞提供非特定支援，且可僅用以緩衝來自微流體器件之矽或鋁氧化物表面之細胞。可 期望在完成細胞培養之一週期之後破壞調節表面以促進微流體器件之一生長室內之擴增細胞群體之輸出。此可用於細胞展現黏著行為時。可破壞調節表面，藉由併入其他肽基元(其係並非由所關注之細胞高度分泌之一蛋白酶之酶基)而部分或全部移除調節表面。在一非限制性實例中，ENLYQS(Glu-Asn-Leu-Tyr-Gln-Ser)之肽基元可依預設計時間間隔併入至一調節表面中。此基元係高度序列特定且因此用於高度受控裂解之TEV蛋白酶(菸草蝕刻病毒半胱胺酸蛋白酶，Sigma Aldrich catalog no.T4455)之一酶基。在完成培養週期之後，TEV蛋白酶可流動至微流體器件中且被允許擴散至生長室之隔離區域中。接著，破壞調節表面以促進微流體器件內之細胞之輸出。因此，各種其他解蛋白基元可經設計且併入至一調節表面中以藉由熟習此項技術者可想出之一適合特定蛋白酶而裂解。 In some embodiments, the conditioning surface can have a cleavable moiety that is incorporated into a covalent or non-covalently linked molecule of the conditioning surface. The modulating surface can comprise a peptide motif (such as RGD) having one of the functions described above, or it can have another peptide motif that promotes cell growth or provides contact cues for cell proliferation. In other embodiments, the conditioning surface provides non-specific support to the cells and may only be used to buffer cells from the surface of the microfluidic device or the aluminum oxide surface. can It is desirable to disrupt the regulatory surface after one cycle of cell culture is completed to facilitate the output of the expanded population of cells within one of the growth chambers of the microfluidic device. This can be used when cells exhibit adhesive behavior. The regulatory surface can be disrupted by partially or completely removing the regulatory surface by incorporating other peptide motifs that are not secreted by one of the proteases of the cell of interest. In a non-limiting example, the peptide motif of ENLYQS (Glu-Asn-Leu-Tyr-Gln-Ser) can be incorporated into a conditioning surface at pre-designed time intervals. This motif is one of the enzyme groups of the TEV protease (Tobacco Etch Virus cysteine protease, Sigma Aldrich catalog no. T4455) which is highly sequence specific and thus used for highly controlled lysis. After the completion of the culture period, the TEV protease can flow into the microfluidic device and be allowed to diffuse into the isolated regions of the growth chamber. Next, the conditioning surface is disrupted to promote the output of cells within the microfluidic device. Thus, various other decorin motifs can be designed and incorporated into a regulatory surface to be cleaved by one of skill in the art to a particular protease.

流體培養基Fluid medium

關於與具有一通道及一或多個生長室之微流體器件有關之上述討論，一流體培養基(例如一第一培養基及/或一第二培養基)可為能夠使一細胞維持於一實質上可變狀態中之任何流體。該可變狀態將取決於生物微小物體及所執行之培養實驗。 With regard to the above discussion relating to microfluidic devices having one channel and one or more growth chambers, a fluid medium (eg, a first medium and/or a second medium) can be capable of maintaining a cell substantially Any fluid in a state of change. This variable state will depend on the biological microscopic object and the culture experiment performed.

第一流體培養基及/或第二流體培養基可提供細胞存活所需之流體組分及溶解氣體組分兩者，且亦可使用緩衝流體培養基或pH監測或兩者來使pH維持於一所要範圍內。 The first fluid medium and/or the second fluid medium can provide both fluid components and dissolved gas components required for cell survival, and can also maintain pH in a desired range using buffered fluid medium or pH monitoring or both. Inside.

若該細胞係一哺乳動物細胞，則第一流體培養基及/或第二流體培養基可包含此項技術中已知之哺乳動物血清或一無血清合成培養基，其能夠提供必需營養素、內分泌素、生長因子或細胞生長信號。類似於上述調節表面，第一流體培養基及/或第二流體培養基可包含胎牛血清(FBS)或小牛血清(FCS)。替代地，第一流體培養基及/或第二流體培養基可不包含任何動物源血清，但可包含一合成培養基，其 可包含生理相關金屬離子(其包含(但不限於)鈉、鉀、鈣、鎂及/或鋅)抗氧化劑、表面活性劑及/或必需營養素之任何者或全部。合成培養基可為無血清的，同時仍含有一些蛋白質，其中該等蛋白質具有經界定數量及類型。一無血清培養基中之組分之一非限制性例示性列表包含：維生素，諸如B7、α-生育酚、α-生育酚乙酸酯、維生素A及其乙酸酯；蛋白質，諸如BSA、過氧化氫酶、胰島素、轉鐵蛋白、超氧化物歧化酶；小分子，諸如皮質固酮、D-半乳糖、乙醇胺鹽酸鹽、還原谷胱甘肽、L-肉鹼鹽酸鹽、亞油酸、亞麻酸、黃體固酮、腐胺二鹽酸鹽及三碘甲腺原氨酸；及鹽，其包含(但不限於)亞硒酸鈉、磷酸鈉、磷酸鉀、磷酸鈣及/或磷酸鎂。流體培養基可含有上文針對調節表面所描述之抗氧化劑之任何者。 If the cell is a mammalian cell, the first fluid medium and/or the second fluid medium may comprise mammalian serum or a serum-free synthetic medium known in the art that is capable of providing essential nutrients, endocrine, growth factors Or cell growth signal. Similar to the conditioning surface described above, the first fluid medium and/or the second fluid medium may comprise fetal bovine serum (FBS) or calf serum (FCS). Alternatively, the first fluid medium and/or the second fluid medium may not contain any animal source serum, but may comprise a synthetic medium, Any or all of physiologically relevant metal ions including, but not limited to, sodium, potassium, calcium, magnesium and/or zinc, antioxidants, surfactants and/or essential nutrients may be included. The synthetic medium can be serum free while still containing some proteins, wherein the proteins are of defined quantity and type. A non-limiting, illustrative list of one of the components in a serum-free medium comprises: vitamins such as B7, alpha-tocopherol, alpha-tocopheryl acetate, vitamin A and its acetate; proteins such as BSA, Hydrogenase, insulin, transferrin, superoxide dismutase; small molecules such as corticosterone, D-galactose, ethanolamine hydrochloride, reduced glutathione, L-carnitine hydrochloride, linoleum Acid, linolenic acid, luteinone, putrescine dihydrochloride, and triiodothyronine; and salts, including but not limited to sodium selenite, sodium phosphate, potassium phosphate, calcium phosphate, and/or Magnesium phosphate. The fluid medium can contain any of the antioxidants described above for the conditioning surface.

可透過一0.22微米過濾器單元(VWR,Cat.No.73520-986)而無菌過濾流體培養基。 The fluid medium can be sterile filtered through a 0.22 micron filter unit (VWR, Cat. No. 73520-986).

在一些實施例中，一適合培養基可包含下列之任何者或可完全由下列之任何者組成：杜爾貝科氏改質伊格爾培養基(ThermoFisher Scientific,Cat # 11960-051)、FreeStyle^TM培養基(Invitrogen,ThermoFisher Scientific,Cat.No.11960-051)、RPMI-1640(GIBCO®,ThermoFisher Scientific,Cat.No.11875-127)、Hybridoma-SFM(ThermoFisher Scientific,Cat.No.12045-076)、培養基E(Stem Cell,Cat.No.3805)、1X CD CHO培養基(ThermoFisher Scientific,Cat.No.10743-011)、Iscove改質杜爾貝科氏培養基(ThermoFisher Scientific,Cat.No.12440-061)或CD DG44培養基(ThermoFisher Scientific,Cat.No.10743-011)。 In some embodiments, a suitable medium may comprise any of those of the following or may consist entirely by any of the following composition: Dulbecco's modified Eagle medium (ThermoFisher Scientific, Cat # 11960-051) , FreeStyle TM medium (Invitrogen, ThermoFisher Scientific, Cat. No. 11960-051), RPMI-1640 (GIBCO®, ThermoFisher Scientific, Cat. No. 11875-127), Hybridoma-SFM (ThermoFisher Scientific, Cat. No. 12045-076), Medium E (Stem Cell, Cat. No. 3805), 1X CD CHO medium (ThermoFisher Scientific, Cat. No. 10743-011), Iscove modified Dulbecco's medium (ThermoFisher Scientific, Cat. No. 12440-061) Or CD DG44 medium (ThermoFisher Scientific, Cat. No. 10743-011).

培養基可另外包含胎牛血清(FBS，購自GIBCO®,ThermoFisher Scientific)、熱失活胎牛血清或小牛血清(FCS，Sigma-Aldrich Cat Nos.F2442、F6176、F4135及其他)。FBS可以下列之一濃度存在：約 1% v/v至約20% v/v、約1% v/v至約15% v/v、約1% v/v至約10% v/v或約1% v/v至約5% v/v、或該等範圍之任何者內之任何數值。培養基可另外包含人類AB血清(Sigma-Aldrich,Cat.No.S2146)且可以下列之一濃度存在：約1% v/v至約20% v/v、約1% v/v至約15% v/v、約1% v/v至約10% v/v或約1% v/v至約5% v/v、或該等範圍之任何者內之任何數值。 The medium may additionally contain fetal bovine serum (FBS, available from GIBCO®, ThermoFisher Scientific), heat-inactivated fetal bovine serum or calf serum (FCS, Sigma-Aldrich Cat Nos. F 2442, F6176, F4135 and others). FBS can exist in one of the following concentrations: approximately 1% v/v to about 20% v/v, about 1% v/v to about 15% v/v, about 1% v/v to about 10% v/v or about 1% v/v to about 5 % v/v, or any value within any of these ranges. The medium may additionally comprise human AB serum (Sigma-Aldrich, Cat. No. S2146) and may be present in one of the following concentrations: from about 1% v/v to about 20% v/v, from about 1% v/v to about 15%. v/v, from about 1% v/v to about 10% v/v or from about 1% v/v to about 5% v/v, or any value within any of the ranges.

培養基可另外包含青黴素-鏈黴素(ThermoFisher Scientific,Cat.No.15140-163)。筆鏈球菌可以下列範圍內之一濃度存在：約0.01% v/v至約10% v/v、約0.1% v/v至約10% v/v、約0.01% v/v至約5% v/v、約0.1% v/v至約5% v/v、約0.1% v/v至約3% v/v、約0.1% v/v至約2% v/v、約0.1% v/v至約1% v/v或該等範圍之任何者內之任何值。在其他實施例中，培養基可包含遺傳黴素(ThermoFisher Scientific,Cat.No.101310-035)。遺傳黴素可以下列之一濃度存在：約0.5微克/毫升、約1.0微克/毫升、約5.0微克/毫升、約10.0微克/毫升、約15微克/毫升、約20微克/毫升、約30微克/毫升、約50微克/毫升、約70微克/毫升、約100微克/毫升或此等範圍內之任何值。 The medium may additionally contain penicillin-streptomycin (ThermoFisher Scientific, Cat. No. 15140-163). Streptococcus penicillium can be present in one of the following ranges: from about 0.01% v/v to about 10% v/v, from about 0.1% v/v to about 10% v/v, from about 0.01% v/v to about 5% v/v, from about 0.1% v/v to about 5% v/v, from about 0.1% v/v to about 3% v/v, from about 0.1% v/v to about 2% v/v, about 0.1% v /v to about 1% v/v or any value within any of these ranges. In other embodiments, the medium may comprise geneticin (ThermoFisher Scientific, Cat. No. 101310-035). Geneticin can be present in one of the following concentrations: about 0.5 micrograms per milliliter, about 1.0 micrograms per milliliter, about 5.0 micrograms per milliliter, about 10.0 micrograms per milliliter, about 15 micrograms per milliliter, about 20 micrograms per milliliter, about 30 micrograms per minute. ML, about 50 μg/ml, about 70 μg/ml, about 100 μg/ml or any value within these ranges.

培養基可包含一緩衝液。該緩衝液可為Good緩衝液之一者。該緩衝液可為(但不限於)(4-(2-羥乙基)-1-哌嗪乙磺酸)(HEPES)(ThermoFisher Scientific,Cat.No.15630-080)。該緩衝液可以下列之一濃度存在：約1毫莫耳、約3毫莫耳、約5毫莫耳、約7毫莫耳、約9毫莫耳、約10毫莫耳、約12毫莫耳、約15毫莫耳、約20毫莫耳、約40毫莫耳、約60毫莫耳、約100毫莫耳或此等範圍內之任何值。 The medium may contain a buffer. The buffer can be one of the Good buffers. The buffer can be, but is not limited to, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) (ThermoFisher Scientific, Cat. No. 15630-080). The buffer may be present in one of the following concentrations: about 1 millimole, about 3 millimolar, about 5 millimolar, about 7 millimolar, about 9 millimolar, about 10 millimolar, about 12 millimolar Ear, about 15 millimolar, about 20 millimolar, about 40 millimolar, about 60 millimolar, about 100 millimolar or any value within the range.

培養基可另外包含穀胺醯胺之二肽取代物，GlutaMAX^TM(GIBCO® ThermoFisher Scientific,Cat No.35050-079)。穀胺醯胺之取代物可以下列之一濃度存在：約0.2毫莫耳、約0.5毫莫耳、約0.7毫莫 耳、約1.0毫莫耳、約1.2毫莫耳、約1.5毫莫耳、約1.7毫莫耳、約2.0毫莫耳、約2.5毫莫耳、約3.0毫莫耳、約4.0毫莫耳、約7.0毫莫耳或約10.0毫莫耳或此等範圍內之任何值。培養基可包含MEM非必需胺基酸(ThermoFisher Scientific,Cat.No.10370-088)。MEM非必需胺基酸可以下列之一濃度存在：約0.2毫莫耳、約0.5毫莫耳、約0.7毫莫耳、約1.0毫莫耳、約1.2毫莫耳、約1.5毫莫耳、約1.7毫莫耳、約2.0毫莫耳、約2.5毫莫耳、約3.0毫莫耳、約4.0毫莫耳、約7.0毫莫耳或約10.0毫莫耳或此等範圍內之任何值。 The medium may additionally comprise a dipeptide substitution of glutamine, GlutaMAX ^(TM) (GIBCO® ThermoFisher Scientific, Cat No. 35050-079). The substitution of glutamine can be present in one of the following concentrations: about 0.2 millimolar, about 0.5 millimolar, about 0.7 millimolar, about 1.0 millimolar, about 1.2 millimolar, about 1.5 millimolar, About 1.7 millimolar, about 2.0 millimolar, about 2.5 millimolar, about 3.0 millimolar, about 4.0 millimolar, about 7.0 millimolar or about 10.0 millimolar or any value within these ranges. The medium may comprise MEM non-essential amino acids (ThermoFisher Scientific, Cat. No. 10370-088). The MEM non-essential amino acid can be present in one of the following concentrations: about 0.2 millimolar, about 0.5 millimolar, about 0.7 millimolar, about 1.0 millimolar, about 1.2 millimolar, about 1.5 millimolar, about 1.7 millimolar, about 2.0 millimolar, about 2.5 millimolar, about 3.0 millimolar, about 4.0 millimolar, about 7.0 millimolar or about 10.0 millimolar or any value within these ranges.

培養基可另外含有葡萄糖(ThermoFisher Scientific,Cat.No.15023-021)。葡萄糖可以下列之一濃度存在：約0.1g/L、約0.3g/L、約0.5g/L、約0.8g/L、約1.0g/L、約1.5g/L、約2.0g/L、約2.5g/L、約3.0g/L、約3.5g/L、約4.0g/L、約5.0g/L、約7.0g/L、約10.0g/L或此等範圍內之任何值。 The medium may additionally contain glucose (ThermoFisher Scientific, Cat. No. 15023-021). Glucose may be present in one of the following concentrations: about 0.1 g/L, about 0.3 g/L, about 0.5 g/L, about 0.8 g/L, about 1.0 g/L, about 1.5 g/L, about 2.0 g/L, Any value in the range of about 2.5 g/L, about 3.0 g/L, about 3.5 g/L, about 4.0 g/L, about 5.0 g/L, about 7.0 g/L, about 10.0 g/L, or the like.

培養基可另外包含巰基乙醇(ThermoFisher Scientific,Cat.No.31350-010)。巰基乙醇可以下列之一濃度存在：約0.001% v/v至約1.5% v/v、約0.005% v/v至約1.0% v/v、約0.01% v/v至約1.0% v/v、約0.15% v/v至約1.0% v/v、約0.2% v/v至約1% v/v或此等範圍內之任何值。 The medium may additionally contain mercaptoethanol (ThermoFisher Scientific, Cat. No. 31350-010). Mercaptoethanol can be present in one of the following concentrations: from about 0.001% v/v to about 1.5% v/v, from about 0.005% v/v to about 1.0% v/v, from about 0.01% v/v to about 1.0% v/v From about 0.15% v/v to about 1.0% v/v, from about 0.2% v/v to about 1% v/v or any value within such ranges.

培養基可包含OPI培養基添加物，其包含草醯乙酸、丙酮酸及胰島素(Sigma-Aldrich,Cat.No.O-5003)。OPI培養基添加物可以下列之一濃度存在：約0.001% v/v至約1.5% v/v、約0.005% v/v至約1.0% v/v、約0.01% v/v至約1.0% v/v、約0.15% v/v至約1.0% v/v、約0.2% v/v至約1% v/v或此等範圍內之任何值。培養基可含有B-27補充劑(50倍)、無血清培養基(ThermoFisher Scientific,Cat.No.17504-163)。B-27補充劑可以下列之一濃度存在：約0.01% v/v至約10.5% v/v、約0.05% v/v至約5.0% v/v、約0.1% v/v至約5.0% v/v、約0.5% v/v至約5% v/v或此等範圍內之任何值。 The medium may comprise an OPI medium supplement comprising oxalic acid, pyruvic acid and insulin (Sigma-Aldrich, Cat. No. O-5003). The OPI medium supplement may be present in one of the following concentrations: from about 0.001% v/v to about 1.5% v/v, from about 0.005% v/v to about 1.0% v/v, from about 0.01% v/v to about 1.0% v /v, from about 0.15% v/v to about 1.0% v/v, from about 0.2% v/v to about 1% v/v or any value within these ranges. The medium may contain B-27 supplement (50-fold), serum-free medium (ThermoFisher Scientific, Cat. No. 17504-163). The B-27 extender can be present in one of the following concentrations: from about 0.01% v/v to about 10.5% v/v, from about 0.05% v/v to about 5.0% v/v, from about 0.1% v/v to about 5.0%. v/v, about 0.5% v/v to about 5% v/v or any value within these ranges.

如本文中所描述，一培養基或一培養基之一添加物可包含用於產生一調節表面之一或多個Pluronic®聚合物，且可包含Pluronic® L44、L64、P85、F68及F127(其包含F127NF)。Pluronic®聚合物可以下列之一濃度存在於培養基中：約0.001% v/v至約10% v/v、約0.01% v/v至約5% v/v、約0.01% v/v至約1% v/v或約0.05% v/v至約1% v/v。針對可經提供為一套組之一培養基添加物，濃度可為最終培養基濃度之1倍、5倍、10倍、100倍或約100倍。 As described herein, a medium or a medium supplement may comprise one or more Pluronic® polymers for producing a conditioning surface, and may comprise Pluronic® L44, L64, P85, F68 and F127 (including F127NF). The Pluronic® polymer can be present in the medium at a concentration of from about 0.001% v/v to about 10% v/v, from about 0.01% v/v to about 5% v/v, from about 0.01% v/v to about 1% v/v or from about 0.05% v/v to about 1% v/v. The concentration may be one, five, ten, 100 or about 100 times the concentration of the final medium for one of the media supplements that may be provided as a set.

培養基可包含IL 6(Sigma-Aldrich,Cat.No.SRP3096-20UG)。IL 6可以下列之一濃度存在：約1nM、約5nM、約10nM、約15nM、約20nM、約25nM、約30nM、約40nM、約50nM或此等範圍內之任何值。 The medium may comprise IL 6 (Sigma-Aldrich, Cat. No. SRP 3096-20 UG). IL 6 can be present in one of the following concentrations: about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, or any value within the range.

培養基可另外包含丙酮酸鈉(ThermoFisher Scientific,Cat.No.11360-070)。穀胺醯胺之取代物可以下列之一濃度存在：約0.1毫莫耳、約0.02毫莫耳、約0.04毫莫耳、約0.06毫莫耳、約0.08毫莫耳、約0.1毫莫耳、約0.5毫莫耳、約0.7毫莫耳、約1.0毫莫耳、約1.2毫莫耳、約1.5毫莫耳、約1.7毫莫耳、約2.0毫莫耳、約2.5毫莫耳、約3.0毫莫耳、約4.0毫莫耳、約7.0毫莫耳或約10.0毫莫耳或此等範圍內之任何值。 The medium may additionally contain sodium pyruvate (ThermoFisher Scientific, Cat. No. 11360-070). The substitution of glutamine can be present in one of the following concentrations: about 0.1 millimolar, about 0.02 millimolar, about 0.04 millimolar, about 0.06 millimolar, about 0.08 millimolar, about 0.1 millimolar, About 0.5 millimolar, about 0.7 millimolar, about 1.0 millimolar, about 1.2 millimolar, about 1.5 millimolar, about 1.7 millimolar, about 2.0 millimolar, about 2.5 millimolar, about 3.0 Millo-ohms, about 4.0 millimolar, about 7.0 millimolar or about 10.0 millimoles or any value in the range.

氣體環境Gas environment

系統提供細胞存活所需氣體之一混合物，其包含(但不限於)氧氣及二氧化碳。兩種氣體溶解至流體培養基中，且可由細胞使用，因此隨時間更改一生長室之一隔離區域中之流體培養基之氣體含量。特定言之，二氧化碳含量可隨時間改變，其影響微流體器件中之流體培養基之pH。在一些實驗條件中，可使用非最佳氧氣分壓。 The system provides a mixture of gases required for cell survival, including but not limited to oxygen and carbon dioxide. The two gases are dissolved into the fluid medium and can be used by the cells, thus changing the gas content of the fluid medium in the isolated region of one of the growth chambers over time. In particular, the carbon dioxide content can change over time, which affects the pH of the fluid medium in the microfluidic device. In some experimental conditions, a non-optimal oxygen partial pressure can be used.

溫度控制temperature control

在一些實施例中，藉由控制至少一調節表面之溫度而調節(若干)生長室及/或(若干)流動區域之至少一調節表面。系統可包含一組件，其可控制及調變微流體器件之生長室及/或流動區域之至少一調節表面之溫度。系統可包含用於對微流體器件提供溫度調變之帕耳帖加熱、電阻加熱或任何其他適合方法。系統亦可包含感測器及/或回饋組件來將熱輸入控制至一預定範圍。在一些實施例中，至少一調節表面具有至少約25℃、約26℃、約27℃、約28℃、約29℃、約30℃、約31℃、約32℃、約33℃、約34℃、約35℃、約36℃、約37℃、約38℃、約39℃或約40℃之一溫度，且穩定於該溫度處。在一些實施例中，至少一表面具有大於約25℃之一溫度。在其他實施例中，至少一表面具有自約30℃至約40℃、自約35℃至約38℃或自約36℃至約37℃範圍內之一溫度。在一些實施例中，至少一調節表面具有至少約30℃之一溫度。 In some embodiments, at least one conditioning surface of the growth chamber and/or the flow area(s) is adjusted by controlling the temperature of the at least one conditioning surface. The system can include a component that can control and modulate the temperature of at least one conditioning surface of the growth chamber and/or flow region of the microfluidic device. The system can include Peltier heating, resistance heating, or any other suitable method for providing temperature modulation to the microfluidic device. The system can also include a sensor and/or a feedback component to control the heat input to a predetermined range. In some embodiments, the at least one conditioning surface has at least about 25 ° C, about 26 ° C, about 27 ° C, about 28 ° C, about 29 ° C, about 30 ° C, about 31 ° C, about 32 ° C, about 33 ° C, about 34 One of a temperature of ° C, about 35 ° C, about 36 ° C, about 37 ° C, about 38 ° C, about 39 ° C or about 40 ° C, and is stable at this temperature. In some embodiments, at least one surface has a temperature greater than about 25 °C. In other embodiments, at least one surface has a temperature ranging from about 30 ° C to about 40 ° C, from about 35 ° C to about 38 ° C, or from about 36 ° C to about 37 ° C. In some embodiments, the at least one conditioning surface has a temperature of at least about 30 °C.

在培育期間提供灌注之流量控制器Flow controller that provides perfusion during incubation

流量控制器可在培育週期期間將第一流體培養基灌注於流動區域中(如上文所描述)以將營養素提供至生長室中之細胞且自生長室帶走廢棄物，其中營養素之交換及廢棄物之移除實質上經由擴散而發生。控制器可為與微流體器件分離之一組件或可經併入為微流體器件之部分。流量控制器可經構形以非連續地將培養基灌注於流動區域中。流量控制器可經構形以依一週期性方式或一不規律方式灌注(若干)培養基。 The flow controller can infuse the first fluid medium into the flow region (as described above) during the incubation cycle to provide nutrients to the cells in the growth chamber and carry away the waste from the growth chamber, wherein the nutrient exchange and waste The removal occurs substantially through diffusion. The controller can be one component separate from the microfluidic device or can be incorporated as part of the microfluidic device. The flow controller can be configured to non-continuously infuse the medium into the flow region. The flow controller can be configured to perfuse the medium (several) in a periodic manner or in an irregular manner.

在一些其他實施例中，控制器可經構形以約每隔4小時、3小時、2小時、60分鐘、57分鐘、55分鐘、53分鐘、50分鐘、47分鐘、45分鐘、43分鐘、40分鐘、37分鐘、35分鐘、33分鐘、30分鐘、27分鐘、25分鐘、23分鐘、20分鐘、17分鐘、15分鐘、13分鐘、10分鐘、7分鐘或5分鐘將(若干)流體培養基灌注於流動區域中一次。在一些實 施例中，控制器可經構形以約每隔5分鐘至約每隔20分鐘灌注流體培養基一次。在其他實施例中，控制器可經構形以約每隔15分鐘至約每隔45分鐘灌注流體培養基一次。在其他實施例中，控制器可經構形以每隔30分鐘至約每隔60分鐘灌注流體培養基一次。在其他實施例中，控制器可經構形以每隔45分鐘至約每隔90分鐘灌注流體培養基一次。在一些其他實施例中，控制器可經構形以每隔60分鐘至約120分鐘灌注流體培養基一次。替代地，控制器可經構形以每隔2小時至每隔6小時灌注流體培養基一次。 In some other embodiments, the controller can be configured to be approximately every 4 hours, 3 hours, 2 hours, 60 minutes, 57 minutes, 55 minutes, 53 minutes, 50 minutes, 47 minutes, 45 minutes, 43 minutes, Fluid medium (several) at 40 minutes, 37 minutes, 35 minutes, 33 minutes, 30 minutes, 27 minutes, 25 minutes, 23 minutes, 20 minutes, 17 minutes, 15 minutes, 13 minutes, 10 minutes, 7 minutes or 5 minutes Infuse in the flow area once. In some real In an embodiment, the controller can be configured to perfuse the fluid medium once every about 5 minutes to about every 20 minutes. In other embodiments, the controller can be configured to perfuse the fluid medium once every about 15 minutes to about every 45 minutes. In other embodiments, the controller can be configured to perfuse the fluid medium once every 30 minutes to about every 60 minutes. In other embodiments, the controller can be configured to perfuse the fluid medium once every 45 minutes to about every 90 minutes. In some other embodiments, the controller can be configured to perfuse the fluid medium once every 60 minutes to about 120 minutes. Alternatively, the controller can be configured to perfuse the fluid medium once every 2 hours to every 6 hours.

在一些實施例中，控制器226可經構形以在可為約5秒、約10秒、約15秒、約20秒、約25秒、約30秒、約35秒、約40秒、約45秒、約50秒、約55秒、約60秒、約65秒或約70秒之一時間週期內灌注培養基。在其他實施例中，控制器可經構形以灌注培養基達約1分鐘、約1.2分鐘、約1.4分鐘、約1.5分鐘、約1.6分鐘、約1.8分鐘、約2.0分鐘、約2.2分鐘、約2.4分鐘、約2.5分鐘、約2.6分鐘、約2.8分鐘、約3.0分鐘、約3.2分鐘、約3.4分鐘、約3.5分鐘、約3.6分鐘、約3.8分鐘或約4.0分鐘。 In some embodiments, controller 226 can be configured to be about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about The medium is perfused for a period of 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 65 seconds, or about 70 seconds. In other embodiments, the controller can be configured to perfuse the medium for about 1 minute, about 1.2 minutes, about 1.4 minutes, about 1.5 minutes, about 1.6 minutes, about 1.8 minutes, about 2.0 minutes, about 2.2 minutes, about 2.4. Minutes, about 2.5 minutes, about 2.6 minutes, about 2.8 minutes, about 3.0 minutes, about 3.2 minutes, about 3.4 minutes, about 3.5 minutes, about 3.6 minutes, about 3.8 minutes, or about 4.0 minutes.

在各種實施例中，控制器可經構形以灌注培養基達約5秒至約4分鐘、約10秒至約3.5分鐘、約15秒至約3分鐘、約15秒至約2分鐘、約25秒至約90秒、約30秒至約75秒、約40秒至約2.0分鐘、約60秒至約2.5分鐘、約90秒至約3.0分鐘或1.8分鐘至約4分鐘。 In various embodiments, the controller can be configured to perfuse the medium for from about 5 seconds to about 4 minutes, from about 10 seconds to about 3.5 minutes, from about 15 seconds to about 3 minutes, from about 15 seconds to about 2 minutes, about 25 Seconds to about 90 seconds, from about 30 seconds to about 75 seconds, from about 40 seconds to about 2.0 minutes, from about 60 seconds to about 2.5 minutes, from about 90 seconds to about 3.0 minutes, or from 1.8 minutes to about 4 minutes.

流量控制器(圖中未展示)可經構形以依一速率將第一流體培養基灌注於流動區域中，該速率遠大於組分自生長室之隔離區域擴散至流動通道之平均速率。例如，流動區域中之流體流動速率可為約0.009微升/秒、約0.01微升/秒、約0.02微升/秒、約0.03微升/秒、約0.05微升/秒、約0.1微升/秒、約0.2微升/秒、約0.3微升/秒、約0.4微升/秒、約0.5微升/秒、約0.6微升/秒、約0.7微升/秒、約0.8微升/秒、約0.9微 升/秒、約1.0微升/秒、約1.1微升/秒、約1.2微升/秒、約1.3微升/秒、約1.4微升/秒、約1.5微升/秒、約1.7微升/秒、約1.9微升/秒、約2.0微升/秒、約2.2微升/秒、約2.4微升/秒、約2.6微升/秒、約2.8微升/秒、約3.0微升/秒、約3.2微升/秒、約3.4微升/秒、約3.6微升/秒、約3.8微升/秒、約4.0微升/秒、約4.2微升/秒、約4.4微升/秒、約4.6微升/秒、約4.8微升/秒、約5.0微升/秒、約6.0微升/秒、約7.0微升/秒、約8.0微升/秒或約9.0微升/秒，其等之任何者係將掃及生長室之一連接區域(但不會掃及(若干)生長室之一隔離區域)之一速率。控制器能夠提供第一流體培養基之一速度，其係流體培養基速度之一非掃及速率，即，低於V_max(微流體器件之最大速度，其避免微流體器件歸因於過度壓力而破裂且限制生長室中之一第二流體培養基與流動區域中之一第一流體培養基之間的組分移動至擴散區)之任何適合速率。在一些實施例中，控制器可經構形以依下列各者透過流動區域而灌注第一流體培養基：約0.05微升/秒、約0.06微升/秒、約0.07微升/秒、約0.08微升/秒、約0.09微升/秒、約0.10微升/秒、約0.11微升/秒、約0.12微升/秒、約0.13微升/秒、約0.14微升/秒、約0.15微升/秒、約0.16微升/秒、約0.17微升/秒、約0.18微升/秒、約0.19微升/秒、約0.20微升/秒、約0.30微升/秒、約0.40微升/秒、約0.50微升/秒、約0.60微升/秒、約0.70微升/秒、約0.80微升/秒、約0.90微升/秒、約1.00微升/秒、約1.10微升/秒、約1.20微升/秒、約1.30微升/秒、約1.40微升/秒、約1.50微升/秒、約1.60微升/秒、約1.70微升/秒、約1.80微升/秒、約1.90微升/秒、約2.00微升/秒、約2.10微升/秒、約2.20微升/秒、約2.30微升/秒、約2.40微升/秒、約2.50微升/秒、約2.60微升/秒、約2.70微升/秒、約2.80微升/秒、約2.90微升/秒或約3.00微升/秒。在一些實施例中，控制器可經構形以依下列各者透過複數個流動區域之各者而灌注第一流體培養基：約0.01微升/秒、約0.02微升/ 秒、約0.03微升/秒、約0.04微升/秒、約0.05微升/秒、約0.06微升/秒、約0.07微升/秒、約0.08微升/秒、約0.09微升/秒、約0.10微升/秒或約0.11微升/秒。 A flow controller (not shown) can be configured to infuse the first fluid medium into the flow region at a rate that is substantially greater than the average rate at which the components diffuse from the isolation region of the growth chamber to the flow channel. For example, the fluid flow rate in the flow region can be about 0.009 microliters per second, about 0.01 microliters per second, about 0.02 microliters per second, about 0.03 microliters per second, about 0.05 microliters per second, about 0.1 microliters. /second, about 0.2 microliters/second, about 0.3 microliters/second, about 0.4 microliters/second, about 0.5 microliters/second, about 0.6 microliters/second, about 0.7 microliters/second, about 0.8 microliters/ Seconds, about 0.9 microliters per second, about 1.0 microliters per second, about 1.1 microliters per second, about 1.2 microliters per second, about 1.3 microliters per second, about 1.4 microliters per second, about 1.5 microliters per second. , about 1.7 microliters per second, about 1.9 microliters per second, about 2.0 microliters per second, about 2.2 microliters per second, about 2.4 microliters per second, about 2.6 microliters per second, about 2.8 microliters per second, About 3.0 microliters per second, about 3.2 microliters per second, about 3.4 microliters per second, about 3.6 microliters per second, about 3.8 microliters per second, about 4.0 microliters per second, about 4.2 microliters per second, about 4.4 microliters per second, about 4.6 microliters per second, about 4.8 microliters per second, about 5.0 microliters per second, about 6.0 microliters per second, about 7.0 microliters per second, about 8.0 microliters per second, or about 9.0. Microliters per second, any of which would sweep one of the growth zones of one of the growth chambers (but not one of the isolation zones of one of the growth chambers). The controller is capable of providing a velocity of one of the first fluid media, which is one of the velocity of the fluid media, non-sweeping rate, ie, below _Vmax (maximum velocity of the microfluidic device, which prevents the microfluidic device from rupturing due to excessive pressure And limiting any suitable rate of movement of one of the second fluid medium in the growth chamber to the first fluid medium in the flow region to the diffusion zone. In some embodiments, the controller can be configured to perfuse the first fluid medium through the flow region by: about 0.05 microliters per second, about 0.06 microliters per second, about 0.07 microliters per second, about 0.08 Microliters per second, about 0.09 microliters per second, about 0.10 microliters per second, about 0.11 microliters per second, about 0.12 microliters per second, about 0.13 microliters per second, about 0.14 microliters per second, about 0.15 microliters. L/s, about 0.16 microliters/second, about 0.17 microliters/second, about 0.18 microliters/second, about 0.19 microliters/second, about 0.20 microliters/second, about 0.30 microliters/second, about 0.40 microliters. / sec, about 0.50 microliters per second, about 0.60 microliters per second, about 0.70 microliters per second, about 0.80 microliters per second, about 0.90 microliters per second, about 1.00 microliters per second, about 1.10 microliters per second. Seconds, about 1.20 microliters per second, about 1.30 microliters per second, about 1.40 microliters per second, about 1.50 microliters per second, about 1.60 microliters per second, about 1.70 microliters per second, about 1.80 microliters per second. , about 1.90 microliters per second, about 2.00 microliters per second, about 2.10 microliters per second, about 2.20 microliters per second, about 2.30 microliters per second, about 2.40 microliters per second, about 2.50 microliters per second, About 2.60 microliters per second, about 2.70 microliters per second, about 2.80 microliters per second, about 2.90 microliters per second, or about 3.00 microliters per second. In some embodiments, the controller can be configured to perfuse the first fluid medium through each of the plurality of flow regions in each of: about 0.01 microliters per second, about 0.02 microliters per second, about 0.03 microliters / sec, about 0.04 microliters per second, about 0.05 microliters per second, about 0.06 microliters per second, about 0.07 microliters per second, about 0.08 microliters per second, about 0.09 microliters per second, about 0.10 microliters per second. Seconds or about 0.11 microliters per second.

在各種實施例中，流動速率及灌注之持續時間提供流動通道之至少約3個、約4個、約5個、約6個、約7個、約8個、約9個、約10個、約11個、約15個、約20個、約25個、約30個、約35個、約50個、約75個、約100個、約200個、約300個或300個以上體積之第一流體培養基之總量。 In various embodiments, the flow rate and the duration of the infusion provide at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, of the flow channels, About 11, about 15, about 20, about 25, about 30, about 35, about 50, about 75, about 100, about 200, about 300, or more than 300 volumes The total amount of a fluid medium.

在各種實施例中，可使用變動持續時間、變動流動速率及灌注停止持續時間來完成灌注，如圖7及圖8之方法中所展示及如下文所討論。 In various embodiments, the perfusion can be accomplished using a varying duration, a varying flow rate, and a perfusion stop duration, as shown in the methods of Figures 7 and 8, and as discussed below.

儲液器、培養基調節及引入組分Reservoir, medium conditioning and introduction of components

系統可進一步包含經構形以含有流體培養基之一儲液器，流體培養基可在微流體器件之入口124處引入且可藉由流量控制器而灌注。儲液器可在一上流位置處流體地連接至如本文中所描述之微流體器件之任何者(非限制性實例包含100、200、240、290或400)(圖5A至圖5E)。可在儲液器中調節流體培養基以含有所要氣體平衡(即，針對一非限制性實例，含有5%二氧化碳之一混合物)，其對受培養之細胞提供最佳化生長，且亦可緩和微流體器件中之pH。 The system can further comprise a reservoir configured to contain a fluid medium that can be introduced at the inlet 124 of the microfluidic device and can be infused by a flow controller. The reservoir can be fluidly coupled to any of the microfluidic devices as described herein at an upflow location (non-limiting examples include 100, 200, 240, 290 or 400) ( Figs. 5A-5E ). The fluid medium can be adjusted in the reservoir to contain the desired gas balance (i.e., for a non-limiting example, a mixture of 5% carbon dioxide) that provides optimized growth to the cultured cells and can also moderate The pH in the fluid device.

在一些實施例中，儲液器可進一步含有不同於微流體器件中之受研究細胞之一細胞群體。此細胞群體可為餵食細胞，其產生微流體器件中之細胞生長及/或存活所需之可溶傳信或生長因子。依此方式，流體培養基可在引入至微流體器件之前針對最佳化生長及/或存活性而調節。使用儲液器來容置餵食細胞群體可防止污染微流體器件中之受培養細胞群體；可將來自餵食細胞之可溶分泌物併入至經傳遞至微流體器件中之流體培養基中，但餵食細胞可不與流體培養基一起 向上抽吸。 In some embodiments, the reservoir may further comprise a population of cells other than the studied cells in the microfluidic device. This population of cells can be a feeding cell that produces a soluble signaling or growth factor required for cell growth and/or survival in a microfluidic device. In this manner, the fluid medium can be adjusted for optimal growth and/or viability prior to introduction into the microfluidic device. The use of a reservoir to house a population of feeder cells prevents contamination of the cultured cell population in the microfluidic device; soluble secretions from the feeder cells can be incorporated into the fluid medium that is delivered to the microfluidic device, but fed Cells may not be with fluid medium Pump up.

圖5A中展示系統之一儲液器、調節及引入組分之一實施例。在此實施例中，儲液器可為另一微流體器件502，其含有在微流體器件502內調節之流體培養基202(圖中未展示)。微流體器件502具有一封閉體510及一基底512，其等之至少一者係可透氣的。微流體器件502亦可含有一餵食細胞群體，其經維持使得餵食細胞產生微流體器件500A中之(若干)細胞生長及/或存活所需之可溶生長因子或其他細胞傳信組分。儲液器502可容置於一室516內以提供一5%二氧化碳氣體環境，例如(但不限於)一氣體環境。儲液器502中之流體培養基202吸收穿過儲液器之可透氣壁之氣體混合物(例如空氣中5%二氧化碳)，且亦自餵食細胞吸收可溶分泌物。培養基202藉由泵514而自儲液器502，通過不可透氣連接導管506，經由入口124灌注至微流體器件500A中，且在微流體器件500A之流動通道134中形成流212。在此實施例中，泵連接導管504(未標記)、轉移連接導管506、基底104或封閉體102均為不可透氣的。流體培養基流212掃及通過微流體器件500A之生長室且容許流體培養基204之廢棄組分自生長室(圖中未展示)擴散出，同時容許組分自流動通道134中之流體培養基202擴散至生長室中。最後，廢流體培養基202'(圖中未展示)經由出口連接導管508中之出口124'而退出微流體器件500A。 One embodiment of one of the reservoir, conditioning and introduction components of the system is shown in Figure 5A. In this embodiment, the reservoir can be another microfluidic device 502 that contains a fluid medium 202 (not shown) that is conditioned within the microfluidic device 502. The microfluidic device 502 has an enclosure 510 and a substrate 512, at least one of which is breathable. The microfluidic device 502 can also contain a population of feeding cells that are maintained such that the feeding cells produce soluble growth factors or other cellular signaling components required for cell growth and/or survival in the microfluidic device 500A. The reservoir 502 can be housed in a chamber 516 to provide a 5% carbon dioxide gas environment, such as, but not limited to, a gaseous environment. Fluid medium 202 in reservoir 502 absorbs a gas mixture through the gas permeable wall of the reservoir (e.g., 5% carbon dioxide in air) and also absorbs soluble secretions from the feeding cells. The medium 202 is perfused from the reservoir 502 by the pump 514, through the non-breathable connection conduit 506, into the microfluidic device 500A via the inlet 124, and a stream 212 is formed in the flow channel 134 of the microfluidic device 500A. In this embodiment, the pump connection conduit 504 (not labeled), the transfer connection conduit 506, the substrate 104, or the enclosure 102 are all non-breathable. The fluid medium stream 212 sweeps through the growth chamber of the microfluidic device 500A and allows the waste components of the fluid medium 204 to diffuse out of the growth chamber (not shown) while allowing the components to diffuse from the fluid medium 202 in the flow channel 134 to In the growth room. Finally, waste fluid medium 202' (not shown) exits microfluidic device 500A via outlet 124' in outlet connection conduit 508.

在另一實施例中，流體培養基202經由泵連接導管504且透過可透氣塊518而轉移至微流體器件500B中，如圖5B中所展示。可透氣塊518併入至封閉體102之上表面中且形成封閉體102之上表面之一部分。由可透氣塊518形成之封閉體102之上表面之部分可位於微流體器件500B之生長室之上流。微流體器件500B容置於一室516內，室516提供經交換至微流體器件500B中之流體培養基中之一氣體環境(例如5%二氧化碳)。另外，室516可對微流體器件500B提供溫度及/或濕度 調節。泵連接導管504、封閉體102或基底104均為不可透氣的，且透過可透氣塊518之交換可充當微流體器件500B之「肺」且適當地調節微流體器件500B內之培養基。在此實施例中，流體培養基202可在裝載至泵514中之前於另一組件中經另外調節，且亦可因此含有(例如)來自一餵食細胞培養之分泌物質。 In another embodiment, fluid medium 202 is transferred to microfluidic device 500B via pump connection conduit 504 and through gas permeable block 518, as shown in Figure 5B. The gas permeable block 518 is incorporated into the upper surface of the enclosure 102 and forms a portion of the upper surface of the enclosure 102. Portions of the upper surface of the enclosure 102 formed by the gas permeable block 518 can be located above the growth chamber of the microfluidic device 500B. The microfluidic device 500B is housed in a chamber 516 that provides a gaseous environment (e.g., 5% carbon dioxide) that is exchanged into the fluid medium in the microfluidic device 500B. Additionally, chamber 516 can provide temperature and/or humidity to microfluidic device 500B. Adjustment. The pump connection conduit 504, closure 102 or substrate 104 are both non-breathable and exchangeable through the gas permeable block 518 can act as a "lung" for the microfluidic device 500B and properly condition the medium within the microfluidic device 500B. In this embodiment, the fluid medium 202 can be additionally adjusted in another component prior to loading into the pump 514, and can therefore also contain, for example, a secreted material from a fed cell culture.

在另一實施例中，可透氣塊整合至微流體器件500C之封閉體102之上表面以形成一可透氣區段518'，如圖5C中所展示。流體培養基可如上文針對圖5B之實施例所討論般經調節及經引入且可進一步包含來自一餵食細胞群體之分泌物質。微流體器件500C可容置於含有一氣體環境(例如空氣中5%二氧化碳)之一室516中。氣體環境可橫跨可透氣區段518'交換，可透氣區段518'可為封閉體102之上表面中之一個區段或複數個區段。室516可進一步適當調節器件500C之溫度及濕度。在此實施例中，泵連接導管504、封閉體102(除可透氣塊518'之外)及基底104可為不可透氣的。在一些實施例中，至少一可透氣區段518'定位於微流體器件500C之一生長室上方。在另一實施例中，至少一可透氣區段518'定位於微流體器件500C之流動區域134上方。在其他實施例中，可透氣區段518'可定位於至少一生長室及至少一流動區域134兩者上方。 In another embodiment, the gas permeable block is integrated into the upper surface of the enclosure 102 of the microfluidic device 500C to form a gas permeable section 518', as shown in Figure 5C. The fluid medium can be adjusted and introduced as discussed above for the embodiment of Figure 5B and can further comprise a secreted material from a feeding cell population. The microfluidic device 500C can be housed in a chamber 516 containing a gaseous environment, such as 5% carbon dioxide in air. The gaseous environment can be exchanged across the gas permeable section 518', which can be one or more of the upper surfaces of the enclosure 102. Chamber 516 can further appropriately adjust the temperature and humidity of device 500C. In this embodiment, the pump connection conduit 504, the enclosure 102 (other than the gas permeable block 518'), and the substrate 104 may be non-breathable. In some embodiments, at least one gas permeable section 518' is positioned above one of the growth chambers of the microfluidic device 500C. In another embodiment, at least one gas permeable section 518' is positioned above the flow area 134 of the microfluidic device 500C. In other embodiments, the gas permeable section 518' can be positioned over both the at least one growth chamber and the at least one flow region 134.

在一進一步實施例中，可透氣管道504'可用以在將培養基引入至微流體器件500D中之前調節(例如，平衡)流體培養基，如圖5D中所展示。可透氣管道504'之長度可經選擇以提供足以容許一封閉體516內之有效氣體交換及平衡之表面面積，封閉體516可含有諸如(但不限於)空氣中5%二氧化碳之一氣體環境。516之環境可進一步調節可透氣泵連接導管504'內之培養基之溫度及/或濕度。可用於可透氣連接導管之一可透氣材料之一非限制性實例係Teflon® AF。流體培養基可在引入至泵組件514之前藉由與一餵食細胞群體接觸而調節且由此可含 有可最佳化微流體器件500D中之(若干)受培養細胞之生長及/或存活性之分泌物質。使用餵食細胞群體之先前調節可發生於室516內或可執行於自身具有對溫度、濕度、pH及/或氣體環境之任何者之環境控制之另一培養組分中。在此實施例中，微流體器件500D之封閉體102及基底104可為不可透氣的。 In a further embodiment, the gas permeable conduit 504' can be used to condition (eg, equilibrate) the fluid culture medium prior to introduction of the culture medium into the microfluidic device 500D, as shown in Figure 5D. The length of the gas permeable conduit 504' can be selected to provide a surface area sufficient to permit efficient gas exchange and balance within an enclosure 516, which can contain a gaseous environment such as, but not limited to, 5% carbon dioxide in air. The environment of 516 can further adjust the temperature and/or humidity of the medium in the gas permeable pump connection conduit 504'. One non-limiting example of a breathable material that can be used for a gas permeable connecting conduit is Teflon® AF. The fluid medium can be adjusted prior to introduction to the pump assembly 514 by contact with a feeding cell population and can thereby be included There is a secretory material that optimizes the growth and/or viability of the cultured cells (several) in the microfluidic device 500D. Previous adjustments using a population of feeding cells can occur within chamber 516 or can be performed in another culture component that has its own environmental control for any of temperature, humidity, pH, and/or gaseous environments. In this embodiment, the enclosure 102 and substrate 104 of the microfluidic device 500D can be non-breathable.

在系統之儲液器、培養基調節及引入組分之又一實施例中，可在能夠放置於一適當氣體環境下之一儲液器502'中調節培養基，如圖5E中所展示。儲液器502'無需為一微流體器件或任何特定類型之培養組件。儲液器502'藉由自一氣體環境源524提供一連接供給526而放置於諸如(例如)空氣中5%二氧化碳之一適當氣體環境下。儲液器502'內之流體培養基具有與自源524提供之氣體環境之氣體交換，且藉此經調節。儲液器502'中之流體培養基亦可含有餵食細胞之一培養基來提供可最佳化微流體器件500E中之受培養細胞之生長及/或存活性之分泌物質。調節流體培養基可自儲液器502'經由轉移連接導管522(其連接至一泵514上之一閥520)而轉移且可經由連接導管504而由泵514注入至微流體器件500E之通道134中。注入至微流體器件500E中之流體培養基形成流體流212。在通過流動通道134之後，廢流體培養基202'經由出口導管508而退出微流體器件500E。在此實施例中，轉移連接導管522、連接導管504、閥520、泵514、封閉體102及基底104可全部為不可透氣的。在一些實施例中，將源524連接至儲液器502'之連接導管526可實質上為不可透氣的。在其他實施例中，連接導管526無需為實質上不可透氣的，而是可為相對不可透氣的。 In yet another embodiment of the system of reservoir, medium conditioning, and introduction of components, the medium can be adjusted in one of the reservoirs 502' that can be placed in a suitable gaseous environment, as shown in Figure 5E. The reservoir 502' need not be a microfluidic device or any particular type of culture assembly. The reservoir 502' is placed in a suitable gaseous environment such as, for example, 5% carbon dioxide in air by providing a connection supply 526 from a gaseous environment source 524. The fluid medium within the reservoir 502' has gas exchange with the gaseous environment provided by the source 524 and is thereby regulated. The fluid medium in the reservoir 502' may also contain a medium of the feeding cells to provide a secretory material that optimizes the growth and/or viability of the cultured cells in the microfluidic device 500E. The conditioning fluid medium can be transferred from the reservoir 502' via a transfer connection conduit 522 (which is coupled to one of the valves 520 on a pump 514) and can be injected by the pump 514 into the channel 134 of the microfluidic device 500E via the connection conduit 504. . The fluid medium injected into the microfluidic device 500E forms a fluid stream 212. After passing through the flow channel 134, the spent fluid medium 202' exits the microfluidic device 500E via the outlet conduit 508. In this embodiment, transfer connection conduit 522, connection conduit 504, valve 520, pump 514, enclosure 102, and substrate 104 may all be gas impermeable. In some embodiments, the connection conduit 526 that connects the source 524 to the reservoir 502' can be substantially gas impermeable. In other embodiments, the connecting conduit 526 need not be substantially gas impermeable, but may be relatively non-breathable.

在圖5E所展示之一些實施例中，氣體(圖中未展示)可為連續流動的或可為脈衝式的，例如，自源524輸入之週期性替換氣體(圖中未展示)可為空氣中5%二氧化碳。在其他實施例中，自源524輸入之氣體可為100%二氧化碳。當使用100%二氧化碳氣體時，少量二氧化碳氣 體可注入至儲液器502'之頂部空間(圖中未展示)中以使頂部空間維持一5%二氧化碳混合物。在一些實施例中，當氣體注入至儲液器502'之頂部空間中時，儲液器502'可進一步包含一風扇(圖中未展示)來使注入氣體與已存在於頂部空間(圖中未展示)中之其他氣體組分(圖中未展示)混合。在一些實施例中，當氣體之輸入係脈衝式時，微流體器件500E之蓋102可具有併入或附接於其內之二氧化碳感測器(圖中未展示)。在一些實施例中，100%二氧化碳氣體可自源524輸入以比使用市售空氣中5%二氧化碳氣體混合物節約成本。在其他實施例中，100%二氧化碳氣體可引入至源524中且與空氣混合以製備空氣中之一5%二氧化碳混合物。 In some embodiments shown in FIG. 5E, the gas (not shown) may be continuously flowing or may be pulsed, for example, a periodic replacement gas (not shown) input from source 524 may be air. 5% carbon dioxide. In other embodiments, the gas input from source 524 can be 100% carbon dioxide. When using 100% carbon dioxide gas, a small amount of carbon dioxide gas The body can be injected into the headspace (not shown) of the reservoir 502' to maintain a 5% carbon dioxide mixture in the headspace. In some embodiments, when gas is injected into the headspace of the reservoir 502', the reservoir 502' can further include a fan (not shown) to allow the injected gas to be present in the headspace (in the figure) Other gas components (not shown) are mixed in the undisplayed). In some embodiments, when the input of the gas is pulsed, the cover 102 of the microfluidic device 500E can have a carbon dioxide sensor (not shown) incorporated or attached thereto. In some embodiments, 100% carbon dioxide gas may be input from source 524 to save cost compared to using a 5% carbon dioxide gas mixture in commercial air. In other embodiments, 100% carbon dioxide gas can be introduced into source 524 and mixed with air to produce a 5% carbon dioxide mixture in air.

在上述實施例之任何者中，室516可進一步經加濕使得室之氣體環境不改變微流體器件及/或儲液器中之流體培養基之滲透壓。 In any of the above embodiments, chamber 516 may be further humidified such that the gaseous environment of the chamber does not alter the osmotic pressure of the fluid medium in the microfluidic device and/or reservoir.

在另一實施例中，對培養於生長室中之細胞提供適當氣體交換之一替代方法可提供氣體流動通過微流體器件(圖中未展示)之流動區域。適當氣體(例如5%二氧化碳)可直接泵抽或脈動透過流動通道。因為生長室之隔離區域經設計為大多數未被掃及體積，所以定位於隔離區域內之細胞不受移動通過流動通道(掃及區域)之空氣或氣泡干擾。此將提供流動通道中之氣體與生長室內之流體培養基之間的非常快速氣體交換，因為擴散距離相較於(例如)一50mL錐形管內之擴散距離而言係非常小的。接著，氣體可在任何選定時間量之後由流體培養基替換。氣體流動可依任何所要頻率重複以使溶解氣體組分保持一穩定濃度，該濃度亦對流體培養基之pH產生影響。替代地，非最佳氣體組合物或重複可用以擾動細胞之環境。 In another embodiment, an alternative method of providing suitable gas exchange for cells cultured in the growth chamber provides for flow of gas through the flow region of the microfluidic device (not shown). A suitable gas (eg 5% carbon dioxide) can be pumped or pulsed through the flow channel. Because the isolated areas of the growth chamber are designed to be largely unswept, the cells positioned within the isolated area are not disturbed by air or bubbles moving through the flow channels (sweeping areas). This will provide a very fast gas exchange between the gas in the flow channel and the fluid medium in the growth chamber because the diffusion distance is very small compared to, for example, the diffusion distance within a 50 mL conical tube. The gas can then be replaced by fluid medium after any selected amount of time. The gas flow can be repeated at any desired frequency to maintain the dissolved gas component at a stable concentration which also affects the pH of the fluid medium. Alternatively, the non-optimal gas composition or repetition can be used to disturb the environment of the cells.

總言之，存在可用以將調節培養基提供至本文中所描述之微流體器件之生長室中之細胞之各種組件及構形。微流體器件100、200、240、290或400之任何者可與圖5A至圖5E之實施例之任何者一起使 用。系統及套組可包含經構形以連接至微流體器件之入口及/或出口之連接導管。連接導管亦可經構形以連接至儲液器及/或泵組件。 In summary, there are various components and configurations that can be used to provide conditioning media to cells in the growth chamber of the microfluidic devices described herein. Any of the microfluidic devices 100, 200, 240, 290 or 400 can be combined with any of the embodiments of Figures 5A-5E use. The system and kit can include a connecting conduit configured to connect to an inlet and/or an outlet of the microfluidic device. The connecting conduit can also be configured to connect to the reservoir and/or pump assembly.

據此，提供一種用於培養一或多個生物細胞之微流體器件，其包含：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包括經調節以支援該微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之至少一表面，其中該至少一生長室包含一隔離區域及一連接區域，該隔離區域與該連接區域流體地連接且該連接區域包含至該流動區域之一近端開口。在各種實施例中，該微流體器件之該隔離區域可經構形以含有一第二流體培養基。當該流動區域及該至少一生長室實質上分別填充有該第一流體培養基及該第二流體培養基時，該第二流體培養基之組分可擴散至該第一流體培養基中及/或該第一流體培養基之組分可擴散至該第二流體培養基中；及該第一流體培養基實質上不流動至該隔離區域中。在各種實施例中，該至少一調節表面可經調節以支援該微流體器件內之該一或多個生物細胞之可移植性。在一些實施例中，該調節表面之部分可經構形以支援該微流體器件內之該等生物細胞之可移植性。 Accordingly, a microfluidic device for culturing one or more biological cells is provided, comprising: a flow region configured to flow with one of the first fluid media; and at least one growth chamber including the Adjusting to support at least one surface of cell growth, viability, portability, or any combination thereof, within the microfluidic device, wherein the at least one growth chamber comprises an isolation region and a connection region, the isolation region being coupled to the connection region The region is fluidly connected and the connection region includes a proximal opening to one of the flow regions. In various embodiments, the isolation region of the microfluidic device can be configured to contain a second fluid medium. When the flow area and the at least one growth chamber are substantially filled with the first fluid medium and the second fluid medium, respectively, components of the second fluid medium may diffuse into the first fluid medium and/or the first A component of a fluid medium can diffuse into the second fluid medium; and the first fluid medium does not substantially flow into the isolation region. In various embodiments, the at least one conditioning surface can be adjusted to support portability of the one or more biological cells within the microfluidic device. In some embodiments, portions of the conditioning surface can be configured to support the portability of the biological cells within the microfluidic device.

在一些實施例中，該微流體器件之該至少一調節表面可包含一聚合物，其包含伸烷基醚部分。在其他實施例中，該微流體器件之該至少一調節表面可包含一聚合物，其包括羧酸部分、磺酸部分、核酸部分或膦酸部分。在其他實施例中，該微流體器件之該至少一調節表面可包含一聚合物，其包含糖類部分。在一些實施例中，包含糖類部分之聚合物可為聚葡萄糖。在一些其他實施例中，該微流體器件之該至少一調節表面可包含一聚合物，其包括胺基酸部分。 In some embodiments, the at least one conditioning surface of the microfluidic device can comprise a polymer comprising an alkyl ether moiety. In other embodiments, the at least one conditioning surface of the microfluidic device can comprise a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety, or a phosphonic acid moiety. In other embodiments, the at least one conditioning surface of the microfluidic device can comprise a polymer comprising a carbohydrate moiety. In some embodiments, the polymer comprising a carbohydrate moiety can be polydextrose. In some other embodiments, the at least one conditioning surface of the microfluidic device can comprise a polymer comprising an amino acid moiety.

替代地，該微流體器件之該至少一調節表面可包含哺乳動物血清之一或多個組分。哺乳動物血清之該等組分可為一培養基之補充劑。在一些實施例中，該哺乳動物血清可為胎牛血清(FBS)或小牛血 清(FCS)。 Alternatively, the at least one conditioning surface of the microfluidic device can comprise one or more components of mammalian serum. These components of the mammalian serum can be a supplement to a medium. In some embodiments, the mammalian serum can be fetal bovine serum (FBS) or calf blood. Clear (FCS).

在該微流體器件之各種實施例中，該至少一調節表面可包含糖類部分。在一些實施例中，該至少一調節表面可包含伸烷基醚部分。在其他實施例中，該至少一調節表面可包含胺基酸部分。在一些其他實施例中，該至少一調節表面可包含烷基或全氟烷基部分。在一些實施例中，該等烷基或全氟烷基部分可具有大於10個碳之一主鏈長度。在一些實施例中，該至少一調節表面可包含一部分，其可為：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。 In various embodiments of the microfluidic device, the at least one conditioning surface can comprise a carbohydrate moiety. In some embodiments, the at least one conditioning surface can comprise an alkyl ether moiety. In other embodiments, the at least one conditioning surface can comprise an amino acid moiety. In some other embodiments, the at least one conditioning surface can comprise an alkyl or perfluoroalkyl moiety. In some embodiments, the alkyl or perfluoroalkyl moiety can have a backbone length of greater than 10 carbons. In some embodiments, the at least one conditioning surface can comprise a portion, which can be: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or polysaccharide (which can include, but is not limited to, polydextrose) An alcohol (including but not limited to, propargyl alcohol); a polyol comprising, but not limited to, a polyvinyl alcohol; an alkyl ether comprising, but not limited to, polyethylene glycol; a polyelectrolyte (which includes, but is not limited to, polyacrylic acid or polyvinylphosphonic acid); an amine group (which includes derivatives thereof such as, but not limited to, alkylated amine groups, hydroxyalkylated amine groups, phosphonium salts, and one containing no a heterocyclic group of an aromatized nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid comprising, but not limited to, a propiolic acid (which provides a carboxylate anion surface); a phosphonic acid , which includes, but is not limited to, ethynylphosphonic acid (which provides a phosphonate anion surface); a sulfonic acid anion; a carboxybetaine; a sulfobetaine; an amine sulfonic acid; or an amino acid.

在該微流體器件之各種實施例中，該至少一調節表面可包含共價鍵聯至該微流體器件之一表面之一鍵聯基團，且該鍵聯基團可鍵聯至經構形以支援該微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之一部分。在一些實施例中，該鍵聯基團可為矽烷氧基鍵聯基團。在其他實施例中，該鍵聯基團可為膦酸酯鍵聯基團。在一些實施例中，該鍵聯基團可直接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在一些實施例中，該調節表面之該部分可經構形以支援該微流體器件內之該等生物細胞之可移植性。在其他實施例中，該鍵聯基團可直接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在其他實施例 中，該鍵聯基團可經由一鍵聯子而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在各種實施例中，該鍵聯子可包含伸***基部分。 In various embodiments of the microfluidic device, the at least one conditioning surface can comprise a linkage group covalently bonded to one of the surfaces of the microfluidic device, and the linking group can be bonded to the conformation To support part of any combination of cell growth, viability, portability, or the like within the microfluidic device. In some embodiments, the linking group can be a decyloxy linking group. In other embodiments, the linking group can be a phosphonate linkage group. In some embodiments, the linking group can be directly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the portion of the conditioning surface can be configured to support the portability of the biological cells within the microfluidic device. In other embodiments, the linking group can be directly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. In other embodiments The linking group can be indirectly linked via a linkage to the portion configured to support cell growth, viability, portability, or any combination thereof. In various embodiments, the linker can comprise a triazolyl moiety.

在該微流體器件之各種實施例中，該至少一調節表面可包含兩性離子。在其他實施例中，該至少一調節表面可包含膦酸部分或羧酸部分。在其他實施例中，該調節表面可包含陰離子。在一些其他實施例中，該至少一調節表面可包含胺基或胍部分。在其他實施例中，該至少一調節表面可包含陽離子。 In various embodiments of the microfluidic device, the at least one conditioning surface can comprise zwitterions. In other embodiments, the at least one conditioning surface can comprise a phosphonic acid moiety or a carboxylic acid moiety. In other embodiments, the conditioning surface can comprise an anion. In some other embodiments, the at least one conditioning surface can comprise an amine or anthracene moiety. In other embodiments, the at least one conditioning surface can comprise a cation.

在該微流體器件之各種實施例中，該至少一調節表面可包含至少一細胞黏著阻斷分子。該至少一細胞黏著阻斷分子可破壞肌動蛋白纖維形成，阻斷整合素受體，或減弱細胞與DNA污損表面之結合。該至少一細胞黏著阻斷分子可為細胞鬆弛素B、一含RGD之肽或一DNase 1蛋白。在其他實施例中，該至少一細胞黏著阻斷分子可包含多於一種類型之細胞黏著阻斷分子之一組合。 In various embodiments of the microfluidic device, the at least one conditioning surface can comprise at least one cell adhesion blocking molecule. The at least one cell adhesion blocking molecule disrupts actin fiber formation, blocks integrin receptors, or attenuates binding of cells to DNA stained surfaces. The at least one cell adhesion blocking molecule can be cytochalasin B, an RGD-containing peptide or a DNase 1 protein. In other embodiments, the at least one cell adhesion blocking molecule can comprise a combination of one of more than one type of cell adhesion blocking molecule.

在該微流體器件之各種實施例中，該至少一調節表面經構形以被加熱至至少約30℃之一溫度。該至少一調節表面可經構形以穩定於至少約30℃之一溫度處。 In various embodiments of the microfluidic device, the at least one conditioning surface is configured to be heated to a temperature of at least about 30 °C. The at least one conditioning surface can be configured to stabilize at a temperature of at least about 30 °C.

在該微流體器件之各種實施例中，該微流體器件可進一步包含一微流體通道，其包括該流動區域之至少一部分。在一些實施例中，該至少一生長室之該連接區域可直接通至該微流體通道中。在一些實施例中，該微流體器件之該至少一生長室之該隔離區域可具有足以支援細胞擴增至約100個細胞之一範圍之尺寸。在一些實施例中，可在該至少一生長室中維持不超過1×10²個生物細胞。在一些實施例中，該至少一生長室之體積可小於或等於約2×10⁶立方微米。在其他實施例中，可在該至少一生長室中維持不超過1×10²個生物細胞，且該至少一生長室之體積可小於或等於約1×10⁷立方微米。 In various embodiments of the microfluidic device, the microfluidic device can further comprise a microfluidic channel comprising at least a portion of the flow region. In some embodiments, the connection region of the at least one growth chamber can pass directly into the microfluidic channel. In some embodiments, the isolated region of the at least one growth chamber of the microfluidic device can have a size sufficient to support cell expansion to a range of about 100 cells. In some embodiments, no more than 1 x 10 ² biological cells can be maintained in the at least one growth chamber. In some embodiments, the volume of the at least one of the growth chamber may be less than or equal to about 2 × 10 ⁶ cubic microns. In other embodiments, can be maintained is not more than 1 × 10 ² cells bio the at least one growth chamber and the growth chamber of a volume of at least be less than or equal to about 1 × 10 ⁷ cubic microns.

在該微流體器件之各種實施例中，該微流體器件可進一步包含經構形以將該第一流體培養基或該第二流體培養基輸入至該流動區域中之至少一入口及經構形以在該第一培養基自該流動區域退出時接收該第一培養基之至少一出口。 In various embodiments of the microfluidic device, the microfluidic device can further comprise a configuration to input the first fluid medium or the second fluid medium into at least one of the inlet regions and configured to The first medium receives at least one outlet of the first medium upon withdrawal from the flow region.

在該微流體器件之各種實施例中，該微流體器件可進一步包含具有一介電泳(DEP)構形之一基板，其經構形以將一或多個生物細胞引入至該生長室中或將該一或多個生物細胞移出該生長室。可光學地致動該DEP構形。 In various embodiments of the microfluidic device, the microfluidic device can further comprise a substrate having a dielectrophoretic (DEP) configuration configured to introduce one or more biological cells into the growth chamber or The one or more biological cells are removed from the growth chamber. The DEP configuration can be optically actuated.

在該微流體器件之各種實施例中，該微流體器件可進一步包含在該至少一生長室或其之該隔離區域上方之一可變形蓋區域，藉此按壓該可變形蓋區域施加足以使該生物細胞自該隔離區域輸出至該流動區域之一力。在一些實施例中，該微流體器件可包含一蓋，其中該蓋之至少一部分係可透氣的，藉此將一氣體分子源提供至定位於該微流體器件中之一流體培養基。在一些實施例中，該蓋之該可透氣部分可定位於該至少一生長室上方。在一些實施例中，該蓋之該可透氣部分可定位於該流動區域上方。在一些實施例中，該微流體器件可進一步包含在該至少一生長室或其之該隔離區域上方之一可變形蓋區域，藉此按壓該可變形蓋區域施加足以使該生物細胞自該隔離區域輸出至該流動區域之一力。 In various embodiments of the microfluidic device, the microfluidic device can further comprise a deformable cover region over the at least one growth chamber or the isolation region thereof, whereby pressing the deformable cover region is sufficient to cause the The biological cell outputs a force from the isolated region to the flow region. In some embodiments, the microfluidic device can include a lid, wherein at least a portion of the lid is gas permeable, thereby providing a source of gas molecules to a fluid medium positioned in the microfluidic device. In some embodiments, the gas permeable portion of the lid can be positioned over the at least one growth chamber. In some embodiments, the gas permeable portion of the lid can be positioned above the flow region. In some embodiments, the microfluidic device can further comprise a deformable cover region above the at least one growth chamber or the isolation region thereof, whereby pressing the deformable cover region is applied to isolate the biological cell from the isolation The zone outputs a force to one of the flow zones.

在該微流體器件之各種實施例中，該調節表面可包含一可裂解部分。該可裂解部分可經構形以容許破壞該調節表面，藉此促進該一或多個生物細胞在培養之後之可移植性。 In various embodiments of the microfluidic device, the conditioning surface can comprise a cleavable portion. The cleavable moiety can be configured to permit disruption of the modulating surface, thereby facilitating portability of the one or more biological cells after culture.

在該微流體器件之各種實施例中，該至少一生長室可包含複數個生長室。 In various embodiments of the microfluidic device, the at least one growth chamber can comprise a plurality of growth chambers.

在該微流體器件之各種實施例中，該一或多個生物細胞可包含複數個生物細胞。在一些實施例中，該一或多個生物細胞可包含一或 多個哺乳動物細胞。在一些實施例中，該一或多個生物細胞可包含一或多個融合瘤細胞。在一些實施例中，該一或多個生物細胞可包含一或多個淋巴細胞或白血球細胞。在其他實施例中，該一或多個生物細胞可包含一B細胞、一T細胞、NK細胞、一巨噬細胞或其等之一組合。在各種實施例中，該一或多個生物細胞可包含一或多個貼附細胞。在一些實施例中，該生長室中之該一或多個生物細胞可為一單一細胞且群落可為生物細胞之一選殖群落。 In various embodiments of the microfluidic device, the one or more biological cells can comprise a plurality of biological cells. In some embodiments, the one or more biological cells can comprise one or Multiple mammalian cells. In some embodiments, the one or more biological cells can comprise one or more fusion tumor cells. In some embodiments, the one or more biological cells can comprise one or more lymphocytes or white blood cells. In other embodiments, the one or more biological cells can comprise a B cell, a T cell, an NK cell, a macrophage, or a combination thereof. In various embodiments, the one or more biological cells can comprise one or more adherent cells. In some embodiments, the one or more biological cells in the growth chamber can be a single cell and the colony can be a colony of one of the biological cells.

pH感測器pH sensor

系統可進一步包含連接至微流體器件600(如圖6中所展示)之至少一入口124及/或至少一出口124'之至少一感測器。替代地，器件600可為器件100、200、240、290、400或500A至500E之任何者。感測器可經構形以在第一流體培養基進入微流體器件600時偵測其之一pH。替代地，感測器可經構形以在第一流體培養基退出微流體器件600時偵測其之一pH。感測器可併入至微流體器件中或其可為能夠附接至微流體器件之一入口124及/或一出口124'或與微流體器件之一入口124及/或一出口124'排成一行之一分離組件。 The system can further include at least one sensor coupled to at least one inlet 124 and/or at least one outlet 124' of the microfluidic device 600 (as shown in FIG. 6). Alternatively, device 600 can be any of devices 100, 200, 240, 290, 400 or 500A through 500E. The sensor can be configured to detect one of the pH of the first fluid medium as it enters the microfluidic device 600. Alternatively, the sensor can be configured to detect one of the pH of the first fluid medium as it exits the microfluidic device 600. The sensor can be incorporated into the microfluidic device or it can be attachable to one of the inlets 124 and/or an outlet 124' of the microfluidic device or to one of the inlets 124 and/or one of the outlets 124' of the microfluidic device Separate components in one line.

在一些實施例中，pH感測器係一光學感測器。一光學感測器可提供相較於基於電極之台式裝置之一優點，此係因為基於電極之裝置可包含龐大探針以使小量(微升)流體之pH難以或無法量測。類似地，線上流通溶液可歸因於微電極之特性而具有非常長沈降時間(5分鐘至15分鐘)，且可在各使用之前需要外延校準程序。此外，電極會快速劣化以因此需要更多維護。 In some embodiments, the pH sensor is an optical sensor. An optical sensor can provide an advantage over an electrode-based benchtop device because the electrode-based device can include bulky probes to make the pH of a small amount (microliter) of fluid difficult or impossible to measure. Similarly, the in-line flow solution can have very long settling times (5 minutes to 15 minutes) due to the characteristics of the microelectrodes, and an epitaxial calibration procedure can be required prior to each use. In addition, the electrodes deteriorate rapidly so that more maintenance is required.

光學感測器可為一整合無電極器件，其包含用於照明之一LED及用於可見色彩偵測之一整合比色感測器。該比色感測器可為一色敏光電晶體。該比色感測器可在可見光波長區域(例如約390奈米至約700奈米)中偵測。經一pH相依染料(諸如(但不限於)酚紅)染色之培養基可 提供瞬間且無接觸之光學信號。使用此一光學感測器之一光學無電極量測方法無需與培養基接觸且無需對使用者部分進行校準。光學量測可經校準以消除溫度相依性。另外，一光學感測器之使用最小化使感測器污損之風險以因此減少維護或替換。光源(LED)及色彩感測器之小型化亦使此經得起測試非常小體積液體(<1微升)及整合至可攜式或手持式儀器中之檢驗。系統可包含藉由LED及光電晶體感測器之控制/監測設備180之驅動電子器件，且可在pH之偵測判定pH在所要範圍外時由控制模組172進一步提供一報警組件。另外，由於色彩偵測之安定時間較快(亞秒)，所以可將此感測器***於一回饋迴路中以經由調變培養基周圍之一氣體環境中之二氧化碳含量而調節培養基之pH。替代地，控制模組172或控制/監測設備180可進一步提供組件以藉由添加緩衝液及/或酸性或鹼性培養基組分而調變引入流體培養基之pH以將pH校正回所要範圍。 The optical sensor can be an integrated electrodeless device that includes one of the LEDs for illumination and one integrated colorimetric sensor for visible color detection. The colorimetric sensor can be a color-sensitive photoelectric crystal. The colorimetric sensor can be detected in the visible wavelength region (eg, from about 390 nm to about 700 nm). A medium dyed with a pH dependent dye such as, but not limited to, phenol red Provides an instantaneous, contactless optical signal. One of the optical sensors used in this optical sensor does not require contact with the culture medium and does not require calibration of the user portion. Optical measurements can be calibrated to eliminate temperature dependence. In addition, the use of an optical sensor minimizes the risk of fouling the sensor to thereby reduce maintenance or replacement. The miniaturization of the light source (LED) and color sensor also allows for the testing of very small volumes of liquid (<1 microliter) and integration into portable or handheld instruments. The system can include drive electronics for the control/monitoring device 180 of the LED and optoelectronic sensor, and can further provide an alarm component by the control module 172 when the pH is detected to be outside the desired range. In addition, since the color detection stabilization time is faster (sub-second), the sensor can be inserted into a feedback loop to adjust the pH of the medium via the carbon dioxide content in a gaseous environment surrounding the modulation medium. Alternatively, control module 172 or control/monitoring device 180 may further provide components to modulate the pH of the introduced fluid medium to adjust the pH back to the desired range by adding a buffer and/or an acidic or alkaline medium component.

在一些實施例中，感測器610連接至流體培養基入口管道606，接近於微流體器件之至少一入口124。管道606可為透明、實質上透明或半透明的。LED 614照射管道606及管道606內之染色流體培養基202a'。整合比色感測器612可監測引入流體培養基之pH，確定pH具有一特定培養實驗之一所要範圍內之一值，且在pH超出該所要範圍時報警。 In some embodiments, the sensor 610 is coupled to the fluid medium inlet conduit 606 proximate to at least one inlet 124 of the microfluidic device. The conduit 606 can be transparent, substantially transparent, or translucent. LED 614 illuminates conduit 606 and dye fluid medium 202a' within conduit 606. The integrated colorimetric sensor 612 can monitor the pH of the fluid medium introduced, determine that the pH has one of the desired ranges for one of the particular culture experiments, and alert the pH when the pH is outside of the desired range.

在一些實施例中，感測器610'連接至流體培養基出口管道608，接近於微流體器件之至少一出口124'。管道608可為透明、實質上透明或半透明的。LED 614'照射管道608及管道608內之染色出流流體培養基202a"。整合比色感測器612'可監測引入流體培養基之pH，確定pH具有一特定培養實驗之一所要範圍內之一值，且在pH超出該所要範圍時報警。 In some embodiments, the sensor 610' is coupled to the fluid medium outlet conduit 608 proximate to at least one outlet 124' of the microfluidic device. The conduit 608 can be transparent, substantially transparent, or translucent. The LED 614' illuminates the dyed outflow fluid medium 202a" in the conduit 608 and the conduit 608. The integrated colorimetric sensor 612' monitors the pH of the fluid medium introduced and determines that the pH has a value within one of the desired ranges for a particular culture experiment. And alarm when the pH exceeds the desired range.

細胞cell

能夠用於本發明之系統及方法中之一細胞可為任何類型之細胞。例如，細胞可為一胚胎細胞、卵母細胞或***細胞、幹細胞、先驅細胞或自一組織解離之一細胞、一血細胞、一融合瘤細胞、一培養細胞、來自一細胞系之一細胞、一癌細胞、一受感染細胞、一轉染及/或轉形細胞(細胞系(其包含(但不限於)中國倉鼠卵巢(CHO)細胞)、一報導體細胞或其類似者。細胞可為一哺乳動物細胞或細胞可為非哺乳動物細胞。細胞可包含一細菌、一真菌、一原生動物或受一寄生物種(例如，利什曼原蟲或惡性瘧原蟲)感染之一哺乳動物細胞。在一些實施例中，哺乳動物細胞可為人類、鼠類、豬或所關注之任何其他哺乳動物。 One of the cells and methods that can be used in the present invention can be any type of cell. For example, the cell can be an embryonic cell, an oocyte or a sperm cell, a stem cell, a precursor cell or a cell dissociated from a tissue, a blood cell, a fusion tumor cell, a culture cell, a cell from a cell line, a cell Cancer cells, an infected cell, a transfected and/or transformed cell (cell line (including but not limited to) Chinese hamster ovary (CHO) cells), a reporter cell or the like. The cell can be a The mammalian cell or cell can be a non-mammalian cell. The cell can comprise a bacterium, a fungus, a protozoan or a mammalian cell infected with a parasitic species (eg, Leishmania or Plasmodium falciparum). In some embodiments, the mammalian cell can be a human, a murine, a pig, or any other mammal of interest.

在一些實施例中，細胞可來自在培養基中積極生長之一細胞群體或獲得自一新鮮組織樣本(例如，藉由一固體組織樣本之解離，諸如一活體組織檢查或細針抽吸)、血液、唾液、尿液或其他體液。替代地，一或多個生物細胞可來自先前冷凍之其他樣本之一培養基。 In some embodiments, the cells may be derived from a population of cells actively growing in the culture medium or obtained from a fresh tissue sample (eg, by dissociation of a solid tissue sample, such as a biopsy or fine needle aspiration), blood. , saliva, urine or other body fluids. Alternatively, one or more biological cells may be from one of the other samples previously frozen.

在一些實施例中，一或多個生物細胞可包含一或多個融合瘤細胞。在其他實施例中，一或多個生物細胞可包含一或多個淋巴細胞或白血球細胞。在一些實施例中，細胞係一B細胞、一T細胞、一NK細胞、一樹突細胞、一巨噬細胞或其他免疫細胞類型或其等之一前驅體，諸如一先驅細胞或一造血幹細胞。 In some embodiments, one or more biological cells can comprise one or more fusion tumor cells. In other embodiments, one or more biological cells can comprise one or more lymphocytes or white blood cells. In some embodiments, the cell line is a B cell, a T cell, an NK cell, a dendritic cell, a macrophage or other immune cell type or a precursor thereof, such as a precursor cell or a hematopoietic stem cell.

在各種實施例中，一或多個生物細胞係一或多個貼附細胞。當將一或多個貼附細胞引入至微流體器件時，額外調節處理可經提供以對貼附細胞提供適當可溶或不可溶環境因子(例如一或多個細胞外間質組分)以容許持久存活及/或細胞增殖。 In various embodiments, one or more biological cell lines are one or more attached cells. When one or more adherent cells are introduced into the microfluidic device, additional conditioning treatments can be provided to provide the attached cells with a suitable soluble or insoluble environmental factor (eg, one or more extracellular interstitial components). Allow for long-lasting survival and/or cell proliferation.

取決於實驗之特定目標，可將僅一個細胞或複數個細胞引入至微流體器件中來培養及/或選殖。當僅一個細胞被引入至系統之一生長室中且根據本文中所描述之方法而培育時，所得擴增群體係最初引 入至該生長室中之該細胞之一選殖群落。 Depending on the particular goal of the experiment, only one cell or multiple cells can be introduced into the microfluidic device for cultivation and/or colonization. When only one cell is introduced into a growth chamber of the system and incubated according to the methods described herein, the resulting amplified population system is initially introduced One of the cells entering the growth chamber is colonized.

方法method

本發明提供一種用於在一系統中培養至少一生物細胞之方法，該系統包含具有至少一生長室及一流動區域之一微流體器件。在亦具有一流動區域之一微流體器件之一生長室中培養一(或若干)細胞可允許營養素、生長因子或其他細胞傳信物種在選定時間週期特定引入以達成細胞生長、存活性或可移植性參數之控制。將該至少一生物細胞引入至具有至少一調節表面之該至少一生長室中，其中該調節表面支援細胞生長、存活性、可移植性或其等之任何組合。在一些實施例中，該調節表面支援該微流體器件內之細胞可移植性。在一些實施例中，可移植性包含防止細胞黏著至該微流體器件。在其他實施例中，可移植性包含對貼附細胞提供將支援細胞生長、存活性、可移植性或其等之任何組合之一調節表面，同時亦允許在一培養週期之後於該微流體器件內移動細胞。該至少一調節表面可為如本文中所描述之任何調節表面。可使用如本文中所描述之數種不同動力來完成該至少一生物細胞之引入，該等動力之部分可容許對將一特定生物細胞放置至該微流體器件上之一特定位置中(例如，放置至一預選定生長室中)進行精確控制。可由本文中所描述之方法完成之細胞放置/移除及營養素/傳信/環境刺激之精確控制難以或無法使用大尺度或其他微流體培養方法來達成。 The present invention provides a method for culturing at least one biological cell in a system comprising a microfluidic device having at least one growth chamber and a flow region. Incubating one (or several) cells in a growth chamber that also has one of the microfluidic devices in a flow region may allow for the specific introduction of nutrients, growth factors, or other cell signaling species over a selected time period to achieve cell growth, viability, or Control of transplantability parameters. The at least one biological cell is introduced into the at least one growth chamber having at least one conditioning surface, wherein the conditioning surface supports cell growth, viability, portability, or any combination thereof. In some embodiments, the conditioning surface supports cell portability within the microfluidic device. In some embodiments, portability comprises preventing cells from adhering to the microfluidic device. In other embodiments, portability comprises modulating the surface of the attached cells to provide support for cell growth, viability, portability, or the like, while also permitting the microfluidic device after a culture period. Move cells inside. The at least one conditioning surface can be any conditioning surface as described herein. The introduction of the at least one biological cell can be accomplished using a plurality of different kinetics as described herein, the portion of the motility permitting placement of a particular biological cell into a particular location on the microfluidic device (eg, Place it in a pre-selected growth chamber for precise control. Accurate control of cell placement/removal and nutrient/signal/environmental stimulation that can be accomplished by the methods described herein is difficult or impossible to achieve using large scale or other microfluidic culture methods.

在放置之後，在至少足以使該至少一生物細胞擴增而產生生物細胞之一群落之一長時間週期內培育該至少一生物細胞。當將生物細胞引入至分離生長室中時，所得擴增群落可精確地經識別以進一步用作生物細胞之可分離群組。當將僅一個生物細胞引入至一生長室且允許該生物細胞擴增時，所得群落係生物細胞之一選殖群體。任何適當細胞(其包含(但不限於)如上文所描述之細胞)可用於該等方法中。 After placement, the at least one biological cell is incubated for a prolonged period of time that is at least sufficient to cause the at least one biological cell to expand to produce one of the biological cells. When biological cells are introduced into the isolated growth chamber, the resulting expanded population can be accurately identified for further use as a separable group of biological cells. When only one biological cell is introduced into a growth chamber and the biological cell is allowed to expand, the resulting community is one of the colonized population of biological cells. Any suitable cell, including but not limited to cells as described above, can be used in such methods.

該微流體器件可為如本文中所描述之微流體器件100、300、400、500A至500E或600之任何者，且該微流體器件可為具有如本文中所描述之組件之任何者之一系統之部分。該至少一生長室可包含複數個生長室，且可使用如本文中所討論之任何適合數目個生長室。在該等方法之一些實施例中，該微流體器件可具有約500個至約1500個生長室、約1000個至約2000個生長室、約1000個至約3500個生長室、約2000個至約5000個生長室、約3000個至約7000個生長室、約5000個至約10000個生長室、約7500個至約15000個生長室、約10000個至約17500個生長室或約12500個至約20000個生長室。 The microfluidic device can be any of the microfluidic devices 100, 300, 400, 500A to 500E or 600 as described herein, and the microfluidic device can be any of the components having the components as described herein. Part of the system. The at least one growth chamber can comprise a plurality of growth chambers, and any suitable number of growth chambers as discussed herein can be used. In some embodiments of the methods, the microfluidic device can have from about 500 to about 1500 growth chambers, from about 1000 to about 2000 growth chambers, from about 1000 to about 3500 growth chambers, from about 2000 to About 5,000 growth chambers, from about 3,000 to about 7,000 growth chambers, from about 5,000 to about 10,000 growth chambers, from about 7,500 to about 15,000 growth chambers, from about 10,000 to about 17,500 growth chambers or from about 12,500 to About 20,000 growth chambers.

在培養一或多個生物細胞之方法中，該至少一調節表面可為如本文中所描述之任何調節表面。該調節表面可共價鍵聯至該微流體器件。在一些實施例中，該調節表面可包含共價鍵聯至該表面之一鍵聯基團，且該鍵聯基團亦可鍵聯至經構形以支援該微流體器件內之該一或多個生物細胞之細胞生長、存活性、可移植性或其等之任何組合之一部分。在一些實施例中，可在輸入該一或多個生物細胞之前提供具有一調節表面之一微流體器件。 In methods of culturing one or more biological cells, the at least one conditioning surface can be any conditioning surface as described herein. The conditioning surface can be covalently bonded to the microfluidic device. In some embodiments, the conditioning surface can comprise a covalently bonded to one of the bonding groups on the surface, and the linking group can also be bonded to the conformation to support the one or Part of any combination of cell growth, viability, portability, or the like of a plurality of biological cells. In some embodiments, a microfluidic device having one of the conditioning surfaces can be provided prior to inputting the one or more biological cells.

引入至少一生物細胞Introducing at least one biological cell

在一些實施例中，將該至少一生物細胞引入至該至少一生長室中可包含：使用具有足以移動該至少一生物細胞之力量之一介電泳(DEP)力。可使用電鑷子(諸如光電鑷子(OET))來產生該DEP力。在一些其他實施例中，將一或多個生物細胞引入至該至少一生長室中可包含：使用流體流動及/或重力(例如，藉由使該微流體器件傾斜，使得該(等)細胞降落至定位於該(等)細胞下方之一生長室中)。 In some embodiments, introducing the at least one biological cell into the at least one growth chamber can comprise: using a dielectrophoresis (DEP) force having a force sufficient to move the at least one biological cell. Electrical forceps, such as photoelectric tweezers (OET), can be used to generate the DEP force. In some other embodiments, introducing one or more biological cells into the at least one growth chamber can comprise: using fluid flow and/or gravity (eg, by tilting the microfluidic device such that the (etc) cells Landing into a growth chamber positioned below the cell.

在一些實施例中，透過至該微流體器件之一流動區域(例如流動通道)中之一入口124而將該至少一生物細胞引入至該微流體器件中。該流動通道中之培養基流可將該細胞運送至接近於一生長室之一開口 之一位置。在將該生物細胞定位於一生長室之一開口接近處之後，可使用本文中所描述之動力(其包含介電泳或重力)之任何者來將該生物細胞移動至該生長室中。介電泳力可包含電致動力或光學致動力，且該等DEP力可進一步由光電鑷子(OET)提供。該至少一生物細胞可移動通過該流動通道而至至少一生長室之一連接區域之近端開口，其中該連接區域直接通至該流動通道/區域且流體地連接至該流動通道/區域。該至少一生長室之該連接區域亦流體地連接至該至少一生長室之一隔離區域。該至少一生物細胞可進一步移動通過該連接區域而至該至少一生長室之該隔離區域中。該至少一生長室之該隔離區域可具有足以支援細胞擴增之尺寸。然而，通常，該生長室之尺寸會將此擴增限制於不超過約1×10³個、約5×10²個、約4×10²個、約3×10²個、約2×10²個、約1×10²個、約50個、約25個、約15個或甚至僅10個培養細胞。在一些實施例中，該隔離區域可具有足以支援細胞擴增至不超過約1×10²個、約50個、約25個、約15個或約10個培養細胞之尺寸。吾人已驚奇地發現，可在具有不超過下列各者之一體積之一隔離區域中成功地執行高達約1×10²個細胞之細胞培育及/或擴增：約1.0×10⁷立方微米、約6×10⁶立方微米、約2×10⁶立方微米、約1.5×10⁶立方微米或約1.0×10⁶立方微米。在一些其他實施例中，可在具有不超過約4×10⁵立方微米之一體積之一隔離區域中成功地執行高達約1×10²個細胞之細胞培育及/或擴增。該生物細胞之大小可取決於細胞類型而自下列各者大幅變動：具有約1微米之一直徑及約1立方微米之一體積之細菌、具有約7微米至約8微米之一直徑及約100立方微米之一體積之一小人類細胞(諸如一血紅細胞)、具有約40微米(非匯合)之一直徑及約2000立方微米之一體積之一永生化細胞系(諸如海拉細胞(HeLa))、具有約25微米至高達約60微米之一直徑及約4700立方微米至約100,000立方微米之一體積之一巨核細胞、或具有約120微米 之一直徑及約900,000立方微米之一體積之一人類卵細胞。據此，具有約4×10⁵立方微米之一體積之一生長室可容許較大品種之巨核細胞(約1×10⁵立方微米之體積)擴增非常少，例如，總共至多不到5個細胞。替代地，相同小生長室(約4×10⁵立方微米之體積)可容許細菌細胞(約1立方微米之體積)擴增至約400,000個細菌細胞。 In some embodiments, the at least one biological cell is introduced into the microfluidic device through one of the inlets 124 in one of the flow regions (eg, flow channels) of the microfluidic device. The flow of medium in the flow channel can transport the cells to a location close to one of the openings of one of the growth chambers. After positioning the biological cell in proximity to one of the openings of a growth chamber, any of the kinetics described herein, including dielectrophoresis or gravity, can be used to move the biological cell into the growth chamber. Dielectrophoretic forces may include electrodynamic or optical actuation, and such DEP forces may be further provided by photoelectric tweezers (OET). The at least one biological cell is movable through the flow channel to a proximal opening of a connection region of at least one growth chamber, wherein the connection region is directly connected to the flow channel/region and is fluidly connected to the flow channel/region. The connection region of the at least one growth chamber is also fluidly coupled to one of the at least one growth chamber isolation regions. The at least one biological cell can be further moved through the connection region to the isolated region of the at least one growth chamber. The isolated region of the at least one growth chamber can have a size sufficient to support cell expansion. However, in general, the size of the growth chamber limits this amplification to no more than about 1 x 10 ³ , about 5 x 10 ² , about 4 x 10 ² , about 3 x 10 ² , about 2 x 10 ² , about 1 x 10 ² , about 50, about 25, about 15, or even only 10 cultured cells. In some embodiments, the isolated region can be of a size sufficient to support cell expansion to no more than about 1 x 10 ² , about 50, about 25, about 15 or about 10 cultured cells. I have surprisingly found, may have no more than one of each by volume of one of the following isolation region is successfully performed up to about 1 × 10 ² cells and the cells incubated / or amplification: about 1.0 × 10 ⁷ cubic microns, About 6 x 10 ⁶ cubic microns, about 2 x 10 ⁶ cubic microns, about 1.5 x 10 ⁶ cubic microns, or about 1.0 x 10 ⁶ cubic microns. In some other embodiments, one may have no more than about one volume of the isolation region 4 × 10 ⁵ cubic microns is successfully performed up to about 1 × 10 ² cells and the cells incubated / or amplification. The size of the biological cell can vary widely depending on the cell type: bacteria having a diameter of about 1 micron and a volume of about 1 cubic micron, having a diameter of about 7 micrometers to about 8 micrometers, and about 100 One of cubic micrometers of small human cells (such as a red blood cell), one of about 40 micrometers (non-confluent) diameter, and one of about 2000 cubic micrometers of immortalized cell line (such as HeLa cells). a megakaryocyte having a diameter of from about 25 microns up to about 60 microns and a volume of from about 4700 cubic microns to about 100,000 cubic microns, or one having a diameter of about 120 microns and a volume of about 900,000 cubic microns Human egg cells. Accordingly, a growth chamber having a volume of about 4 × 10 ⁵ cubic micrometers can allow a large variety of megakaryocytes (a volume of about 1 × 10 ⁵ cubic micrometers) to be expanded very little, for example, up to less than 5 in total. cell. Alternatively, the same growth chamber small (about 4 × 10 ⁵ volumes of cubic microns) allowable bacterial cell (about 1 volume of cubic microns) to about 400,000 amplified bacterial cells.

方法可進一步包含：將一第一流體培養基引入至該微流體器件之該流動區域之一微流體通道中。在一些實施例中，在引入該至少一生物細胞之前執行引入該第一流體培養基。當在引入該至少一生物細胞之前引入該第一流體培養基時，一流動速率可經選擇使得該第一流體培養基(例如)依任何適合速率自該微流體器件之該流動通道流動至該生長室中。替代地，若該微流體器件已預充有含有過多之一或多個調節試劑之一培養基，則該第一流體培養基依一速率流動至該微流體通道中，使得該第一流體培養基替換該流動區域中之含有(若干)過多調節試劑之任何剩餘培養基。 The method can further comprise introducing a first fluid medium into the microfluidic channel of the flow region of the microfluidic device. In some embodiments, introducing the first fluid medium is performed prior to introducing the at least one biological cell. When the first fluid medium is introduced prior to introduction of the at least one biological cell, a flow rate can be selected such that the first fluid medium flows, for example, from the flow channel of the microfluidic device to the growth chamber at any suitable rate in. Alternatively, if the microfluidic device has been prefilled with a medium containing one or more conditioning agents, the first fluid medium flows into the microfluidic channel at a rate such that the first fluid medium replaces the Any remaining medium in the flow zone containing (several) excess conditioning reagents.

當在將該至少一生物細胞引入至該生長室之後引入該第一流體培養基之流時，該第一流體培養基之流動速率可經選擇以不掃及該隔離區域，其不會使該至少一生物細胞自該隔離區域位移。包圍該至少一生長室之該隔離區域中之該至少一生物細胞的流體培養基係第二流體培養基，其可相同於或不同於該第一流體培養基。在一些實施例中，該第二流體培養基可相同於該第一流體培養基，但在培育步驟期間，細胞廢棄物及耗乏培養基組分可使該第二流體培養基表現為不同於該第一流體培養基。 When the flow of the first fluid medium is introduced after introducing the at least one biological cell into the growth chamber, the flow rate of the first fluid medium may be selected so as not to sweep the isolation region, which does not cause the at least one The biological cells are displaced from the isolated area. The fluid medium surrounding the at least one biological cell in the isolated region of the at least one growth chamber is a second fluid medium that may be the same as or different from the first fluid medium. In some embodiments, the second fluid medium can be the same as the first fluid medium, but during the incubating step, the cell waste and the spent medium component can cause the second fluid medium to behave differently than the first fluid Medium.

培育細胞Nurturing cells

在本文所描述之方法中，在至少足以擴增至少一生物細胞而產生生物細胞之一群落之一長時間週期內培育細胞。該時間週期可經選擇為自約1天至約10天。在其他實施例中，培育週期可延長至超過10 天且可延續任何所要週期。由於生長室之隔離區域中之細胞被提供營養素且具有藉由灌注流體培養基而移除之廢棄物，所以細胞可無限期地生長。因為隔離區域填充有擴增細胞群體，所以任何額外擴增將導致擴增生物細胞棲息於生長室之連接區域，連接區域係生長室之一掃及區域。灌注培養基可使擴增生物細胞自生長室之連接區域且隨後自微流體器件掃出。據此，可使存在於生長室之隔離區域中之細胞之數目穩定於取決於生物細胞之大小及生長室之隔離區域之大小的一最大數目處。使一隔離細胞群體中之細胞之最大數目穩定之能力提供相較於細胞培養之其他當前可用方法之一優點，此係因為可消除冗長細胞群體***。 In the methods described herein, the cells are incubated for a prolonged period of time at least one of the colonies that are at least sufficient to amplify at least one biological cell to produce a biological cell. The time period can be selected from about 1 day to about 10 days. In other embodiments, the incubation period can be extended to over 10 It can continue any desired cycle. Since the cells in the isolated area of the growth chamber are provided with nutrients and have waste removed by perfusion of the fluid medium, the cells can grow indefinitely. Because the isolated region is filled with a population of expanded cells, any additional amplification will result in the augmented biological cells inhabiting the connection region of the growth chamber, which is one of the growth chambers sweeping the region. The perfusion medium allows the expanded biological cells to be swept out of the connection region of the growth chamber and subsequently from the microfluidic device. Accordingly, the number of cells present in the isolated region of the growth chamber can be stabilized at a maximum number depending on the size of the biological cells and the size of the isolated regions of the growth chamber. The ability to stabilize the maximum number of cells in a population of isolated cells provides one of the advantages over other currently available methods of cell culture because it eliminates severance of cell population division.

在一些實施例中，可實施培育達約1天、約2天、約3天、約4天、約5天、約6天、約7天、約8天、約9天、約10天或10天以上。培育週期可在下列範圍內：自約1天至約6天、自約1天至約5天、自約1天至約4天、自約1天至約3天或自約1天至約2天。在其他實施例中，可實施培育達少於約5天、少於約4天、少於約3天或少於約2天。在一些實施例中，可實施培育達少於約3天或少於約2天。在其他實施例中，可實施培育達約3小時、約4小時、約5小時、約6小時、約7小時、約8小時、約9小時、約10小時、約11小時、約12小時、約13小時、約14小時、約15小時、約16小時、約17小時、約18小時、約19小時、約20小時、約21小時、約22小時或約23小時。 In some embodiments, incubation can be performed for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or More than 10 days. The incubation period can be in the range of from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, or from about 1 day to about 2 days. In other embodiments, the incubation can be carried out for less than about 5 days, less than about 4 days, less than about 3 days, or less than about 2 days. In some embodiments, incubation can be performed for less than about 3 days or less than about 2 days. In other embodiments, incubation can be carried out for about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, About 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, or about 23 hours.

在培養步驟期間，可在整個培養步驟之一或多個時間點處監測至少一生長室及其內所含之任何細胞之一影像。可將該影像儲存於系統之一處理組件之記憶體中。 During the culturing step, at least one growth chamber and one of the images of any cells contained therein can be monitored at one or more time points throughout the culturing step. The image can be stored in the memory of one of the processing components of the system.

灌注細胞Perfusion cell

在培育步驟期間，存在於生長室之隔離區域內之第二流體培養基可變成耗乏營養素、生長因子或其他生長刺激劑。第二流體培養基 可累積細胞廢棄物。另外，因為至少一生物細胞在培育週期期間不斷生長，所以可期望在培育開始時將營養素、生長因子或其他生長刺激劑更改為不同於第一培養基或第二培養基之營養素、生長因子或其他生長刺激劑。如本文中所描述，一微流體器件之一生長室中之培養可給予引入及更改由至少一生物細胞感測之化學梯度(其可更密切接近活體內條件)之特定及選擇性能力。替代地，將由至少一生物細胞感測之化學梯度有意更改為非最佳化條件組可容許細胞在經設計以探索疾病或治療路徑之條件下擴增。因此，方法可包含：在培育步驟期間灌注第一流體培養基，其中經由微流體器件之至少一入口124而引入第一流體培養基且其中經由微流體器件之至少一出口而輸出視情況包括來自第二流體培養基之組分之第一流體培養基。 During the incubation step, the second fluid medium present in the isolated region of the growth chamber can become depleted of nutrients, growth factors or other growth stimulators. Second fluid medium Cellular waste can be accumulated. In addition, since at least one biological cell continues to grow during the incubation period, it may be desirable to change the nutrient, growth factor or other growth stimulating agent to a nutrient, growth factor or other growth different from the first or second medium at the beginning of the incubation. Stimulant. As described herein, culture in a growth chamber of a microfluidic device can impart specific and selective capabilities to introduce and modify chemical gradients sensed by at least one biological cell that can be more closely related to in vivo conditions. Alternatively, intentionally changing the chemical gradient sensed by at least one biological cell to a non-optimized condition set may allow the cell to be expanded under conditions designed to explore the disease or treatment pathway. Accordingly, the method can include: priming the first fluid medium during the incubating step, wherein the first fluid medium is introduced via at least one inlet 124 of the microfluidic device and wherein the output is via the at least one outlet of the microfluidic device, optionally from the second A first fluid medium of a component of the fluid medium.

交換第一流體培養基之組分以藉此提供新鮮營養素、可溶生長因子及其類似者及/或交換包圍隔離區域內之(若干)細胞之培養基之廢棄組分發生於實質上在擴散條件下之生長室之掃及區域與未掃及區域之界面處。吾人已驚奇地發現，有效交換產生於實質上無流動條件下。據此，吾人已驚人地發現，成功培育無需連續不斷灌注。因此，灌注可為非連續的。在一些實施例中，灌注係週期性的，且在一些實施例中，灌注係不規律的。灌注週期之間的間斷可具有足以容許隔離區域中之第二流體培養基之組分擴散至流動通道/區域中之第一流體培養基中及/或第一流體培養基之組分擴散至第二流體培養基中之持續時間，第一培養基實質上均不流動至隔離區域中。 Exchanging components of the first fluid medium to thereby provide fresh nutrients, soluble growth factors and the like and/or exchanging waste components of the medium surrounding the cells(s) in the isolated region occurs substantially under diffusion conditions The sweep of the growth chamber and the interface between the area and the unswept area. We have surprisingly found that efficient exchange results from substantially no flow conditions. Based on this, we have surprisingly found that successful cultivation does not require continuous perfusion. Therefore, the perfusion can be discontinuous. In some embodiments, the perfusion is periodic, and in some embodiments, the perfusion is irregular. The discontinuity between the perfusion cycles may be sufficient to allow diffusion of components of the second fluid medium in the isolation region into the first fluid medium in the flow channel/region and/or diffusion of components of the first fluid medium to the second fluid medium For the duration of the medium, the first medium does not substantially flow into the isolated area.

在另一實施例中，低灌注速率亦可用以獲得生長室之未掃及區域內及生長室之未掃及區域外之流體培養基之組分之有效交換。 In another embodiment, the low perfusion rate can also be used to obtain an effective exchange of components of the fluid medium in the unswept region of the growth chamber and outside the unswept region of the growth chamber.

據此，將至少一生物細胞灌注於一微流體器件之至少一生長室中之一方法展示於圖7中且包含一灌注步驟7002，其中第一流體培養基依一第一灌注速率R₁透過該微流體器件之一流動區域而流動至流體 地連接至該生長室之一流動區域中達一第一灌注時間D₁。R₁可經選擇為一非掃及流動速率，如本文中所描述。方法700進一步包含步驟7004：停止流體培養基之流動達一第一灌注停止時間S₁。使步驟7002及7004重複W次(其中W可為選自1至約1000之一整數)，此後，灌注程序700完成。在一些實施例中，W可為2至約1000之一整數。 Accordingly, at least one of the at least one biological cell growth perfusion chamber of a microfluidic device one of the methods shown in FIG. 7 and comprises a filling step 7002, wherein the first fluid medium in accordance with a first rate R ₁ through the filling one microfluidic device to flow to the fluid flow region connected to one of the growth chamber region of the flow of a first perfusion time D _1. R ₁ can be selected to be a non-sweep and flow rate as described herein. The method of 700 further comprising the step 7004: stop the flow of a first fluid medium filling the stopping time S _1. Steps 7002 and 7004 are repeated W times (where W can be an integer selected from 1 to about 1000), after which the perfusion procedure 700 is completed. In some embodiments, W can be an integer from 2 to about 1000.

圖8中展示將至少一生物細胞灌注於一微流體器件之至少一生長室中之另一方法800，其包含一第一灌注循環，該第一灌注循環包含步驟8002：使流體培養基依一第一灌注速率R₁透過該微流體器件之一流動區域而流動至流體地連接至該生長室之一流動區域中達一第一灌注時間D₁。R₁可經選擇為一未掃及流動速率，如本文中所描述。該第一灌注循環包含步驟8004：停止流體培養基之流動達一第一灌注停止時間S₁。可使該第一灌注循環重複W次，其中W係選自1至約1000之一整數。在該第一灌注循環完成W次重複之後，方法800進一步包含一第二灌注循環，其包含步驟8006：使第一流體培養基依一第二灌注速率R₂流動達一第二灌注時間D₂，其中R₂經選擇為一非掃及流動速率。方法800之該第二灌注循環進一步包含步驟8008：停止流體培養基之流動達一第二灌注停止時間S₂。此後，方法返回至該第一灌注循環之步驟8002及8004且使組合兩循環灌注程序重複V次，其中V係1至約5000之一整數。W及V之組合可經選擇以滿足所要培育週期終點。 Another method 800 of infusing at least one biological cell into at least one growth chamber of a microfluidic device is shown in FIG. 8, comprising a first perfusion cycle comprising the step 8002 of accommodating the fluid medium a perfusion rate R ₁ of the through flow area of one of the microfluidic device coupled to the fluid flow to the growth chamber one of a first flow region of perfusion time D _1. R ₁ can be selected to be an unswept and flow rate as described herein. The first filling cycle comprises the step of 8004: stop the flow of a first fluid medium filling the stopping time S _1. The first perfusion cycle can be repeated W times, wherein W is selected from an integer from 1 to about 1000. After the W-repetition is completed in the first perfusion cycle, the method 800 further includes a second perfusion cycle comprising the step 8006 of flowing the first fluid medium to a second perfusion rate R ₂ according to a second perfusion rate R ₂ , Where R ₂ is selected to be a non-sweep and flow rate. The method of filling the second cycle of 800 further comprising the step 8008: stop the flow of a second fluid medium filling the stop time S _2. Thereafter, the method returns to steps 8002 and 8004 of the first perfusion cycle and the combined two-cycle perfusion procedure is repeated V times, wherein V is an integer from 1 to about 5000. The combination of W and V can be selected to meet the end of the desired incubation period.

在方法700或800之各種實施例中，灌注速率R₁可為如上文針對流量控制器構形所描述之流體培養基之任何非掃及流動速率。在一些實施例中，R₁可為約0.009微升/秒、約0.010微升/秒、約0.020微升/秒、約0.030微升/秒、約0.040微升/秒、約0.05微升/秒、約0.06微升/秒、約0.07微升/秒、約0.08微升/秒、約0.09微升/秒、約0.10微升/秒、約0.11微升/秒、約0.12微升/秒、約0.13微升/秒、約0.14微升/秒、約0.15微升/秒、約0.16微升/秒、約0.17微升/秒、約0.18微升/秒、約0.19微 升/秒、約0.20微升/秒、約0.30微升/秒、約0.40微升/秒、約0.50微升/秒、約0.60微升/秒、約0.70微升/秒、約0.80微升/秒、約0.90微升/秒、約1.00微升/秒、約1.10微升/秒、約1.20微升/秒、約1.30微升/秒、約1.40微升/秒、約1.50微升/秒、約1.60微升/秒、約1.70微升/秒、約1.80微升/秒、約1.90微升/秒、約2.00微升/秒、約2.10微升/秒、約2.20微升/秒、約2.40微升/秒、約2.50微升/秒、約2.60微升/秒、約2.70微升/秒、約2.80微升/秒、約2.90微升/秒或約3.00微升/秒。 In the method of this embodiment, the perfusion rate is above R ₁ may be any non-sweep and flow rate of the fluid flow controller as described for the configuration of the medium 700 or 800 of the various embodiments. In some embodiments, R ₁ can be about 0.009 microliters per second, about 0.010 microliters per second, about 0.020 microliters per second, about 0.030 microliters per second, about 0.040 microliters per second, about 0.05 microliters per second. Seconds, about 0.06 microliters per second, about 0.07 microliters per second, about 0.08 microliters per second, about 0.09 microliters per second, about 0.10 microliters per second, about 0.11 microliters per second, about 0.12 microliters per second. , about 0.13 microliters per second, about 0.14 microliters per second, about 0.15 microliters per second, about 0.16 microliters per second, about 0.17 microliters per second, about 0.18 microliters per second, about 0.19 microliters per second, About 0.20 microliters per second, about 0.30 microliters per second, about 0.40 microliters per second, about 0.50 microliters per second, about 0.60 microliters per second, about 0.70 microliters per second, about 0.80 microliters per second, about 0.90 microliters per second, about 1.00 microliters per second, about 1.10 microliters per second, about 1.20 microliters per second, about 1.30 microliters per second, about 1.40 microliters per second, about 1.50 microliters per second, about 1.60. Microliters per second, about 1.70 microliters per second, about 1.80 microliters per second, about 1.90 microliters per second, about 2.00 microliters per second, about 2.10 microliters per second, about 2.20 microliters per second, about 2.40 microseconds. L/s, about 2.50 microliters/second, about 2.60 microliters/second, about 2.70 microliters/second, about 2.80 microliters/second, about 2.90 microliters/second, or about 3.00 microliters/second.

在方法800之各種實施例中，第二灌注速率R₂可為如上文針對流量控制器構形所描述之流體培養基之任何非掃及流動速率。在一些實施例中，R₂可為0.009微升/秒、0.010微升/秒、0.020微升/秒、0.030微升/秒、0.040微升/秒、0.05微升/秒、0.06微升/秒、0.07微升/秒、0.08微升/秒、0.09微升/秒、0.10微升/秒、0.11微升/秒、0.12微升/秒、0.13微升/秒、0.14微升/秒、0.15微升/秒、0.16微升/秒、0.17微升/秒、0.18微升/秒、0.19微升/秒、0.20微升/秒、0.30微升/秒、0.40微升/秒、0.50微升/秒、0.60微升/秒、0.70微升/秒、0.80微升/秒、0.90微升/秒、1.00微升/秒、1.10微升/秒、1.20微升/秒、1.30微升/秒、1.40微升/秒、1.50微升/秒、1.60微升/秒、1.70微升/秒、1.80微升/秒、1.90微升/秒、2.00微升/秒、2.10微升/秒、2.20微升/秒、2.40微升/秒、2.50微升/秒、2.60微升/秒、2.70微升/秒、2.80微升/秒、2.90微升/秒或3.00微升/秒。可選擇流動速率R₁及/或R₂之任何組合。通常，灌注速率R₂可大於灌注速率R₁，且可為R₁之約5倍、約10倍、約20倍、約30倍、約40倍、約50倍、約60倍、約70倍、約80倍、約90倍、約100倍或100倍以上。在一些實施例中，R₂比R₁快至少10倍。在其他實施例中，R₂比R₁快至少20倍。在又一實施例中，R₂係R₁之速率之至少100倍。 In various embodiments of method 800, the second perfusion rate above R ₂ may be any non-sweep and flow rate of the fluid flow controller as described for the configuration of the medium. In some embodiments, R ₂ can be 0.009 μl/sec, 0.010 μl/sec, 0.020 μl/sec, 0.030 μl/sec, 0.040 μl/sec, 0.05 μl/sec, 0.06 μl/ Seconds, 0.07 microliters per second, 0.08 microliters per second, 0.09 microliters per second, 0.10 microliters per second, 0.11 microliters per second, 0.12 microliters per second, 0.13 microliters per second, 0.14 microliters per second, 0.15 μl/sec, 0.16 μl/sec, 0.17 μl/sec, 0.18 μl/sec, 0.19 μl/sec, 0.20 μl/sec, 0.30 μl/sec, 0.40 μl/sec, 0.50 μm L/s, 0.60 μl/sec, 0.70 μl/sec, 0.80 μl/sec, 0.90 μl/sec, 1.00 μL/sec, 1.10 μl/sec, 1.20 μl/sec, 1.30 μl/ Seconds, 1.40 microliters/second, 1.50 microliters/second, 1.60 microliters/second, 1.70 microliters/second, 1.80 microliters/second, 1.90 microliters/second, 2.00 microliters/second, 2.10 microliters/second, 2.20 microliters per second, 2.40 microliters per second, 2.50 microliters per second, 2.60 microliters per second, 2.70 microliters per second, 2.80 microliters per second, 2.90 microliters per second, or 3.00 microliters per second. Any combination of flow rates R ₁ and/or R ₂ can be selected. Typically, the perfusion rate R ₂ may be greater than the infusion rates R _1, and can be R ₁ of about 5-fold, about 10 fold, about 20 fold, about 30 fold, about 40 fold, about 50 fold, about 60 fold, about 70-fold , about 80 times, about 90 times, about 100 times or more than 100 times. In some embodiments, R ₂ is at least 10 times faster than R ₁ . In other embodiments, R ₂ is at least 20 times faster than R ₁ . In yet another embodiment, R ₂ is at least 100 times the rate of R ₁ .

在方法700或800之各種實施例中，第一灌注時間D₁可為如上文針對流量控制器構形所描述之任何適合灌注持續時間。在各種實施例中，D₁可為約5秒、約10秒、約15秒、約20秒、約25秒、約30秒、約35秒、約40秒、約45秒、約50秒、約55秒、約60秒、約65秒、約70秒、約80秒、約90秒、約100秒、約110秒、約120秒、約130秒、約140秒、約150秒、約160秒、約170秒或約180秒。在其他實施例中，D₁可為一時間範圍，例如約10秒至約40秒，如上文所描述。在一些實施例中，D₁可為約30秒至約75秒。在其他實施例中，D₁可為約100秒。在其他實施例中，D₁可在自約60秒至約150秒之一範圍內。在其他實施例中，D₁可為約20分鐘、約30分鐘、約40分鐘、約50分鐘、約60分鐘、約80分鐘、約90分鐘、約110分鐘、約120分鐘、約140分鐘、約160分鐘、約180分鐘、約200分鐘、約220分鐘、約240分鐘、約250分鐘、約260分鐘、約270分鐘、約290分鐘或約300分鐘。在一些實施例中，D₁係約40分鐘至約180分鐘。 In various embodiments of method 700 or 800, the first perfusion time D ₁ can be any suitable perfusion duration as described above for the flow controller configuration. In various embodiments, D ₁ can be about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, About 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 80 seconds, about 90 seconds, about 100 seconds, about 110 seconds, about 120 seconds, about 130 seconds, about 140 seconds, about 150 seconds, about 160 Seconds, about 170 seconds or about 180 seconds. In other embodiments, D ₁ can be a time range, such as from about 10 seconds to about 40 seconds, as described above. In some embodiments, D ₁ can be from about 30 seconds to about 75 seconds. In other embodiments, D ₁ can be about 100 seconds. In other embodiments, D ₁ can range from about 60 seconds to about 150 seconds. In other embodiments, D ₁ can be about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 80 minutes, about 90 minutes, about 110 minutes, about 120 minutes, about 140 minutes, About 160 minutes, about 180 minutes, about 200 minutes, about 220 minutes, about 240 minutes, about 250 minutes, about 260 minutes, about 270 minutes, about 290 minutes, or about 300 minutes. In some embodiments, D ₁ is from about 40 minutes to about 180 minutes.

在方法700或800之各種實施例中，第二灌注時間D₂可為如上文針對流量控制器構形所描述之任何適合灌注持續時間。在各種實施例中，D₂可為約5秒、約10秒、約15秒、約20秒、約25秒、約30秒、約35秒、約40秒、約45秒、約50秒、約55秒、約60秒、約65秒、約70秒、約80秒、約90秒或約100秒。在其他實施例中，D₂可為一時間範圍，例如約5秒至約20秒，如上文所描述。在其他實施例中，D₂可為約30秒至約70秒。在其他實施例中，D₂可為約60秒。 In various embodiments of method 700 or 800, the second perfusion time D ₂ can be any suitable perfusion duration as described above for the flow controller configuration. In various embodiments, D ₂ can be about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, About 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 80 seconds, about 90 seconds, or about 100 seconds. In other embodiments, D ₂ can be a time range, such as from about 5 seconds to about 20 seconds, as described above. In other embodiments, D ₂ can be from about 30 seconds to about 70 seconds. In other embodiments, D ₂ can be about 60 seconds.

在方法700或800之各種實施例中，第一灌注時間D₁可相同於或不同於第二灌注時間D₂。可選擇D₁及D₂之任何組合。在一些實施例中，D₁及/或D₂之灌注持續時間可經選擇為短於停止週期S₁及/或S₂。 In various embodiments of method 700 or 800, the first perfusion time D ₁ may be the same as or different than the second perfusion time D ₂ . Any combination of D ₁ and D ₂ can be selected. In some embodiments, the perfusion duration of D ₁ and/or D ₂ may be selected to be shorter than the stop period S ₁ and/or S ₂ .

在方法700或800之各種實施例中，第一灌注停止時間S₁可經選擇為如上文針對流量控制器構形之灌注週期之間的一時間間隔所描述之 任何適合時間週期。在一些實施例中，S₁可為約0分鐘、約5分鐘、約10分鐘、約15分鐘、約20分鐘、約25分鐘、約30分鐘、約35分鐘、約40分鐘、約45分鐘、約60分鐘、約65分鐘、約80分鐘、約90分鐘、約100分鐘、約120分鐘、約150分鐘、約180分鐘、約210分鐘、約240分鐘、約270分鐘或約300分鐘。在各種實施例中，S₁可為如上文針對流量控制器構形之灌注之間的時間間隔所描述之任何適當時間範圍，例如約20分鐘至約60分鐘。在一些實施例中，S₁可為約10分鐘至約30分鐘。在其他實施例中，S₁可為約15分鐘。在其他實施例中，S₁可為約0秒、約5秒、約10秒、約20秒、約30秒、約40秒、約50秒、約60秒、約70秒、約80秒或約90秒。在一些實施例中，S₁係約0秒。 700 or 800. In various embodiments of the method, the first stop time S ₁ infusion can be any suitable time period described above of for the interval between the time a casting cycle configuration of the flow controller selected. In some embodiments, S ₁ can be about 0 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, About 60 minutes, about 65 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 120 minutes, about 150 minutes, about 180 minutes, about 210 minutes, about 240 minutes, about 270 minutes, or about 300 minutes. In various embodiments, S ₁ can be any suitable time range as described above for the time interval between perfusions configured for the flow controller, such as from about 20 minutes to about 60 minutes. In some embodiments, S ₁ can be from about 10 minutes to about 30 minutes. In other embodiments, S ₁ can be about 15 minutes. In other embodiments, S ₁ can be about 0 seconds, about 5 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 80 seconds, or About 90 seconds. In some embodiments, S ₁ is about 0 seconds.

在方法700或800之各種實施例中，第二灌注停止時間S₂可經選擇為如上文針對流量控制器構形之灌注週期之間的一時間間隔所描述之任何適合時間週期。在一些實施例中，S₂可為約0分鐘、約5分鐘、約6分鐘、約7分鐘、約8分鐘、約9分鐘、約10分鐘、約20分鐘、約30分鐘、約45分鐘、約50分鐘、約60分鐘、約90分鐘、約120分鐘、約180分鐘、約240分鐘、約270分鐘或約300分鐘。在各種實施例中，S₂可為如上文針對流量控制器構形之灌注之間的時間間隔所描述之任何適當時間範圍，例如約15分鐘至約45分鐘。在一些實施例中，S₂可為約10分鐘至約30分鐘。在其他實施例中，S₂可為約8分鐘或約9分鐘。在其他實施例中，S₂係約0分鐘。 In various embodiments method 700 or 800 of the embodiment, the infusion is stopped a second time S ₂ may be any suitable time period described above of for the interval between the time a casting cycle configuration of the flow controller selected. In some embodiments, S ₂ can be about 0 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, About 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 270 minutes, or about 300 minutes. In various embodiments, S ₂ can be any suitable time range as described above for the time interval between perfusions configured for the flow controller, such as from about 15 minutes to about 45 minutes. In some embodiments, S ₂ can be from about 10 minutes to about 30 minutes. In other embodiments, S ₂ can be about 8 minutes or about 9 minutes. In other embodiments, the S ₂ is about 0 minutes.

在方法700或800之各種實施例中，第一灌注停止時間S₁及第二灌注停止時間S₂可獨立地選自任何適合值。S₁可相同於或不同於S₂。 700 or 800. In various embodiments of the method, a first infusion is stopped a second time S ₁ and S ₂ perfusion stop time may be independently selected from any suitable value. S ₁ may be the same as or different from S ₂ .

在方法800之各種實施例中，重複次數W可經選擇為相同於或不同於重複次數V。 In various embodiments of method 800, the number of repetitions W can be selected to be the same or different from the number of repetitions V.

在方法700或800之各種實施例中，W可為約1、約4、約5、約6、約8、約10、約12、約15、約18、約20、約24、約30、約36、約40、 約45或約50。在一些實施例中，W可經選擇為約1至約20。在一些實施例中，W可為1。 In various embodiments of method 700 or 800, W can be about 1, about 4, about 5, about 6, about 8, about 10, about 12, about 15, about 18, about 20, about 24, about 30, About 36, about 40, About 45 or about 50. In some embodiments, W can be selected from about 1 to about 20. In some embodiments, W can be one.

在方法800之各種實施例中，V可為約5、約10、約20、約25、約30、約35、約40、約50、約60、約80、約100、約120、約240、約300、約350、約400、約450、約500、約600、約750、約900或約1000。在一些實施例中，V可經選擇為約10至約120。在其他實施例中，V可經選擇為約5至約24。在一些實施例中，V可為約30至約50或可為約400至約500。 In various embodiments of method 800, V can be about 5, about 10, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 80, about 100, about 120, about 240. About 300, about 350, about 400, about 450, about 500, about 600, about 750, about 900, or about 1000. In some embodiments, V can be selected from about 10 to about 120. In other embodiments, V can be selected from about 5 to about 24. In some embodiments, V can be from about 30 to about 50 or can be from about 400 to about 500.

在方法800之各種實施例中，重複次數W可經選擇為相同於或不同於重複次數V。 In various embodiments of method 800, the number of repetitions W can be selected to be the same or different from the number of repetitions V.

在方法700或800之各種實施例中，灌注之第一步驟(由步驟7002/7004或8002/8004表示)之一總時間係約1小時至約10小時且W係一整數1。在各種實施例中，灌注之第一步驟之總時間係約9分鐘至約15分鐘。 In various embodiments of method 700 or 800, the total time of one of the first steps of perfusion (represented by steps 7002/7004 or 8002/8004) is from about 1 hour to about 10 hours and W is an integer one. In various embodiments, the total time of the first step of infusion is from about 9 minutes to about 15 minutes.

在方法800之各種實施例中，一灌注循環之第二步驟(由步驟8006/8008表示)之一總時間係約1分鐘至約15分鐘或約1分鐘至約20分鐘。 In various embodiments of method 800, a total time of a second step of a perfusion cycle (represented by steps 8006/8008) is from about 1 minute to about 15 minutes or from about 1 minute to about 20 minutes.

在方法700或800之任何者中，灌注方法可持續達生物細胞之整個培育週期，例如約1天、約2天、約3天、約4天、約5天、約6天、約7天、約8天、約9天、約10天或10天以上。 In any of methods 700 or 800, the perfusion method can last for the entire incubation period of the biological cells, such as about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days. , about 8 days, about 9 days, about 10 days or more than 10 days.

在圖8之方法800之另一非限制性實施例中，控制器可經構形以在灌注步驟8002期間將(若干)流體培養基灌注於流動區域中，其具有較長灌注週期D₁。控制器可依一第一速率灌注流體培養基達下列之一週期：約45分鐘、約60分鐘、約75分鐘、約90分鐘、約105分鐘、約120分鐘、約2.25小時、約2.5小時、約2.45小時、約3.0小時、約3.25小時、約3.5小時、約3.75小時、約4.0小時、約4.25小時、約4.5小 時、約4.75小時、約5小時或約6小時。在第一灌注週期D₁結束時，可停止流體培養基之流動達一停止時間週期S₁，S₁可為約0秒、約15秒、約30秒、約45秒、約1分鐘、約1.25分鐘、約1.5分鐘、約2.0分鐘、約3.0分鐘、約4分鐘、約5分鐘或約6分鐘。在一些實施例中，第一流動速率R₁可經選擇為約0.009微升/秒、約0.01微升/秒、約0.02微升/秒、約0.03微升/秒、約0.05微升/秒、約0.1微升/秒、約0.2微升/秒、約0.3微升/秒、約0.4微升/秒或約0.5微升/秒。可停止流體培養基之流動達少於約1分鐘之一灌注停止週期S₁或S₁可為0秒。替代地，S₁可為約30秒、約1.5分鐘、約2.0分鐘、約2.5分鐘或約3分鐘。接著，可使用一不同灌注速率進行一第二灌注週期D₂。在一些實施例中，第二灌注速率可高於第一灌注速率。在一些實施例中，第二灌注速率R₂可選自約1.0微升/秒、約1.1微升/秒、約1.2微升/秒、約1.3微升/秒、約1.4微升/秒、約1.5微升/秒、約1.7微升/秒、約1.9微升/秒、約2.0微升/秒、約2.2微升/秒、約2.4微升/秒、約2.6微升/秒、約2.8微升/秒、約3.0微升/秒、約3.2微升/秒、約3.4微升/秒、約3.6微升/秒、約3.8微升/秒、約4.0微升/秒、約4.2微升/秒、約4.4微升/秒、約4.6微升/秒、約4.8微升/秒、約5.0微升/秒、約6.0微升/秒、約7.0微升/秒、約8.0微升/秒或約9.0微升/秒。第二灌注週期D₂可為約1秒、約2秒、約3秒、約4秒、約5秒、約6秒、約10秒、約15秒、約30秒、約45秒、約60秒、約65秒、約75秒、約80秒或約90秒。接著可停止灌注達一第二灌注停止週期S₂，S₂可為約0秒、約10秒、約20秒、約30秒、約40秒、約50秒、約60秒、約1.5分鐘、約1.75分鐘、約2.0分鐘、約2.5分鐘、約2.75分鐘、約3.0分鐘或約4.0分鐘。在一些實施例中，D₁可為約2小時、約3小時或約4小時。在各種實施例中，D₁可為約4小時。在各種實施例中，S₁可為0秒或少於約1分鐘。第二灌注週期D₂可為約1秒至約6秒。在一些實施例中，第二灌注停止週期S₂可為約40秒至約1.5分 鐘。 In another method 800 of FIG. 8 of non-limiting embodiments, the controller may be configured to during the filling step 8002 (s) in the perfusion fluid medium flow region, which has a longer casting cycle D _1. The controller can perfuse the fluid medium at a first rate for one of the following cycles: about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 2.25 hours, about 2.5 hours, about 2.45 hours, about 3.0 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4.0 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, or about 6 hours. When the first end of the perfusion period D _1, to stop the flow of the fluid medium of a stopping time period S _1, S ₁ may be between about 0 seconds and about 15 seconds, about 30 seconds, about 45 seconds, about 1 minute, about 1.25 Minutes, about 1.5 minutes, about 2.0 minutes, about 3.0 minutes, about 4 minutes, about 5 minutes, or about 6 minutes. In some embodiments, the first flow rate R ₁ can be selected to be about 0.009 microliters per second, about 0.01 microliters per second, about 0.02 microliters per second, about 0.03 microliters per second, about 0.05 microliters per second. About 0.1 microliters per second, about 0.2 microliters per second, about 0.3 microliters per second, about 0.4 microliters per second, or about 0.5 microliters per second. The flow of the fluid medium can be stopped for less than about 1 minute. One of the perfusion stop periods S ₁ or S ₁ can be 0 seconds. Alternatively, S ₁ can be about 30 seconds, about 1.5 minutes, about 2.0 minutes, about 2.5 minutes, or about 3 minutes. Next, a second perfusion cycle D ₂ can be performed using a different perfusion rate. In some embodiments, the second perfusion rate can be higher than the first perfusion rate. In some embodiments, the second perfusion rate R _{2 can be} selected from about 1.0 microliters per second, about 1.1 microliters per second, about 1.2 microliters per second, about 1.3 microliters per second, about 1.4 microliters per second, About 1.5 microliters per second, about 1.7 microliters per second, about 1.9 microliters per second, about 2.0 microliters per second, about 2.2 microliters per second, about 2.4 microliters per second, about 2.6 microliters per second, about 2.8 microliters per second, about 3.0 microliters per second, about 3.2 microliters per second, about 3.4 microliters per second, about 3.6 microliters per second, about 3.8 microliters per second, about 4.0 microliters per second, about 4.2. Microliters per second, about 4.4 microliters per second, about 4.6 microliters per second, about 4.8 microliters per second, about 5.0 microliters per second, about 6.0 microliters per second, about 7.0 microliters per second, about 8.0 micrometers. L / s or about 9.0 μl / sec. The second perfusion cycle D ₂ can be about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 10 seconds, about 15 seconds, about 30 seconds, about 45 seconds, about 60 Seconds, about 65 seconds, about 75 seconds, about 80 seconds, or about 90 seconds. Infusion may then be stopped up to a second stop reperfusion period S _2, S ₂ may be from about 0 seconds and about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds to about 1.5 minutes, About 1.75 minutes, about 2.0 minutes, about 2.5 minutes, about 2.75 minutes, about 3.0 minutes, or about 4.0 minutes. In some embodiments, D ₁ can be about 2 hours, about 3 hours, or about 4 hours. In various embodiments, D ₁ can be about 4 hours. In various embodiments, S ₁ can be 0 seconds or less than about 1 minute. The second perfusion cycle D ₂ can be from about 1 second to about 6 seconds. In some embodiments, the second perfusion stop period S ₂ can be from about 40 seconds to about 1.5 minutes.

據此，提供一種用於將至少一生物細胞灌注於一微流體器件之至少一生長室中之方法，其包含下列步驟：使用一第一灌注步驟來灌注該至少一生物細胞，該第一灌注步驟包含：使一第一流體培養基依一第一灌注速率R₁流動通過該微流體器件之一流動區域達一第一灌注時間D₁，其中該流動區域流體地連接至該生長室，其中R₁經選擇為一非掃及流動速率；停止該第一流體培養基之流動達一第一灌注停止時間S₁；及使該第一灌注步驟重複W次，其中W係選自1至1000之一整數。該方法可進一步包含下列步驟：使用一第二灌注步驟來灌注該至少一生物細胞，該第二灌注步驟包括：使該第一流體培養基依一第二灌注速率R₂流動達一第二灌注時間D₂，其中R₂經選擇為一非掃及流動速率；停止該第一流體培養基之流動達一第二灌注停止時間S₂；及使該第一灌注步驟之後該第二灌注步驟重複V次，其中V係1至1000之一整數。 Accordingly, a method for infusing at least one biological cell into at least one growth chamber of a microfluidic device is provided, comprising the steps of perfusing the at least one biological cell using a first perfusion step, the first perfusion comprising the step of: causing a first flow of a first fluid medium perfusion rate by _{R. 1} of a first perfusion time D ₁ by one of the flow area of the microfluidic device, wherein the flow area is fluidly connected to the growth chamber, wherein R ₁ being selected as a non-sweeping and flow rate; stopping the flow of the first fluid medium to a first perfusion stop time S ₁ ; and repeating the first infusion step W times, wherein the W system is selected from one of 1 to 1000 Integer. The method may further comprise the steps of: perfusing the at least one biological cell using a second perfusion step, the second infusing step comprising: flowing the first fluid medium to a second perfusion rate according to a second perfusion rate R ₂ D ₂ , wherein R ₂ is selected to be a non-sweeping and flowing rate; stopping the flow of the first fluid medium to a second perfusion stop time S ₂ ; and repeating the second infusion step V times after the first infusion step Where V is an integer from 1 to 1000.

第二灌注速率R₂可大於第一灌注速率R₁。第一灌注時間D₁可相同於或不同於第二灌注時間D₂。第一灌注停止時間S₁可相同於或不同於第二灌注停止時間S₂。當執行第二灌注步驟時，重複次數W可相同於或不同於重複次數V。R₂可比R₁快至少10倍。替代地，R₂可比R₁快至少20倍。R₂可比R₁快至少100倍。D₁可為約30秒至約75秒。在其他實施例中，D₁可為約40分鐘至約180分鐘或約180分鐘至約300分鐘。在一些其他實施例中，D₁可為約60秒至約150秒。S₁可為約10分鐘至約30分鐘。在其他實施例中，S₁可為約5分鐘至約10分鐘。在其他實施例中，S₁可為零。在一些實施例中，D₁可為約40分鐘至約180分鐘，且S₁可為零。在其他實施例中，D₁可為約60秒至約150秒，且S₁可為約5分鐘至約10分鐘。在其他實施例中，D₁可為約180分鐘至約300分鐘，且S₁可為零。第一灌注步驟之總時間可為約1小時至約10小 時。在其他實施例中，第一灌注步驟之總時間可為約2小時至約4小時。在一些實施例中，W可為大於2之一整數。在一些實施例中，W可為約1至約20。在一些實施例中，D₂可為約10秒至約25秒。在其他實施例中，D₂可為約10秒至約90秒。在一些實施例中，S₂可為約10分鐘至約30分鐘。在其他實施例中，S₂可為約15分鐘。在一些實施例中，V可為約10至約120。在一些實施例中，V可為約30至約50或可為約400至約500。在一些實施例中，D₂可為約1秒至約6秒，且S₂可為0秒。在一些實施例中，D₂可為約10秒至約90秒且S₂可為約40秒至約1.5分鐘。在一些實施例中，第二灌注步驟之一重複之一總時間可為約1分鐘至約15分鐘。 The second perfusion rate R ₂ can be greater than the first perfusion rate R ₁ . The first perfusion time D ₁ may be the same as or different from the second perfusion time D ₂ . The first perfusion stop time S ₁ may be the same as or different from the second perfusion stop time S ₂ . When the second infusion step is performed, the number of repetitions W may be the same as or different from the number of repetitions V. R ₂ can be at least 10 times faster than R ₁ . Alternatively, R ₂ can be at least 20 times faster than R ₁ . R ₂ can be at least 100 times faster than R ₁ . D ₁ can be from about 30 seconds to about 75 seconds. In other embodiments, D ₁ may be from about 40 minutes to about 180 minutes to about 180 minutes or about 300 minutes. In some other embodiments, D ₁ can be from about 60 seconds to about 150 seconds. S ₁ can be from about 10 minutes to about 30 minutes. In other embodiments, S ₁ can be from about 5 minutes to about 10 minutes. In other embodiments, S ₁ may be zero. In some embodiments, D ₁ can be from about 40 minutes to about 180 minutes, and S ₁ can be zero. In other embodiments, D ₁ can be from about 60 seconds to about 150 seconds, and S ₁ can be from about 5 minutes to about 10 minutes. In other embodiments, D ₁ can be from about 180 minutes to about 300 minutes, and S ₁ can be zero. The total time of the first infusion step can range from about 1 hour to about 10 hours. In other embodiments, the total time of the first infusion step can range from about 2 hours to about 4 hours. In some embodiments, W can be an integer greater than one of two. In some embodiments, W can be from about 1 to about 20. In some embodiments, D ₂ can be from about 10 seconds to about 25 seconds. In other embodiments, D ₂ can be from about 10 seconds to about 90 seconds. In some embodiments, S ₂ can be from about 10 minutes to about 30 minutes. In other embodiments, S ₂ can be about 15 minutes. In some embodiments, V can be from about 10 to about 120. In some embodiments, V can be from about 30 to about 50 or can be from about 400 to about 500. In some embodiments, D ₂ can be from about 1 second to about 6 seconds, and S ₂ can be 0 seconds. In some embodiments, D ₂ can be from about 10 seconds to about 90 seconds and S ₂ can be from about 40 seconds to about 1.5 minutes. In some embodiments, one of the second infusion steps may be repeated for a total time of from about 1 minute to about 15 minutes.

調節培養基Regulatory medium

為提供維持及增強至少一生物細胞之生長及/或存活性之一培養基(例如第一培養基或第二培養基)，第一流體培養基可含有液體組分及氣體組分兩者(例如，氣體組分可溶解於液體組分中)。另外，流體培養基可包含溶解於液體組分中之其他組分，諸如生物分子、維生素及礦物質。任何適合組分可用於流體培養基中，如熟習此項技術者所知。一些非限制性實例係如上文所討論，但可在不背離本文中所描述之方法之情況下使用諸多其他培養基組合物。培養基可或可不含有動物源血清。在一些實施例中，流體培養基可包含一化學成分確定之培養基(至少在接觸細胞或一含細胞之流體之前)，且可進一步為一無蛋白質或無肽之化學成分確定培養基。在一些實施例中，流體培養基可包含一減少血清培養基。 In order to provide a medium (eg, a first medium or a second medium) that maintains and enhances the growth and/or viability of at least one biological cell, the first fluid medium may contain both a liquid component and a gas component (eg, a gas group) The fraction can be dissolved in the liquid component). Additionally, the fluid medium can contain other components, such as biomolecules, vitamins, and minerals, dissolved in the liquid component. Any suitable component can be used in the fluid medium, as is known to those skilled in the art. Some non-limiting examples are as discussed above, but many other media compositions can be used without departing from the methods described herein. The medium may or may not contain animal derived serum. In some embodiments, the fluid medium can comprise a chemically defined medium (at least prior to contacting the cells or a cell-containing fluid), and the medium can be further defined as a protein-free or peptide-free chemical component. In some embodiments, the fluid medium can comprise a reduced serum medium.

在將第一流體培養基引入至微流體器件中之前，可藉由使一初始流體培養基飽含溶解氣體分子而製備第一流體培養基。另外，使初始流體培養基飽含溶解氣體分子可一直持續至將第一流體培養基引入至微流體器件中之時間點。使初始流體培養基飽和可包含：使微流體 器件與能夠使初始流體培養基飽含溶解氣體分子之一氣體環境接觸。可使初始流體培養基飽和之氣體分子包含(但不限於)氧氣、二氧化碳及氮氣。 The first fluid medium can be prepared by subjecting an initial fluid medium to dissolved gas molecules prior to introduction of the first fluid medium into the microfluidic device. Additionally, subjecting the initial fluid medium to the dissolved gas molecules can continue until the point at which the first fluid medium is introduced into the microfluidic device. Saturating the initial fluid medium can include: making the microfluid The device is in contact with a gaseous environment capable of rendering the initial fluid medium saturated with one of the dissolved gas molecules. Gas molecules that can saturate the initial fluid medium include, but are not limited to, oxygen, carbon dioxide, and nitrogen.

第一流體培養基可進一步包含緩和第一流體培養基之一pH。緩和第一流體培養基之pH可(例如)發生於引入溶解氣體分子之前及/或發生於引入溶解氣體分子期間。可藉由添加一緩衝物而實現此緩和。一適合緩衝物之一非限制性實例係HEPES。其他緩衝物可存在於培養基中且可或可不取決於二氧化碳之存在(諸如碳酸緩衝系統)，且可由熟習技術者選擇。細胞生長所需之鹽、蛋白質、碳水化合物、脂類、維生素及其他小分子亦可形成第一流體培養基組合物之部分。 The first fluid medium can further comprise buffering one of the pH of the first fluid medium. Alleviating the pH of the first fluid medium can occur, for example, prior to introduction of dissolved gas molecules and/or during introduction of dissolved gas molecules. This relaxation can be achieved by adding a buffer. One non-limiting example of a suitable buffer is HEPES. Other buffers may be present in the culture medium and may or may not depend on the presence of carbon dioxide (such as a carbonation buffer system) and may be selected by a skilled artisan. Salts, proteins, carbohydrates, lipids, vitamins and other small molecules required for cell growth may also form part of the first fluid medium composition.

在一些實施例中，可在經由入口而引入第一流體培養基之前於一儲液器中執行使第一流體培養基飽含氣體組分。在其他實施例中，可在儲液器與入口之間的一可透氣連接導管中執行使第一流體培養基飽含氣體組分。在其他實施例中，可經由微流體器件之一蓋之一可透氣部分而執行使第一流體培養基飽含氣體組分。在一些實施例中，流體培養基之氣體飽和亦包含：維持氣體交換環境中之濕度，使得微流體器件內之流體培養基之滲透壓在培育期間不改變。 In some embodiments, the first fluid medium may be saturated with a gas component in a reservoir prior to introduction of the first fluid medium via the inlet. In other embodiments, the first fluid medium may be saturated with a gas component in a gas permeable connecting conduit between the reservoir and the inlet. In other embodiments, the first fluid medium may be saturated with a gas component via a gas permeable portion of one of the microfluidic devices. In some embodiments, gas saturation of the fluid medium also includes maintaining the humidity in the gas exchange environment such that the osmotic pressure of the fluid medium within the microfluidic device does not change during incubation.

第一流體培養基之組合物亦可包含來自一餵食細胞培養基之至少一分泌組分。分泌餵食細胞組分可包含生長因子、內分泌素、細胞激素、小分子、蛋白多糖及其類似者。可在其中執行使第一流體培養基飽含氣體組分之相同儲液器中執行自餵食細胞培養基引入至少一分泌組分，或可在飽和步驟之前完成將至少一分泌組分自餵食細胞培養基引入至第一流體培養基。 The composition of the first fluid medium may also comprise at least one secretory component from a feeding cell culture medium. Secreted feeder cell components can include growth factors, endocrine, cytokines, small molecules, proteoglycans, and the like. The introduction of at least one secretory component from the feeding cell culture medium may be performed in the same reservoir in which the first fluid medium is saturated with the gas component, or the at least one secretory component may be introduced from the feeding cell culture medium before the saturation step First fluid medium.

在一些其他實施例中，第一培養基之組合物亦可包含一(或若干)添加物，其(等)經設計以提供更改流體培養基來測試細胞對該(等)添加物之回應。此(等)添加物可(例如)增強或減弱細胞存活性或生長。 In some other embodiments, the composition of the first medium may also comprise one (or several) additives that are (or) designed to provide a modified fluid medium to test the response of the cells to the (etc.) additive. This (etc.) additive can, for example, enhance or attenuate cell viability or growth.

在一些實施例中，方法可包含：在經由至少一入口而引入第一流體培養基時偵測第一流體培養基之pH。可在直接接近於入口之一位置處執行偵測pH。在一些實施例中，方法可包含：在經由一出口而輸出第一流體培養基時偵測第一流體培養基之pH。可在直接接近於出口之一位置處執行偵測pH。用於偵測pH之偵測器之任一者或兩者可為一光學感測器。在一些實施例中，若pH偏離一可接受範圍，則偵測器能夠提供一警報。在一些其他實施例中，當由偵測器量測之一pH值偏離一可接受範圍時，可更改第一流體培養基之組合物。 In some embodiments, the method can include detecting a pH of the first fluid medium upon introduction of the first fluid medium via the at least one inlet. The detection of the pH can be performed at a position directly adjacent to the inlet. In some embodiments, the method can include detecting a pH of the first fluid medium upon outputting the first fluid medium via an outlet. The detection of the pH can be performed at a position directly adjacent to the outlet. Either or both of the detectors for detecting pH may be an optical sensor. In some embodiments, the detector can provide an alert if the pH deviates from an acceptable range. In some other embodiments, the composition of the first fluid medium can be altered when one of the pH values measured by the detector deviates from an acceptable range.

在培育步驟期間，可監測至少一生長室及其內所含之任何細胞之一影像。 During the incubation step, at least one growth chamber and an image of any of the cells contained therein can be monitored.

輸出至少一生物細胞Output at least one biological cell

在培育步驟完成之後，可自生長室或其隔離區域輸出至少一生物細胞或細胞群落。輸出可包含：使用足以移動一或多個生物細胞/細胞群落之一強介電泳(DEP)力。可光學地致動或電子地致動該DEP力。例如，微流體器件可包含具有一DEP構形(諸如一光電鑷子(OET)構形)之一基板。在其他實施例中，可使用流體流動及/或重力來使至少一生物細胞或細胞群落自生長室或隔離區域輸出。在其他實施例中，可使用作用於生長室或其隔離區域上方之一可變形蓋區域上之壓縮力來使至少一生物細胞或細胞群落自生長室或隔離區域輸出，藉此致使流體自生長室或隔離區域局部流出。 After the incubation step is completed, at least one biological cell or cell population can be exported from the growth chamber or its isolated region. The output can include: using a strong dielectrophoresis (DEP) force sufficient to move one or more biological cells/cell communities. The DEP force can be actuated optically or electronically. For example, the microfluidic device can comprise a substrate having a DEP configuration, such as an optoelectronic force (OET) configuration. In other embodiments, fluid flow and/or gravity may be used to output at least one biological cell or cell population from the growth chamber or isolated region. In other embodiments, the compressive force on the deformable lid region above the growth chamber or its isolation region can be used to output at least one biological cell or cell population from the growth chamber or isolation region, thereby causing fluid self-growth. The chamber or isolated area partially flows out.

在自生長室或隔離區域輸出至少一生物細胞或細胞群落之後，細胞可自流動區域(例如通道)輸出而離開微流體器件。在一些實施例中，使細胞自流動區域輸出包含：使用足以移動一或多個生物細胞/細胞群落之一強DEP力。可如上文所描述般產生該DEP力。在一些其他實施例中，使細胞自流動區域輸出而離開微流體器件包含：使用流體流動及/或重力來移動細胞。 After outputting at least one biological cell or cell population from the growth chamber or isolated region, the cells can exit from the flow region (eg, the channel) and exit the microfluidic device. In some embodiments, exporting the cells from the flow region comprises using a strong DEP force sufficient to move one or more biological cells/cell colonies. This DEP force can be generated as described above. In some other embodiments, exiting the cells from the flow region away from the microfluidic device comprises: using fluid flow and/or gravity to move the cells.

在輸出步驟期間，可監測至少一生長室及其內所含之任何細胞之一影像。 During the output step, at least one growth chamber and an image of any of the cells contained therein can be monitored.

調節至少一表面Adjust at least one surface

在一些實施例中，微流體器件具有一調節狀態中之至少一生長室之至少一表面。在其他實施例中，至少一生長室之表面在引入至少一生物細胞之前被調節且可經執行為培養一或多個生物細胞之方法之部分。調節表面可包含：使用一調節試劑(諸如一聚合物)來處理表面。 In some embodiments, the microfluidic device has at least one surface of at least one growth chamber in an adjusted state. In other embodiments, the surface of at least one growth chamber is modulated prior to introduction of at least one biological cell and can be performed as part of a method of culturing one or more biological cells. Adjusting the surface can include treating the surface with a conditioning agent such as a polymer.

在一些實施例中，提供用於處理一微流體器件(100、300、400、500A至500E及600)之至少一生長室之至少一表面之一方法，其包含下列步驟：使包含過量調節試劑之流體培養基流動至流動通道(圖1A至圖1C、圖2、圖3、圖4A至圖4C)中；培育該微流體器件達一選定時間週期；及替換該通道中之該培養基。在其他實施例中，用於預充一微流體器件之一方法包含下列步驟：使含有一調節試劑之一預充溶液流動至流動通道中；培育該器件達一選定時間週期，藉此調節生長室之至少一表面；及使用一流體培養基替換該通道中之該溶液。該預充溶液可含有如本文中所描述之任何流體培養基。替換調節溶液之流體培養基或具有過量調節試劑之流體培養基可為如本文中所描述之任何培養基且可另外含有細胞。 In some embodiments, a method for processing at least one surface of at least one growth chamber of a microfluidic device (100, 300, 400, 500A to 500E, and 600) is provided, comprising the steps of: including an excess of conditioning reagent The fluid medium flows into the flow channel (Figs. 1A-1C, Fig. 2, Fig. 3, Fig. 4A to Fig. 4C); the microfluidic device is incubated for a selected period of time; and the medium in the channel is replaced. In other embodiments, a method for pre-filling a microfluidic device comprises the steps of flowing a pre-filled solution containing one of the conditioning reagents into a flow channel; incubating the device for a selected period of time, thereby modulating growth At least one surface of the chamber; and replacing the solution in the channel with a fluid medium. The pre-filled solution can contain any fluid medium as described herein. The fluid medium in which the conditioning solution is replaced or the fluid medium having the excess conditioning reagent can be any of the media as described herein and can additionally contain cells.

在一些實施例中，可使用包含伸烷基醚部分之一聚合調節試劑來處理至少一表面。具有伸烷基醚部分之該聚合調節試劑可包含任何適合之含伸烷基醚聚合物，其包含(但不限於)上文所討論之含伸烷基醚聚合物之任何者。在一實施例中，可使用兩親性非離子嵌段共聚物來處理生長室之表面，該等兩親性非離子嵌段共聚物包含在聚合物鏈(例如Pluronic®聚合物)內具有不同比率且位於不同位置中之聚環氧乙烷(PEO)及聚環氧丙烷(PPO)亞單元之嵌段。用於產生一調節表面之特 定Pluronic®聚合物包含Pluronic® L44、L64、P85、F68及F127(其包含F127NF)。 In some embodiments, at least one surface can be treated with a polymerization conditioning reagent comprising one of the alkylene ether moieties. The polymerization modifying agent having an alkylene ether moiety can comprise any suitable alkylene ether containing polymer including, but not limited to, any of the alkylene ether polymers discussed above. In one embodiment, the surface of the growth chamber can be treated with an amphiphilic nonionic block copolymer comprising different ones in the polymer chain (eg, Pluronic® polymer). Blocks of polyethylene oxide (PEO) and polypropylene oxide (PPO) subunits in ratios and located in different locations. Used to create a conditioning surface Pluronic® polymers include Pluronic® L44, L64, P85, F68 and F127 (which contain F127NF).

在其他實施例中，可使用包含羧基部分之一聚合調節試劑來處理表面。適合之含羧酸聚合調節試劑之非限制性實例係如上文所討論且任何適當之含羧酸聚合調節試劑可用以處理表面。 In other embodiments, the surface can be treated with a polymerization conditioning reagent comprising one of the carboxyl moieties. Non-limiting examples of suitable carboxylic acid polymerization modifying agents are as discussed above and any suitable carboxylic acid containing polymerization conditioning reagent can be used to treat the surface.

在其他實施例中，可使用包含糖類部分之一聚合調節試劑來處理表面。適合之含糖類聚合調節試劑之非限制性實例係如上文所討論且任何適當之含糖類聚合調節試劑可用以處理表面。 In other embodiments, the surface may be treated with a polymerization conditioning reagent comprising one of the saccharide moieties. Non-limiting examples of suitable sugar-containing polymerization conditioning agents are as discussed above and any suitable sugar-containing polymerization conditioning reagent can be used to treat the surface.

在其他實施例中，可使用包含磺酸部分之一聚合調節試劑來處理表面。適合之含磺酸聚合調節試劑之非限制性實例係如上文所討論且任何適當之含磺酸聚合調節試劑可用以處理表面。 In other embodiments, the surface can be treated with a polymerization conditioning reagent comprising one of the sulfonic acid moieties. Non-limiting examples of suitable sulfonic acid polymerization modifying agents are as discussed above and any suitable sulfonic acid containing polymerization conditioning reagent can be used to treat the surface.

在其他實施例中，可使用包含胺基酸部分之一聚合調節試劑來處理表面。適合之含胺基酸聚合調節試劑之非限制性實例係如上文所討論且任何適當之含胺基酸聚合調節試劑可用以處理表面。含胺基酸聚合調節試劑可包含一蛋白質。在一些實施例中，使用一蛋白質來處理表面，其中該蛋白質可包含發現於一哺乳動物血清中之一組分或包含一哺乳動物血清之部分。在其他實施例中，使用一哺乳動物血清之組分來處理表面。在一些實施例中，可使用一細胞培養基補充劑(諸如B-27®補充劑((50倍)，來自ThermoFisher Scientific,Cat# 17504044之無血清培養基))來處理表面。該哺乳動物血清可為胎牛血清(FBS)。替代地，該哺乳動物血清可為小牛血清(FCS)。 In other embodiments, the surface may be treated with a polymerization conditioning reagent comprising one of the amino acid moieties. Non-limiting examples of suitable amino acid-containing polymerization conditioning reagents are as discussed above and any suitable amino acid-containing polymerization conditioning reagent can be used to treat the surface. The amino acid-containing polymerization regulating agent may comprise a protein. In some embodiments, a protein is used to treat a surface, wherein the protein can comprise a component found in a mammalian serum or a portion comprising a mammalian serum. In other embodiments, a component of a mammalian serum is used to treat the surface. In some embodiments, a cell culture medium supplement (such as B-27® supplement (50 times), serum free medium from ThermoFisher Scientific, Cat # 17504044) can be used to treat the surface. The mammalian serum can be fetal bovine serum (FBS). Alternatively, the mammalian serum can be calf serum (FCS).

在其他實施例中，可使用包含核酸部分之一聚合調節試劑來處理表面。適合之含核酸聚合調節試劑之非限制性實例係如上文所討論且任何適當之含核酸聚合調節試劑可用以處理表面。 In other embodiments, the surface can be treated using a polymerization conditioning reagent comprising one of the nucleic acid moieties. Non-limiting examples of suitable nucleic acid containing polymerization conditioning reagents are as discussed above and any suitable nucleic acid containing polymerization conditioning reagent can be used to treat the surface.

在一些實施例中，一種以上聚合調節試劑之一混合物可用以處理生長室之表面。 In some embodiments, a mixture of one or more polymerization conditioning agents can be used to treat the surface of the growth chamber.

在一些其他實施例中，調節步驟可包含：使用至少一細胞黏著阻斷分子來處理至少一生長室之至少一表面。在一些實施例中，可在自微流體器件輸出細胞之前執行使用至少一細胞黏著阻斷分子來處理至少一生長室之至少一表面之步驟。在一些實施例中，調節步驟可包含：使用至少一細胞黏著阻斷分子來預培育細胞。在一些實施例中，至少一細胞黏著阻斷分子可用以破壞肌動蛋白纖維形成。在一些實施例中，細胞黏著阻斷分子可為細胞鬆弛素B。在其他實施例中，至少一細胞黏著阻斷分子可阻斷整合素受體。在一些實施例中，細胞黏著阻斷分子可包含肽，其含有一RGD基元。在一些其他實施例中，至少一細胞黏著阻斷分子可減弱細胞與DNA污損表面之結合。可減弱細胞與DNA污損表面之結合之細胞黏著阻斷分子可包含一DNase 1蛋白。在其他實施例中，至少一細胞黏著阻斷分子可包含一小分子纖連蛋白抑制劑。在其他實施例中，至少一細胞黏著阻斷分子可為一抗體，例如一抗B1整合素抗體。在一些實施例中，至少一細胞黏著阻斷分子可包含多於一種類型之細胞黏著阻斷分子之一組合。 In some other embodiments, the adjusting step can include treating at least one surface of the at least one growth chamber with at least one cell adhesion blocking molecule. In some embodiments, the step of treating at least one surface of at least one growth chamber using at least one cell adhesion blocking molecule can be performed prior to outputting the cells from the microfluidic device. In some embodiments, the adjusting step can comprise pre-culturing the cells using at least one cell adhesion blocking molecule. In some embodiments, at least one cell adhesion blocking molecule can be used to disrupt actin fiber formation. In some embodiments, the cell adhesion blocking molecule can be cytochalasin B. In other embodiments, at least one cell adhesion blocking molecule blocks the integrin receptor. In some embodiments, the cell adhesion blocking molecule can comprise a peptide comprising an RGD motif. In some other embodiments, at least one cell adhesion blocking molecule attenuates binding of the cell to the DNA stained surface. A cell adhesion blocking molecule that attenuates binding of a cell to a DNA stained surface can comprise a DNase 1 protein. In other embodiments, at least one cell adhesion blocking molecule can comprise a small molecule of fibronectin inhibitor. In other embodiments, the at least one cell adhesion blocking molecule can be an antibody, such as a primary anti-B1 integrin antibody. In some embodiments, at least one cell adhesion blocking molecule can comprise a combination of one of more than one type of cell adhesion blocking molecule.

在其他實施例中，調節包含：將生長室之表面加熱至約30℃之一溫度。在一些實施例中，方法包含：將表面加熱至至少約25℃、約26℃、約27℃、約28℃、約29℃、約30℃、約31℃、約32℃、約33℃、約34℃、約35℃、約36℃、約37℃、約38℃、約39℃或約40℃之一溫度。在一些實施例中，方法包含：將表面加熱至大於約25℃之一溫度。在其他實施例中，方法包含：將表面加熱至自約30℃至約40℃、自約35℃至約40℃或自約36℃至約38℃之範圍內之一溫度。在一些實施例中，方法包含：將表面加熱至至少約30℃之一溫度。在一些實施例中，加熱表面包含藉由使用一聚合物來處理表面而調節之至少一表面。 In other embodiments, conditioning comprises heating the surface of the growth chamber to a temperature of about 30 °C. In some embodiments, the method comprises: heating the surface to at least about 25 ° C, about 26 ° C, about 27 ° C, about 28 ° C, about 29 ° C, about 30 ° C, about 31 ° C, about 32 ° C, about 33 ° C, A temperature of about 34 ° C, about 35 ° C, about 36 ° C, about 37 ° C, about 38 ° C, about 39 ° C or about 40 ° C. In some embodiments, the method includes heating the surface to a temperature greater than about 25 °C. In other embodiments, the method comprises heating the surface to a temperature ranging from about 30 ° C to about 40 ° C, from about 35 ° C to about 40 ° C, or from about 36 ° C to about 38 ° C. In some embodiments, the method comprises: heating the surface to a temperature of at least about 30 °C. In some embodiments, the heated surface comprises at least one surface that is adjusted by treating the surface with a polymer.

選殖群體Colony

本文中所描述之方法亦包含其中將僅一個生物細胞引入至至少一生長室之方法。提供用於在包含一微流體器件之一系統中選殖一生物細胞之一方法，該微流體器件具有：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包含一隔離區域及一連接區域，該隔離區域與該連接區域流體地連接且該連接區域包含至該流動區域之一近端開口，該方法包含下列步驟：將該生物細胞引入至該至少一生長室中，其中該至少一生長室經構形以具有經調節以支援細胞生長、存活性、可移植性或其等之任何組合之至少一表面；及在至少足以擴增該生物細胞而產生生物細胞之一選殖群體之一長時間週期內培育該生物細胞。在一些實施例中，該系統可為如本文中所描述之任何系統。該微流體器件可為如本文中所描述之任何微流體器件。 The methods described herein also include methods in which only one biological cell is introduced into at least one growth chamber. Providing a method for colonizing a biological cell in a system comprising a microfluidic device, the microfluidic device having: a flow region configured to flow with one of the first fluid media; and at least one a growth chamber comprising an isolation region and a connection region fluidly coupled to the connection region and the connection region comprising a proximal opening to the flow region, the method comprising the steps of: introducing the biological cell to In the at least one growth chamber, wherein the at least one growth chamber is configured to have at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, and at least sufficient to amplify the organism The cell produces one of the colonization groups of one of the biological cells to grow the biological cell for a long period of time. In some embodiments, the system can be any system as described herein. The microfluidic device can be any of the microfluidic devices as described herein.

在用於選殖一生物細胞之該方法之一些實施例中，該至少一調節表面可包含共價鍵聯至該表面之一鍵聯基團，且該鍵聯基團可鍵聯至經構形以支援該微流體器件內之該一或多個生物細胞之細胞生長、存活性或可移植性之一部分。在一些實施例中，該鍵聯基團可包含矽烷氧基鍵聯基團。在其他實施例中，該鍵聯基團可包含膦酸酯鍵聯基團。在一些實施例中，該鍵聯基團可間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。在其他實施例中，該鍵聯基團可直接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯基團可經由連接至一鍵聯子而間接鍵聯至經構形以支援細胞生長、存活性或可移動性之該部分。在一些實施例中，該鍵聯基團可經由連接至一鍵聯子之一第一端而間接鍵聯至經構形以支援細胞生長、存活性或可移動性之該部分。在一些實施例中，該鍵聯子可進一步包含一直鏈部分，其中該直鏈部分之一主鏈包括選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子 之任何組合之1個至200個非氫原子。在一些實施例中，該直鏈部分之該主鏈可包含一或多個伸芳基部分。在其他實施例中，該鍵聯子可包含伸***基部分。在一些實施例中，該伸***基部分可中斷該鍵聯子之該直鏈部分或可連接至該鍵聯子之該直鏈部分之一第二端處。在各種實施例中，經構形以支援細胞生長及/或存活性及/或可移植性之該部分可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。在一些實施例中，該至少一調節表面包括烷基或全氟烷基部分。在其他實施例中，該至少一調節表面包括伸烷基醚部分或聚葡萄糖部分。 In some embodiments of the method for selecting a biological cell, the at least one conditioning surface can comprise a covalently bonded to one of the surface linking groups, and the linking group can be bonded to the conformation Forming to support one of the cell growth, viability, or portability of the one or more biological cells within the microfluidic device. In some embodiments, the linking group can comprise a decyloxy linking group. In other embodiments, the linking group can comprise a phosphonate linkage group. In some embodiments, the linking group can be indirectly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. In other embodiments, the linking group can be directly linked to that portion configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly linked to the moiety configured to support cell growth, viability or mobility via attachment to a linkage. In some embodiments, the linking group can be indirectly linked to the portion configured to support cell growth, viability, or mobility via attachment to one of the first ends of a linkage. In some embodiments, the linker may further comprise a straight chain moiety, wherein one of the linear chain backbones comprises a halogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. 1 to 200 non-hydrogen atoms in any combination. In some embodiments, the backbone of the linear moiety can comprise one or more extended aryl moieties. In other embodiments, the linker can comprise a triazolyl moiety. In some embodiments, the triazole moiety can interrupt the linear portion of the bond or can be attached to the second end of one of the linear portions of the bond. In various embodiments, the portion configured to support cell growth and/or viability and/or portability may comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or a polysaccharide (which may include, but is not limited to, polydextrose); alcohols (including but not limited to, propargyl alcohol); polyols including, but not limited to, polyvinyl alcohol; alkyl ethers, which comprise ( But not limited to) polyethylene glycol; polyelectrolyte (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amine group (which includes derivatives thereof such as, but not limited to, alkylated amine groups, hydroxyalkanes) a amide group, a sulfonium salt, and a heterocyclic group containing an unaromatized nitrogen ring atom, such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid including, but not limited to, propiolic acid ( It may provide a carboxylic acid anion surface; a phosphonic acid comprising, but not limited to, an ethynylphosphonic acid (which may provide a phosphonate anionic surface); a sulfonic acid anion; a carboxybetaine; a sulfobetaine; an amine sulfonic acid; Or an amino acid. In some embodiments, the at least one conditioning surface comprises an alkyl or perfluoroalkyl moiety. In other embodiments, the at least one conditioning surface comprises an alkyl ether moiety or a polydextrose moiety.

在各種實施例中，該方法可進一步包含下列步驟：調節該至少一生長室之該至少一表面。在一些實施例中，調節包含：使用支援該微流體器件內之細胞可移植性之一或多個試劑來處理該至少一表面。在一些實施例中，該調節可包含：使用包含一聚合物之一調節試劑來處理該至少一生長室之該至少一表面。在一些實施例中，該聚合物可包含伸烷基醚部分。在一些實施例中，該聚合物可包含羧酸部分。在一些實施例中，該聚合物可包含糖類部分。在其他實施例中，該聚合物可包含磺酸部分。在其他實施例中，該聚合物可包含胺基酸部分。在進一步實施例中，該聚合物可包含核酸部分。在一些實施例中，該調節可包含：使用哺乳動物血清之一或多個組分來處理該至少一生長 室之該至少一表面。在一些實施例中，該哺乳動物血清可為胎牛血清(FBS)或小牛血清(FCS)。在各種實施例中，調節可包含：使用至少一細胞黏著阻斷分子來處理該至少一生長室之至少一表面。在一些實施例中，該至少一細胞黏著阻斷分子可包含一含RGD之肽。在其他實施例中，該至少一細胞黏著阻斷分子可為細胞鬆弛素B、一整合素抗體、纖連蛋白之一抑制劑或一DNase 1蛋白。在各種實施例中，調節可包含：使用多於一種類型之細胞黏著阻斷分子之一組合來處理該至少一生長室之至少一表面。 In various embodiments, the method can further include the step of adjusting the at least one surface of the at least one growth chamber. In some embodiments, the adjusting comprises treating the at least one surface with one or more reagents that support cell portability within the microfluidic device. In some embodiments, the adjusting can include treating the at least one surface of the at least one growth chamber with a conditioning reagent comprising a polymer. In some embodiments, the polymer can comprise an alkyl ether moiety. In some embodiments, the polymer can comprise a carboxylic acid moiety. In some embodiments, the polymer can comprise a carbohydrate moiety. In other embodiments, the polymer can comprise a sulfonic acid moiety. In other embodiments, the polymer can comprise an amino acid moiety. In further embodiments, the polymer can comprise a nucleic acid moiety. In some embodiments, the adjusting can comprise: treating the at least one growth using one or more components of the mammalian serum At least one surface of the chamber. In some embodiments, the mammalian serum can be fetal bovine serum (FBS) or calf serum (FCS). In various embodiments, the adjusting can include treating at least one surface of the at least one growth chamber with at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule can comprise an RGD-containing peptide. In other embodiments, the at least one cell adhesion blocking molecule can be cytochalasin B, an integrin antibody, an inhibitor of fibronectin, or a DNase 1 protein. In various embodiments, the adjusting can comprise treating at least one surface of the at least one growth chamber using a combination of one of more than one type of cell adhesion blocking molecules.

在各種實施例中，該調節可包含：將該至少一生長室之該至少一表面加熱至約30℃之一溫度。 In various embodiments, the adjusting can include heating the at least one surface of the at least one growth chamber to a temperature of about 30 °C.

在各種實施例中，該方法可進一步包含下列步驟：將一第一流體培養基引入至該微流體器件之該流動區域之一微流體通道中。在一些實施例中，可在引入該生物細胞之前執行引入該第一流體培養基。在一些實施例中，將該生物細胞引入至該至少一生長室中可包含：使用具有足以移動該生物細胞之力量之一介電泳(DEP)力。在一些實施例中，可光學地啟動該DEP力。在一些實施例中，可由光電鑷子(OET)產生該DEP力。在一些其他實施例中，將該生物細胞引入至該至少一生長室中可包含：使用流體流動及/或重力。 In various embodiments, the method can further comprise the step of introducing a first fluid medium into the microfluidic channel of one of the flow regions of the microfluidic device. In some embodiments, introduction of the first fluid medium can be performed prior to introduction of the biological cell. In some embodiments, introducing the biological cell into the at least one growth chamber can comprise: using a dielectrophoresis (DEP) force having a force sufficient to move the biological cell. In some embodiments, the DEP force can be optically activated. In some embodiments, the DEP force can be generated by a photoelectric tweezers (OET). In some other embodiments, introducing the biological cell into the at least one growth chamber can comprise: using fluid flow and/or gravity.

在一些實施例中，將該生物細胞引入至該至少一生長室中可進一步包含：將該生物細胞引入至該至少一生長室之一隔離區域中。在一些實施例中，該至少一生長室之該隔離區域可具有足以支援細胞擴增至不超過1×10²個細胞之尺寸。在一些實施例中，該隔離區域可至少實質上填充有一第二流體培養基。在一些實施例中，該流動區域可流體地連接至該至少一生長室之一連接區域之一近端開口，且其中該連接區域亦可進一步流體地連接至該生長室之該隔離區域。 In some embodiments, introducing the biological cell into the at least one growth chamber can further comprise: introducing the biological cell into an isolated region of the at least one growth chamber. In some embodiments, the isolated region of the at least one growth chamber can have a size sufficient to support cell expansion to no more than 1 x 10 ² cells. In some embodiments, the isolation region can be at least substantially filled with a second fluid medium. In some embodiments, the flow region can be fluidly coupled to a proximal opening of one of the at least one growth chamber connection region, and wherein the connection region can be further fluidly coupled to the isolation region of the growth chamber.

在各種實施例中，該方法可進一步包含下列步驟：在該培育步 驟期間灌注該第一流體培養基，其中可經由該微流體器件之至少一入口而引入該第一流體培養基且其中可經由該微流體器件之至少一出口而輸出視情況包括來自該第二流體培養基之組分之該第一流體培養基。在一些實施例中，灌注可為非連續的。在一些其他實施例中，灌注可為週期性的。在其他實施例中，灌注可為不規律的。在一些實施例中，可依足以容許該隔離區域中之該第二流體培養基之組分擴散至該流動區域中之該第一流體培養基中及/或該第一流體培養基之組分擴散至該隔離區域中之該第二流體培養基中之一速率執行灌注該第一流體培養基；及該第一流體培養基可實質上不流動至該隔離區域中。在一些實施例中，可約每隔10分鐘至約每隔30分鐘執行灌注該第一流體培養基達約45秒至約90秒之一持續時間。在一些實施例中，可執行灌注該第一流體培養基達約2小時至約4小時之一持續時間。在一些實施例中，培育該至少一生物細胞之該時間週期可為自約1天至約10天。 In various embodiments, the method can further comprise the step of: The first fluid medium is perfused during the step, wherein the first fluid medium can be introduced via at least one inlet of the microfluidic device and wherein the output can be via at least one outlet of the microfluidic device, optionally including from the second fluid medium The first fluid medium of the component. In some embodiments, the perfusion can be non-continuous. In some other embodiments, the perfusion can be periodic. In other embodiments, the perfusion can be irregular. In some embodiments, the component of the second fluid medium in the isolation region may be allowed to diffuse into the first fluid medium in the flow region and/or the components of the first fluid medium may be diffused to the The first fluid medium is perfused at a rate in the second fluid medium in the isolated region; and the first fluid medium may not substantially flow into the isolated region. In some embodiments, perfusing the first fluid medium can be performed from about every 10 minutes to about every 30 minutes for a duration of from about 45 seconds to about 90 seconds. In some embodiments, perfusion of the first fluid medium can be performed for a duration of from about 2 hours to about 4 hours. In some embodiments, the time period for culturing the at least one biological cell can be from about 1 day to about 10 days.

在一些實施例中，該第一流體培養基之一組合物可包含液體組分及氣體組分。在各種實施例中，該方法可進一步包含下列步驟：在將該第一流體培養基引入至該微流體器件中之前使該第一流體培養基飽含溶解氣體分子。在各種實施例中，該方法可進一步包含下列步驟：使該微流體器件與能夠使該第一流體培養基或該第二流體培養基飽含溶解氣體分子之一氣體環境接觸。在各種實施例中，該方法可進一步包含下列步驟：在引入溶解氣體分子之後緩和該第一流體培養基之一pH。在一些實施例中，可在經由該入口而引入該第一流體培養基之前於一儲液器中執行使該第一流體培養基飽含該等氣體組分，在該儲液器與該入口之間的一可透氣連接器中執行使該第一流體培養基飽含該等氣體組分，或經由該微流體器件之一蓋之一可透氣部分而執行使該第一流體培養基飽含該等氣體組分。在一些實施例中，該第一 流體培養基之一組合物可包含來自一餵食細胞培養基之至少一分泌組分。 In some embodiments, one of the first fluid media compositions can comprise a liquid component and a gas component. In various embodiments, the method can further comprise the step of saturating the first fluid medium with dissolved gas molecules prior to introducing the first fluid medium into the microfluidic device. In various embodiments, the method can further comprise the step of contacting the microfluidic device with a gaseous environment capable of rendering the first fluid medium or the second fluid medium saturated with a dissolved gas molecule. In various embodiments, the method can further comprise the step of easing the pH of one of the first fluid media after introduction of the dissolved gas molecules. In some embodiments, the first fluid medium may be saturated with the gas component in a reservoir prior to introduction of the first fluid medium via the inlet, between the reservoir and the inlet Performing the first fluid medium with the gas component in a gas permeable connector or performing a gas permeable portion via one of the microfluidic devices is performed to saturate the first fluid medium with the gas components. In some embodiments, the first One of the fluid medium compositions can comprise at least one secretory component from a feeding cell culture medium.

在各種實施例中，該方法可進一步包含下列步驟：在經由該至少一出口而輸出該第一流體培養基時偵測該第一流體培養基之pH。在一些實施例中，可在直接接近於該至少一出口之一位置處執行該偵測步驟。在各種實施例中，該方法可進一步包含下列步驟：在經由該至少一入口而引入該第一流體培養基時偵測該第一流體培養基之PH。在一些實施例中，感測器可為一光學感測器。在各種實施例中，該方法可進一步包含下列步驟：更改該第一流體培養基之一組合物。 In various embodiments, the method can further include the step of detecting the pH of the first fluid medium as it is output via the at least one outlet. In some embodiments, the detecting step can be performed at a location that is directly adjacent to the at least one outlet. In various embodiments, the method can further comprise the step of detecting the pH of the first fluid medium upon introduction of the first fluid medium via the at least one inlet. In some embodiments, the sensor can be an optical sensor. In various embodiments, the method can further comprise the step of modifying one of the compositions of the first fluid medium.

在各種實施例中，該方法可進一步包含下列步驟：監測該至少一生長室及其內所含之任何細胞之一影像。 In various embodiments, the method can further comprise the step of monitoring an image of the at least one growth chamber and any cells contained therein.

在各種實施例中，該生物細胞可為一哺乳動物細胞。在一些實施例中，該生物細胞可為一免疫細胞。在一些實施例中，該生物細胞可為一淋巴細胞或一白血球。在一些實施例中，該生物細胞可為一B細胞、一T細胞、一NK細胞、巨噬細胞或樹突細胞。在一些實施例中，該生物細胞可為一貼附細胞。在一些實施例中，該生物細胞可為一融合瘤細胞。 In various embodiments, the biological cell can be a mammalian cell. In some embodiments, the biological cell can be an immune cell. In some embodiments, the biological cell can be a lymphocyte or a white blood cell. In some embodiments, the biological cell can be a B cell, a T cell, an NK cell, a macrophage, or a dendritic cell. In some embodiments, the biological cell can be a patched cell. In some embodiments, the biological cell can be a fusion tumor cell.

在一些實施例中，該生物細胞可為複數個生物細胞且該至少一生長室係複數個生長室。在各種實施例中，該方法可進一步包含下列步驟：將該複數個生物細胞之僅一者移動至該複數個生長室之各者中。 In some embodiments, the biological cell can be a plurality of biological cells and the at least one growth chamber is a plurality of growth chambers. In various embodiments, the method can further comprise the step of moving only one of the plurality of biological cells into each of the plurality of growth chambers.

在選殖一生物細胞之該方法之一些實施例中，該調節表面可進一步包含一可裂解部分。該方法可包含下列步驟：在使該選殖群體之一或多個生物細胞自該生長室或其之該隔離區域輸出之前使該可裂解部***解。 In some embodiments of the method of selecting a biological cell, the conditioning surface can further comprise a cleavable moiety. The method can include the step of lysing the cleavable moiety prior to exporting one or more of the colonized populations from the growth chamber or the isolated region thereof.

在各種實施例中，該方法可進一步包含下列步驟：使該選殖群體之一或多個生物細胞自該生長室或其之該隔離區域輸出。在一些實施例中，輸出可包含：使用足以移動該一或多個生物細胞之一強介電泳(DEP)力。在一些實施例中，光學地致動該DEP力。在一些實施例中，可由光電鑷子(OET)產生該DEP力。在一些實施例中，輸出可包含：使用流體流動及/或重力。在一些實施例中，輸出可包含：使用作用於該生長室或其之該隔離區域上方之一可變形蓋區域上之壓縮力。在各種實施例中，該方法可進一步包含下列步驟：使該選殖群體之一或多個生物細胞自該流動區域輸出而離開該微流體器件。在一些實施例中，輸出可包含：使用足以移動該一或多個生物細胞之一強DEP力。在一些實施例中，光學地致動該DEP力。在一些實施例中，可由光電鑷子(OET)產生該DEP力。在一些實施例中，輸出可包含：使用流體流動及/或重力。 In various embodiments, the method can further comprise the step of outputting one or more biological cells of the selected population from the growth chamber or the isolated region thereof. In some embodiments, the outputting can comprise: using a strong dielectrophoresis (DEP) force sufficient to move one of the one or more biological cells. In some embodiments, the DEP force is optically actuated. In some embodiments, the DEP force can be generated by a photoelectric tweezers (OET). In some embodiments, the output can include using fluid flow and/or gravity. In some embodiments, the outputting can include using a compressive force acting on the growth chamber or one of the deformable cover regions above the isolation region. In various embodiments, the method can further comprise the step of causing one or more biological cells of the population to be exported from the flow region to exit the microfluidic device. In some embodiments, the outputting can include using a strong DEP force sufficient to move one of the one or more biological cells. In some embodiments, the DEP force is optically actuated. In some embodiments, the DEP force can be generated by a photoelectric tweezers (OET). In some embodiments, the output can include using fluid flow and/or gravity.

套組Set

本發明可提供用於培養一生物細胞之套組，其中該套組包含：一微流體器件，其具有經構形以含有一第一流體培養基之一流動之一流動區域及至少一生長室；及一表面調節試劑。在此實施例中，未預處理該至少一生長室來調節該至少一生長室之至少一表面，而是藉由在引入(若干)細胞之前使用該表面調節試劑來處理而產生該調節表面。本發明亦提供用於培養一生物細胞之其他套組，其中該套組包含一微流體器件，其具有：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包括一隔離區域及一連接區域，其中該隔離區域與該連接區域流體地連接且該連接區域包括至該流動區域之一近端開口；及其中該至少一生長室進一步包括經調節以支援細胞生長、存活性、可移植性或其等之任何組合之至少一表面。本發明亦提供用於培養一生物細胞之其他套組，其包含一微流體器件，該 微流體器件包含：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包含一隔離區域及一連接區域，其中該隔離區域與該連接區域流體地連接且該連接區域具有至該流動區域之一近端開口；其中該至少一生長室具有至少一表面，其具有一表面改質配位基。替代地，本發明可提供用於培養一生物細胞之套組，其中該套組包含：一微流體器件，其具有一流動區域(其經構形以含有一第一流體培養基之一流動)及至少一生長室(其具有可支援細胞生長、存活性、可移植性或其等之任何組合之至少一調節表面)；及一表面調節試劑。該等套組之任何者之微流體器件可為微流體器件100、200、240、290、400、500A至500E或600之任何者且具有上文所描述之特徵之任何者。 The present invention can provide a kit for culturing a biological cell, wherein the kit comprises: a microfluidic device having a flow region configured to contain one of the first fluid medium flows and at least one growth chamber; And a surface conditioning reagent. In this embodiment, the at least one growth chamber is not pretreated to adjust at least one surface of the at least one growth chamber, but the conditioning surface is created by treatment with the surface conditioning agent prior to introduction of the (several) cells. The invention also provides other kits for culturing a biological cell, wherein the kit comprises a microfluidic device having: a flow region configured to flow with one of the first fluid media; and at least one a growth chamber comprising an isolation region and a connection region, wherein the isolation region is fluidly coupled to the connection region and the connection region includes a proximal opening to one of the flow regions; and wherein the at least one growth chamber further comprises an adjustment To support at least one surface of cell growth, viability, portability, or any combination thereof. The invention also provides other kits for culturing a biological cell, comprising a microfluidic device, The microfluidic device comprises: a flow region configured to flow with one of the first fluid media; and at least one growth chamber including an isolation region and a connection region, wherein the isolation region is fluidly associated with the connection region Connecting and the connection region has a proximal opening to one of the flow regions; wherein the at least one growth chamber has at least one surface having a surface modifying ligand. Alternatively, the present invention can provide a kit for culturing a biological cell, wherein the kit comprises: a microfluidic device having a flow region (configured to contain one of the first fluid media flows) and At least one growth chamber (having at least one conditioning surface that supports cell growth, viability, portability, or any combination thereof, etc.); and a surface conditioning agent. The microfluidic device of any of the kits can be any of the microfluidic devices 100, 200, 240, 290, 400, 500A through 500E or 600 and have any of the features described above.

該等套組之任何者之微流體器件可進一步包含一微流體通道，其包含該流動區域之至少一部分；且該器件可進一步包含一生長室，其具有直接通至該微流體通道中之一連接區域。該生長室可進一步包含一隔離區域。該隔離區域可流體地連接至該連接區域且可經構形以含有一第二流體培養基，其中當該流動區域及該至少一生長室實質上分別填充有一第一流體培養基及一第二流體培養基時，該第二流體培養基之組分擴散至該第一流體培養基中及/或該第一流體培養基之組分擴散至該第二流體培養基中；及該第一培養基實質上不流動至該隔離區域中。 The microfluidic device of any of the kits can further comprise a microfluidic channel comprising at least a portion of the flow region; and the device can further comprise a growth chamber having one of the microfluidic channels directly connected thereto Connection area. The growth chamber can further comprise an isolation region. The isolation region can be fluidly coupled to the connection region and can be configured to contain a second fluid medium, wherein the flow region and the at least one growth chamber are substantially filled with a first fluid medium and a second fluid medium, respectively a component of the second fluid medium diffuses into the first fluid medium and/or a component of the first fluid medium diffuses into the second fluid medium; and the first medium does not substantially flow to the isolation In the area.

在該等套組之任何者之各種實施例中，生長室可如同圖1A至圖1C、圖2、圖3及圖4A至圖4C之生長室124、126、128、130、244、246、248或436般構形，其中該生長室之該隔離區域可具有經構形以支援不超過約1×10³個、約5×10²個、約4×10²個、約3×10²個、約2×10²個、約1×10²個、約50個、約25個、約15個或約10個培養細胞之一體積。在其他實施例中，該生長室之該隔離區域具有可支援高達約10 個、約50個或約1×10²個細胞之一體積。如上文所討論，該等生長室之任何構形可用於該等套組之該等微流體器件之該等生長室中。 In various embodiments of any of the sets, the growth chamber can be like the growth chambers 124, 126, 128, 130, 244, 246 of Figures 1A-1C, 2, 3, and 4A-4C. 248 or 436-like configuration, wherein the isolation region of the growth chamber can have a configuration to support no more than about 1×10 ³ , about 5×10 ² , about 4×10 ² , about 3×10 ² One volume of about 2 x 10 ² , about 1 x 10 ² , about 50, about 25, about 15, or about 10 cultured cells. In other embodiments, the isolated region of the growth chamber has a volume that supports up to about 10, about 50, or about 1 x 10 ² cells. As discussed above, any configuration of the growth chambers can be used in the growth chambers of the microfluidic devices of the kits.

在該等套組之任何者之各種實施例中，該等生長室之大小可經構形以維持不超過1×10²個生物細胞，其中該等生長室之體積可不超過1×10⁷立方微米。在其他實施例中，可維持不超過1×10²個生物細胞，該等生長室之體積可不超過5×10⁶立方微米。在其他實施例中，可維持不超過50個生物細胞，且該等生長室之體積可不超過1×10⁶立方微米或不超過5×10⁵立方微米。在該等套組中，該等微流體器件可具有任何數目個如上文所討論之生長室。 In various embodiments of any of the kits, the growth chambers can be sized to maintain no more than 1 x 10 ² biological cells, wherein the growth chambers can have a volume of no more than 1 x 10 ⁷ cubes. Micron. In other embodiments, no more than 1 x 10 ² biological cells may be maintained, and the growth chamber may have a volume of no more than 5 x 10 ⁶ cubic microns. In other embodiments, no more than 50 biological cells may be maintained, and the growth chamber may have a volume of no more than 1 x 10 ⁶ cubic micrometers or no more than 5 x 10 ⁵ cubic micrometers. In such kits, the microfluidic devices can have any number of growth chambers as discussed above.

該等套組之任何者之微流體器件可進一步包含經構形以將流體培養基(例如第一流體培養基或第二流體培養基)輸入至該流動區域中之至少一入口及經構形以在該流體培養基(例如廢第一流體培養基)自該流動區域退出時接收該流體培養基之至少一出口。 The microfluidic device of any of the kits can further comprise a configuration to input a fluid medium (eg, a first fluid medium or a second fluid medium) into at least one inlet of the flow region and configured to The fluid medium (eg, spent first fluid medium) receives at least one outlet of the fluid medium as it exits from the flow region.

該等套組之任何者之微流體器件可進一步包含具有複數個DEP電極之一基板，其中該基板之一表面形成該生長室及該流動區域之一表面。該複數個DEP電極可經構形以產生足以使一或多個生物細胞(例如一選殖群體)移動至一生長室或其之該隔離區域中或使一生物細胞培養基之一或多個細胞移出一生長室或其之該隔離區域之一強介電泳(DEP)力。可光學地致動該等DEP電極且因此光學地致動該DEP力。此等光學致動之DEP電極可為虛擬電極(例如歸因於入射光而具有增強導電性之一非晶矽基板之區域)、光電晶體或由一對應光電晶體接通或切斷之電極。替代地，可電致動該DEP電極且因此電致動該DEP力。在一些其他實施例中，該微流體器件可進一步包含具有複數個電晶體之一基板，其中該基板之一表面形成該生長室及該流動區域之一表面。該複數個電晶體能夠產生足以將該生物細胞引入至該生長室或其之該隔離區域中或將一生物細胞培養基之一或多個細胞移出該生長 室或其之該隔離區域之一強介電泳(DEP)力。可光學地致動該複數個電晶體之各者，且可由光電鑷子產生該DEP力。 The microfluidic device of any of the kits can further comprise a substrate having a plurality of DEP electrodes, wherein a surface of the substrate forms a surface of the growth chamber and the flow region. The plurality of DEP electrodes can be configured to generate one or more cells sufficient to move one or more biological cells (eg, a selection population) into a growth chamber or the isolated region thereof or to a biological cell culture medium A strong dielectric electrophoresis (DEP) force is removed from a growth chamber or one of its isolated regions. The DEP electrodes can be optically actuated and thus optically actuate the DEP force. The optically actuated DEP electrodes can be virtual electrodes (e.g., regions of an amorphous germanium substrate having enhanced conductivity due to incident light), optoelectronic crystals, or electrodes that are turned on or off by a corresponding optoelectronic crystal. Alternatively, the DEP electrode can be electrically actuated and thus electrically actuated. In some other embodiments, the microfluidic device can further comprise a substrate having a plurality of transistors, wherein a surface of the substrate forms a surface of the growth chamber and the flow region. The plurality of transistors are capable of generating sufficient to introduce the biological cell into the growth chamber or the isolated region thereof or to remove one or more cells of a biological cell culture medium from the growth The chamber or one of its isolated regions is a strong dielectrophoresis (DEP) force. Each of the plurality of transistors can be optically actuated and the DEP force can be generated by photo-electric tweezers.

該等套組之任何者之微流體器件可進一步包含在該至少一生長室或其隔離區域上方之一可變形蓋區域，藉此按壓該可變形蓋區域施加使一或多個生物細胞(例如一選殖群體)自該生長區域輸出至該流動區域之一力。 The microfluidic device of any of the kits can further comprise a deformable lid region above the at least one growth chamber or an isolation region thereof, whereby pressing the deformable lid region applies one or more biological cells (eg, A colony population is output from the growth zone to one of the flow zones.

該等套組之任何者之微流體器件可經構形以具有實質上不可透氣之一蓋。替代地，該蓋之一部分之全部可經構形為可透氣的。該蓋之該可透氣部分可滲透二氧化碳、氧氣及氮氣之至少一者。在一些實施例中，該蓋(或其之一部分)可滲透二氧化碳、氧氣或氮氣之一者以上之一組合。 The microfluidic device of any of the kits can be configured to have a substantially gas impermeable cover. Alternatively, all of the portion of the cover can be configured to be breathable. The gas permeable portion of the cover is permeable to at least one of carbon dioxide, oxygen, and nitrogen. In some embodiments, the cover (or a portion thereof) is permeable to one of more than one of carbon dioxide, oxygen, or nitrogen.

該等套組之任何者可進一步包含經構形以含有一流體培養基之一儲液器。該儲液器可流體地連接至本文中所描述之微流體器件之任何者。該儲液器可經構形使得存在於該儲液器中之該流體培養基可由能夠使該流體培養基飽含溶解氣體分子之一氣體環境接觸。該儲液器可進一步經構形以含有與該流體培養基流體接觸之一餵食細胞群體。 Any of the sets may further comprise a reservoir configured to contain a fluid medium. The reservoir can be fluidly coupled to any of the microfluidic devices described herein. The reservoir can be configured such that the fluid medium present in the reservoir can be contacted by a gaseous environment capable of rendering the fluid medium saturated with a dissolved gas molecule. The reservoir can be further configured to contain one of the feeding cell populations in fluid contact with the fluid medium.

該等套組之任何者可包含經構形以連接至該微流體器件之一入口及/或一出口之至少一連接導管。該連接導管亦可經構形以連接至一儲液器或一流量控制器，諸如一泵組件。該連接導管可為可透氣的。該可透氣連接導管可滲透二氧化碳、氧氣及氮氣之至少一者。在一些實施例中，該可透氣導管可滲透二氧化碳、氧氣或氮氣之一者以上之一組合。 Any of the sets may include at least one connecting conduit configured to connect to one of the inlets and/or an outlet of the microfluidic device. The connecting conduit can also be configured to connect to a reservoir or a flow controller, such as a pump assembly. The connecting conduit can be breathable. The gas permeable connecting conduit is permeable to at least one of carbon dioxide, oxygen, and nitrogen. In some embodiments, the gas permeable conduit is permeable to one or more of carbon dioxide, oxygen, or nitrogen.

該等套組之任何者可進一步包含經構形以偵測一第一流體培養基之一pH之一感測器。該感測器可連接至該微流體器件之一入口或其之一附接連接導管。替代地，該感測器可整合至該微流體器件。該感測器可連接至流體培養基進入該微流體器件之點之接近處。該套組 可包含經構形以偵測該微流體器件之該輸出處之流體培養基之一pH之一感測器。該感測器可連接至該微流體器件之一出口或其之一附接連接導管。替代地，該感測器可整合至該微流體器件。該感測器可連接至流體培養基退出該微流體器件之點之接近處。無論該感測器是否附接至該微流體器件之該入口及/或該出口，其均可為一光學感測器。一光學感測器可包含一LED及一整合比色感測器，該整合比色感測器可視情況為一色敏光電晶體。該套組可進一步包含驅動電子組件來控制該pH感測器且自該pH感測器接收輸出。該套組可進一步包含一pH偵測劑。該pH偵測劑可為可在可見光下偵測到之一pH敏感染料。 Any of the sets may further comprise a sensor configured to detect one of the pH of one of the first fluid media. The sensor can be coupled to one of the inlets of the microfluidic device or one of the attachment connection conduits. Alternatively, the sensor can be integrated into the microfluidic device. The sensor can be connected to the point where the fluid medium enters the microfluidic device. The set A sensor that is configured to detect one of the pHs of the fluid medium at the output of the microfluidic device can be included. The sensor can be coupled to one of the outlets of the microfluidic device or one of the attachment connection conduits. Alternatively, the sensor can be integrated into the microfluidic device. The sensor can be connected to the point where the fluid medium exits the microfluidic device. Whether the sensor is attached to the inlet and/or the outlet of the microfluidic device, it can be an optical sensor. An optical sensor can include an LED and an integrated colorimetric sensor, and the integrated colorimetric sensor can be a color-sensitive photoelectric crystal as the case may be. The kit can further include drive electronics to control the pH sensor and receive output from the pH sensor. The kit can further comprise a pH detecting agent. The pH detecting agent can be a pH sensitive dye that can be detected under visible light.

該等套組之任何者亦可包含一培養基，其具有能夠增強該微流體器件上之生物細胞存活性之組分。此等組分可為此項技術中已知之任何適合培養基組分，其包含上文針對流體培養基組分所討論之組分之任何者。 Any of such kits can also comprise a medium having a component that enhances the viability of biological cells on the microfluidic device. These components can be any suitable medium component known in the art, including any of the components discussed above for the fluid medium components.

該等套組之任何者可進一步包含用以偵測一生物細胞或一細胞群體之一狀態之至少一試劑。經構形以偵測細胞之狀態之試劑在此項技術中係熟知的，且可用以(例如)偵測一細胞是否活著或死亡，是否分泌所關注之一物質(諸如抗體、細胞激素或生長因子)，或是否具有所關注之細胞表面標記。此等試劑可用於(但不限於)本文中所描述之套組及方法中。 Any of the sets may further comprise at least one reagent for detecting the status of a biological cell or a population of cells. Agents that are configured to detect the state of a cell are well known in the art and can be used, for example, to detect whether a cell is alive or dead, and to secrete one of the substances of interest (such as antibodies, cytokines, or Growth factor), or whether it has a cell surface marker of interest. Such agents can be used, but are not limited to, in the kits and methods described herein.

針對本文中所提供之套組之任何者，該等套組之組分可位於分離容器中。針對提供於溶液中之套組之組分之任何者，該等組分可以約為用於本發明之方法中之濃度之1倍、5倍、10倍、100倍或1000倍之一濃度存在。 For any of the kits provided herein, the components of the kits can be located in separate containers. Any of the components of the kit provided in the solution may be present at a concentration of one, five, ten, 100 or 1000 times the concentration used in the method of the invention. .

針對其中未預處理微流體器件之至少一生長室來調節至少一生長室之至少一表面之套組，藉由使用表面調節試劑來處理而產生調節 表面；或針對包含一微流體器件(其具有：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其具有可支援細胞生長、存活性、可移植性或其等之任何組合之至少一調節表面)及一表面調節試劑之套組，可使用一表面調節試劑來預調節該生長室之該表面。該表面調節試劑可包含一聚合物，其可為用作一表面調節試劑之上文所描述之聚合物之任何一或多者。在一些實施例中，該表面調節試劑可包含一聚合物，其具有伸烷基醚部分、羧酸部分、磺酸部分、胺基酸部分、核酸部分、糖類部分或其等之任何組合。該表面調節試劑可包含一PEO-PPO嵌段共聚物，諸如Pluronic®聚合物(例如L44、L64、P85或F127)。在一些實施例中，該表面調節試劑可包含哺乳動物血清之一或多個組分。該哺乳動物血清可為胎牛血清(FBS)或小牛血清(FCS)。 Adjusting a set of at least one surface of at least one growth chamber for at least one growth chamber in which the microfluidic device is not pretreated, resulting in adjustment by treatment with a surface conditioning reagent Or a microfluidic device comprising: a flow region configured to flow with one of the first fluid media; and at least one growth chamber having cell growth, viability, and portability A set of at least one conditioning surface of a property or any combination thereof and a surface conditioning agent can be used to pre-condition the surface of the growth chamber using a surface conditioning agent. The surface conditioning agent can comprise a polymer which can be any one or more of the polymers described above for use as a surface conditioning agent. In some embodiments, the surface conditioning agent can comprise a polymer having any combination of an alkyl ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, an amino acid moiety, a nucleic acid moiety, a carbohydrate moiety, or the like. The surface conditioning agent can comprise a PEO-PPO block copolymer such as a Pluronic® polymer (eg, L44, L64, P85 or F127). In some embodiments, the surface conditioning agent can comprise one or more components of a mammalian serum. The mammalian serum can be fetal bovine serum (FBS) or calf serum (FCS).

替代地，用以調節該生長室之該表面之該表面調節試劑可包含於該套組中，與該微流體器件分離。在該套組之其他實施例中，包含一預調節微流體器件及不同於用以調節該生長室之該表面之該表面調節試劑的一表面調節試劑。該不同表面調節試劑可為上文所討論之表面調節試劑之任何者。在一些實施例中，一種以上表面調節試劑包含於該套組中。 Alternatively, the surface conditioning agent used to condition the surface of the growth chamber can be included in the kit and separated from the microfluidic device. In other embodiments of the kit, a pre-conditioning microfluidic device and a surface conditioning reagent different from the surface conditioning reagent for conditioning the surface of the growth chamber are included. The different surface conditioning agents can be any of the surface conditioning agents discussed above. In some embodiments, more than one surface conditioning agent is included in the kit.

在具有一微流體器件之該等套組(其中未預處理該微流體器件之該至少一生長室來調節該至少一表面)之各種實施例中，該套組亦可包含適合用於培養該一或多個生物細胞之一培養基。在一些實施例中，該套組亦可包含一培養基添加物，其包括能夠加強該生長室之一表面之調節之一試劑。該培養基添加物可包含如上文所討論之一調節試劑或增強該至少一生長室之該至少一表面支援細胞生長、存活性、可移植性或其等之任何組合之能力之另一化學物種。此可包含生長因子、內分泌素、抗氧化劑或維生素及其類似者。 In various embodiments having the set of microfluidic devices in which the at least one growth chamber of the microfluidic device is not pretreated to condition the at least one surface, the kit may also comprise a suitable one for culturing the set One of one or more biological cells. In some embodiments, the kit can also include a media supplement that includes an agent that enhances the conditioning of one of the surfaces of the growth chamber. The medium supplement may comprise another chemical species that modulates the agent or enhances the ability of the at least one surface of the at least one growth chamber to support cell growth, viability, portability, or the like, as discussed above. This may include growth factors, endocrine, antioxidants or vitamins and the like.

該套組亦可包含經構形以灌注至少該第一流體培養基之一流量控制器，其可為該微流體器件之一分離組件或可經併入為該微流體器件之部分。該控制器可經構形以非連續地灌注該流體培養基。因此，該控制器可經構形以依一週期性方式或依一不規律方式灌注該流體培養基。 The kit can also include a flow controller configured to perflate at least one of the first fluid medium, which can be a separate component of the microfluidic device or can be incorporated as part of the microfluidic device. The controller can be configured to non-continuously infuse the fluid medium. Thus, the controller can be configured to perfuse the fluid medium in a periodic manner or in an irregular manner.

在另一態樣中，提供一種用於培養一生物細胞之套組，其包含一微流體器件，其具有：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包括一隔離區域及一連接區域，其中該隔離區域與該連接區域流體地連接且該連接區域包括至該流動區域之一近端開口；且其中該至少一生長室進一步包括經調節以支援細胞生長、存活性、可移植性或其等之任何組合之至少一表面。該微流體器件可為如本文中所描述之任何微流體器件，且可具有如本文中所描述之生長室之任何者。該微流體器件可具有一基板，其具有本文中所描述之任何種類之一DEP構形。可光學地致動該DEP構形。該微流體器件之該基板可具有包含式1或式2之基板組合物(如本文中所描述)之一表面，且具有如上文所描述之全部特徵。 In another aspect, a kit for culturing a biological cell is provided, comprising a microfluidic device having: a flow region configured to contain a flow of a first fluid medium; and at least a growth chamber including an isolation region and a connection region, wherein the isolation region is fluidly coupled to the connection region and the connection region includes a proximal opening to one of the flow regions; and wherein the at least one growth chamber further includes a Adjusted to support at least one surface of cell growth, viability, portability, or any combination thereof. The microfluidic device can be any of the microfluidic devices as described herein and can have any of the growth chambers as described herein. The microfluidic device can have a substrate having one of the DEP configurations of any of the types described herein. The DEP configuration can be optically actuated. The substrate of the microfluidic device can have a surface comprising a substrate composition of Formula 1 or Formula 2 (as described herein) and having all of the features as described above.

該套組之該微流體器件之該至少一調節表面可包含糖類部分、伸烷基醚部分、胺基酸部分、烷基部分、氟烷基部分(其可包含全氟烷基部分)、陰離子部分、陽離子部分及/或兩性離子部分。在一些實施例中，該微流體器件之該調節表面可包含糖類部分、伸烷基醚部 分、烷基部分、氟烷基部分或胺基酸部分。該等烷基或全氟烷基部分可具有大於10個碳之一主鏈長度。在一些實施例中，用以支援細胞生長、存活性、可移植性或其等之任何組合之該調節表面可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(其包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸，其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。 The at least one conditioning surface of the microfluidic device of the kit may comprise a saccharide moiety, an alkyl ether moiety, an amino acid moiety, an alkyl moiety, a fluoroalkyl moiety (which may comprise a perfluoroalkyl moiety), an anion Part, cationic moiety and/or zwitterionic moiety. In some embodiments, the conditioning surface of the microfluidic device can comprise a carbohydrate moiety, an alkyl ether moiety a fraction, an alkyl moiety, a fluoroalkyl moiety or an amino acid moiety. The alkyl or perfluoroalkyl moiety can have a backbone length of greater than 10 carbons. In some embodiments, the conditioning surface to support cell growth, viability, portability, or any combination thereof, etc., can comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or a polysaccharide (which may include, but is not limited to, polydextrose); an alcohol (including but not limited to, propargyl alcohol); a polyol comprising, but not limited to, a polyvinyl alcohol; an alkyl ether, which comprises (but not limited to) polyethylene glycol; polyelectrolyte (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amine group (which includes derivatives thereof such as, but not limited to, alkylated amine groups, hydroxyl groups An alkylated amine group, a phosphonium salt, and a heterocyclic group containing an unaromatized nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl; a carboxylic acid comprising, but not limited to, a propiolic acid (which may provide a carboxylate anion surface); a phosphonic acid comprising, but not limited to, an ethynylphosphonic acid (which may provide a phosphonate anion surface); a sulfonic acid anion; a carboxybetaine; a sulfobetaine; an amine sulfonic acid Or amino acid.

在該套組之一些實施例中，該調節表面可包含共價鍵聯至該微流體器件之一表面之一鍵聯基團，且該鍵聯基團可鍵聯至經構形以支援該微流體器件內之該一或多個生物細胞之細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯基團可為矽烷氧基鍵聯基團。替代地，該鍵聯基團可為膦酸酯鍵聯基團。在該套組之一些實施例中，該調節表面之該鍵聯基團可直接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。 In some embodiments of the kit, the conditioning surface can comprise a linkage group covalently bonded to one of the surfaces of the microfluidic device, and the linkage group can be bonded to the conformation to support the The portion of the cell growth, viability, portability, or any combination thereof, of the one or more biological cells within the microfluidic device. The linking group can be a decyloxy linking group. Alternatively, the linking group can be a phosphonate linkage group. In some embodiments of the kit, the linking group of the conditioning surface can be directly linked to the portion configured to support cell growth, viability, portability, or the like.

在其他實施例中，該鍵聯基團可經由一鍵聯子而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯基團可經由連接至一鍵聯子之一第一端而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。該鍵聯子可進一步包含一直鏈部分，其中該直鏈部分之一主鏈包括選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之任何組合之1個至200個非氫原子。在該套組之一些實施例中，該調節表面之該鍵 聯子可進一步包含伸***基部分。該可裂解部分經構形以容許破壞該調節表面，藉此促進該生物分子之可移植性。該套組可進一步包含經構形以使該調節表面之該可裂解部***解之一試劑。 In other embodiments, the linking group can be indirectly linked via a linkage to the portion configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly linked to the portion configured to support cell growth, viability, portability, or the like, via attachment to one of the first ends of a linkage. The linker may further comprise a straight chain moiety, wherein one of the linear chain backbones comprises from 1 to 200 non-selective groups selected from the group consisting of a ruthenium atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. A hydrogen atom. In some embodiments of the kit, the key of the adjustment surface The linker may further comprise a triazole-based moiety. The cleavable portion is configured to permit disruption of the conditioning surface thereby promoting portability of the biomolecule. The kit can further comprise an agent configured to lyse the cleavable portion of the conditioning surface.

在該套組之各種實施例中，該套組可進一步包含一表面調節試劑。在一些實施例中，該表面調節試劑可包含一聚合物，其包括下列之至少一者：伸烷基醚部分、羧酸部分、磺酸部分、膦酸部分、胺基酸部分、核酸部分或糖類部分。在一些其他實施例中，該表面調節試劑包括一聚合物，其包括伸下列之至少一者：烷基醚部分、胺基酸部分或糖類部分。在一些其他實施例中，該調節表面可包含一可裂解部分。 In various embodiments of the kit, the kit can further comprise a surface conditioning agent. In some embodiments, the surface conditioning agent can comprise a polymer comprising at least one of: an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety, or Sugar part. In some other embodiments, the surface conditioning agent comprises a polymer comprising at least one of the group consisting of an alkyl ether moiety, an amino acid moiety, or a carbohydrate moiety. In some other embodiments, the conditioning surface can comprise a cleavable portion.

在該套組之其他實施例中，該表面調節試劑包括至少一細胞黏著阻斷分子。在一些實施例中，該至少一細胞黏著阻斷分子可破壞肌動蛋白纖維形成，阻斷整合素受體，或減弱細胞與DNA污損表面之結合。在一些實施例中，該至少一細胞黏著阻斷分子可為細胞鬆弛素B、一含RGD之肽、一DNase 1蛋白、一纖連蛋白抑制劑或一整合素抗體。在一些實施例中，該至少一細胞黏著阻斷分子可包含多於一種類型之細胞黏著阻斷分子之一組合。 In other embodiments of the kit, the surface conditioning reagent comprises at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule disrupts actin fiber formation, blocks integrin receptors, or attenuates binding of cells to DNA stained surfaces. In some embodiments, the at least one cell adhesion blocking molecule can be cytochalasin B, an RGD-containing peptide, a DNase 1 protein, a fibronectin inhibitor, or an integrin antibody. In some embodiments, the at least one cell adhesion blocking molecule can comprise a combination of one of more than one type of cell adhesion blocking molecule.

在該套組之各種實施例中，該表面調節試劑可包含哺乳動物血清之一或多個組分。該哺乳動物血清可為胎牛血清(FBS)或小牛血清(FCS)。在該套組之各種實施例中，該套組可進一步包含適合用於培養該一或多個生物細胞之一培養基。在一些實施例中，該套組可包含一培養基添加物，其包含經構形以加強生長室之該至少一表面之調節之一試劑。該培養基添加物可包含如上文所討論之一調節試劑或增強該至少一生長室之該至少一表面支援細胞生長、存活性、可移植性或其等之任何組合之能力之另一化學物種。此可包含生長因子、內分泌素、抗氧化劑或維生素及其類似者。 In various embodiments of the kit, the surface conditioning agent can comprise one or more components of a mammalian serum. The mammalian serum can be fetal bovine serum (FBS) or calf serum (FCS). In various embodiments of the kit, the kit can further comprise a medium suitable for culturing the one or more biological cells. In some embodiments, the kit can include a medium supplement comprising one of the agents configured to enhance the conditioning of the at least one surface of the growth chamber. The medium supplement may comprise another chemical species that modulates the agent or enhances the ability of the at least one surface of the at least one growth chamber to support cell growth, viability, portability, or the like, as discussed above. This may include growth factors, endocrine, antioxidants or vitamins and the like.

在該套組之各種實施例中，該套組可包含用以偵測該一或多個生物細胞之一狀態之至少一試劑。 In various embodiments of the kit, the kit can include at least one reagent for detecting the state of one of the one or more biological cells.

在又一態樣中，提供一種用於培養一生物細胞之套組，其包含用於培養一或多個生物細胞之一微流體器件，該微流體器件包含：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包含一隔離區域及一連接區域，其中該隔離區域與該連接區域流體地連接且該連接區域具有至該流動區域之一近端開口；且該至少一生長室具有至少一表面，其具有一表面改質配位基。該微流體器件可為如本文中所描述之任何微流體器件。該表面可包含具有一介電泳(DEP)構形之一基板。該DEP構形可為本文中所描述之任何DEP構形。可光學地致動該DEP構形。該基板係具有如本文中所描述之一表面改質配位基之任何基板，且可具有式3之一結構，且可包含如上文所描述之全部特徵： In yet another aspect, a kit for culturing a biological cell comprising a microfluidic device for culturing one or more biological cells, the microfluidic device comprising: a flow region configured Flowing with one of the first fluid mediums; and at least one growth chamber comprising an isolation region and a connection region, wherein the isolation region is fluidly connected to the connection region and the connection region has a proximity to the flow region The end opening; and the at least one growth chamber has at least one surface having a surface modifying ligand. The microfluidic device can be any of the microfluidic devices as described herein. The surface can comprise a substrate having a dielectrophoresis (DEP) configuration. The DEP configuration can be any of the DEP configurations described herein. The DEP configuration can be optically actuated. The substrate is any substrate having a surface modifying ligand as described herein, and may have a structure of Formula 3, and may include all of the features as described above:

在具有一微流體器件(其具有包含一表面改質配位基之至少一表面)之該套組之各種實施例中，該表面改質配位基可共價鍵聯至該基板之該表面之氧化物部分。該表面改質配位基可包含一反應部分。該表面改質配位基之該反應部分可為疊氮基、胺基、溴基、硫醇基、活性酯基、琥珀醯亞胺基或炔基部分。該表面改質配位基可經由一鍵聯基團而共價鍵聯至該等氧化物部分。在一些實施例中，該鍵聯基團可為矽烷氧基部分。在其他實施例中，該鍵聯基團可為磷酸酯部分。該鍵聯基團可經由一鍵聯子而間接連接至該表面改質配位基之該反應部 分。該鍵聯子可包含一直鏈部分，其中該直鏈部分之一主鏈包含選自矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之任何組合之1個至100個非氫原子。在一些實施例中，該表面改質配位基可包含一或多個可裂解部分。該一或多個可裂解部分可經構形以容許在形成一微流體器件之一調節表面之後破壞該調節表面，藉此促進該一或多個生物細胞在培養之後之可移植性。 In various embodiments of the kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the surface modifying ligand can be covalently bonded to the surface of the substrate The oxide portion. The surface modifying ligand may comprise a reactive moiety. The reactive portion of the surface modifying ligand can be an azide, an amine group, a bromo group, a thiol group, an active ester group, an amber quinone group or an alkynyl moiety. The surface modifying ligand can be covalently bonded to the oxide moieties via a linking group. In some embodiments, the linking group can be a decyloxy moiety. In other embodiments, the linking group can be a phosphate moiety. The linking group may be indirectly attached to the reaction portion of the surface modifying ligand via a bond Minute. The bond may comprise a straight chain moiety, wherein one of the backbone portions comprises from 1 to 100 non-hydrogens selected from the group consisting of a ruthenium atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. atom. In some embodiments, the surface modifying ligand can comprise one or more cleavable moieties. The one or more cleavable portions can be configured to permit disruption of the conditioning surface after formation of a conditioning surface of a microfluidic device, thereby facilitating portability of the one or more biological cells after culture.

在具有一微流體器件(其具有包含一表面改質配位基之至少一表面)之該套組之一些實施例中，該套組可進一步包含一調節改質劑，其包含經構形以支援細胞生長、存活性、可移植性或其等之任何組合之一第一部分及經構形以與該表面改質配位基之反應部分反應之一第二部分，該調節改質劑可具有式5之一結構且具有如本文中所描述之特徵之任何者：部分-(L')_m-R_px 式5 In some embodiments of the kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the kit can further comprise an conditioning modifier comprising a conformation Supporting a first portion of cell growth, viability, portability, or any combination thereof, and a second portion configured to react with a reaction portion of the surface modifying ligand, the conditioning modifier may have One of the structures of Formula 5 and having any of the features as described herein: Part - (L') _{m -} R _px Formula 5

該第二部分可經構形以在與該套組之該微流體器件之該表面改質配位基之該反應部分反應之後將該表面改質配位基轉換成經構形以支援生長室內之一或多個生物細胞之細胞生長、存活性、可移植性或其等之任何組合之一調節表面。該第一部分可包含伸烷基氧化物部分、糖類部分、烷基部分、全氟烷基部分、胺基酸部分、陰離子部分、陽離子部分或兩性離子部分。在一些實施例中，該第一部分可包含：烷基或氟烷基(其包含全氟烷基)部分；單糖或多糖(其可包含(但不限於)聚葡萄糖)；醇類(包含(但不限於)炔丙醇)；多元醇，其包含(但不限於)聚乙烯醇；伸烷基醚，其包含(但不限於)聚乙二醇；聚電解質(其包含(但不限於)聚丙烯酸或聚乙烯膦酸)；胺基(其包含其衍生物，諸如(但不限於)烷化胺基、羥烷基化胺基部分、胍鹽及含有一未芳香化氮環原子之雜環基，諸如(但不限於)嗎啉基或哌嗪基)；羧酸， 其包含(但不限於)丙炔酸(其可提供羧酸陰離子表面)；膦酸，其包含(但不限於)乙炔基膦酸(其可提供膦酸陰離子表面)；磺酸陰離子；羧基甜菜鹼；磺基甜菜鹼；胺磺酸；或胺基酸。該第二部分可為胺基、羧酸、炔基、疊氮基、醛基、溴基或硫醇基部分。在一些實施例中，該調節改質劑之該第一部分或一鍵聯子L'(如上文針對式5所描述)可包含一可裂解部分。該可裂解部分可經構形以容許破壞該調節表面，藉此促進該生物細胞之可移植性。在一些實施例中，該套組可進一步包含經構形以使該調節表面之該可裂解部***解之一試劑。 The second portion can be configured to convert the surface modifying ligand to a conformation to support growth chamber after reacting with the reactive portion of the surface modifying ligand of the microfluidic device of the kit One of the combination of cell growth, viability, portability, or the like of one or more biological cells modulates the surface. The first portion can comprise an alkylene oxide moiety, a saccharide moiety, an alkyl moiety, a perfluoroalkyl moiety, an amino acid moiety, an anionic moiety, a cationic moiety, or a zwitterionic moiety. In some embodiments, the first portion can comprise: an alkyl or fluoroalkyl group (which comprises a perfluoroalkyl group); a monosaccharide or polysaccharide (which can include, but is not limited to, polydextrose); an alcohol (including ( But not limited to) propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkyl ethers, including but not limited to polyethylene glycol; polyelectrolytes (including but not limited to) Polyacrylic acid or polyvinylphosphonic acid); an amine group (including derivatives thereof such as, but not limited to, an alkylated amine group, a hydroxyalkylated amine moiety, a phosphonium salt, and a hetero atom containing an unaromatized nitrogen ring atom) a cyclic group such as, but not limited to, morpholinyl or piperazinyl; carboxylic acid, It includes, but is not limited to, propiolic acid (which provides a carboxylate anion surface); phosphonic acid, which includes, but is not limited to, ethynylphosphonic acid (which provides a phosphonate anion surface); sulfonate anion; carboxyl beet a base; a sulfobetaine; an amine sulfonic acid; or an amino acid. The second moiety can be an amine, carboxylic acid, alkynyl, azido, aldehyde, bromo or thiol moiety. In some embodiments, the first portion or a linkage L' of the conditioning modifier (as described above for Formula 5) can comprise a cleavable moiety. The cleavable moiety can be configured to permit disruption of the conditioning surface thereby promoting portability of the biological cell. In some embodiments, the kit can further comprise an agent configured to lyse the cleavable portion of the conditioning surface.

在具有一微流體器件(其具有包含一表面改質配位基之至少一表面)之該套組之一些實施例中，該套組可進一步包含一表面調節試劑。 In some embodiments of the kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the kit can further comprise a surface conditioning agent.

在具有一微流體器件(其具有包含一表面改質配位基之至少一表面)之該套組之一些實施例中，該表面調節試劑可包含一聚合物，其包括下列之至少一者：伸烷基醚部分、羧酸部分、磺酸部分、膦酸部分、胺基酸部分、核酸部分或糖類部分。在一些其他實施例中，該表面調節試劑包括一聚合物，其包括下列之至少一者：伸烷基醚部分、胺基酸部分或糖類部分。在一些其他實施例中，該調節表面可包含一可裂解部分。 In some embodiments of the kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the surface conditioning agent can comprise a polymer comprising at least one of the following: An alkyl ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety or a carbohydrate moiety. In some other embodiments, the surface conditioning agent comprises a polymer comprising at least one of the alkyl ether moiety, the amino acid moiety, or the carbohydrate moiety. In some other embodiments, the conditioning surface can comprise a cleavable portion.

在具有一微流體器件(其具有包含一表面改質配位基之至少一表面)之該套組之一些實施例中，該表面調節試劑包括至少一細胞黏著阻斷分子。在一些實施例中，該至少一細胞黏著阻斷分子可破壞肌動蛋白纖維形成，阻斷整合素受體，或減弱細胞與DNA污損表面之結合。在一些實施例中，該至少一細胞黏著阻斷分子可為細胞鬆弛素B、一含RGD之肽、一DNase 1蛋白、一纖連蛋白抑制劑或一整合素抗體。在一些實施例中，該至少一細胞黏著阻斷分子可包含多於一種類型之細胞黏著阻斷分子之一組合。 In some embodiments of the kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the surface conditioning reagent comprises at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule disrupts actin fiber formation, blocks integrin receptors, or attenuates binding of cells to DNA stained surfaces. In some embodiments, the at least one cell adhesion blocking molecule can be cytochalasin B, an RGD-containing peptide, a DNase 1 protein, a fibronectin inhibitor, or an integrin antibody. In some embodiments, the at least one cell adhesion blocking molecule can comprise a combination of one of more than one type of cell adhesion blocking molecule.

在具有一微流體器件(其具有包含一表面改質配位基之至少一表面)之該套組之一些實施例中，該表面調節試劑可包含哺乳動物血清之一或多個組分。該哺乳動物血清可為胎牛血清(FBS)或小牛血清(FCS)。 In some embodiments of the kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the surface conditioning agent can comprise one or more components of mammalian serum. The mammalian serum can be fetal bovine serum (FBS) or calf serum (FCS).

在具有一微流體器件(其具有包含一表面改質配位基之至少一表面)之該套組之一些實施例中，該套組可進一步包含適合用於培養該一或多個生物細胞之一培養基。在一些實施例中，該套組可進一步包含一培養基添加物，其包含經構形以加強生長室之該至少一表面之調節之一試劑。該培養基添加物可包含如上文所討論之一連接劑或增強該至少一生長室之該至少一表面支援細胞生長、存活性、可移植性或其等之任何組合之能力之另一化學物種。此可包含生長因子、內分泌素、抗氧化劑或維生素及其類似者。 In some embodiments of the kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the kit can further comprise a substrate suitable for culturing the one or more biological cells. A medium. In some embodiments, the kit can further comprise a medium supplement comprising one of the agents configured to enhance the conditioning of the at least one surface of the growth chamber. The medium supplement may comprise a linker as discussed above or another chemical species that enhances the ability of the at least one surface of the at least one growth chamber to support cell growth, viability, portability, or the like. This may include growth factors, endocrine, antioxidants or vitamins and the like.

在具有一微流體器件(其具有包含一表面改質配位基之至少一表面)之該套組之一些實施例中，該套組可進一步包含用以偵測該一或多個生物細胞之一狀態之至少一試劑。 In some embodiments of the kit having a microfluidic device having at least one surface comprising a surface modifying ligand, the kit can further comprise detecting the one or more biological cells At least one reagent in one state.

實例 Instance

實例1. 一K562紅白血病細胞之培養及生長Example 1. Culture and growth of a K562 erythroleukemia cell

材料：K562細胞(一人類永生化骨髓性白血病細胞系)獲得自美國菌種保存中心(ATCC)(catalog ATCC® CCl-243^TM)且經提供為一懸浮細胞系。藉由播種1×10³個活細胞/mL且在37℃處使用5%二氧化碳氣體環境來培育而維持培養。使細胞***成1×10⁶個細胞/mL或使細胞每隔2天至3天***。在5%二甲基亞碸(DMSO)/95%完全生長培養基中冷凍細胞。 Materials: K562 cells (an immortalized human myeloid leukemia cell line) were obtained from the American Type Culture Collection (ATCC) (catalog ATCC® CCl- 243 TM) was provided as a suspension and a cell line. The culture was maintained by seeding 1 x 10 ³ viable cells/mL and culturing at 37 ° C using a 5% carbon dioxide gas atmosphere. The cells are divided into 1 x 10 ⁶ cells/mL or the cells are divided every 2 to 3 days. Cells were frozen in 5% dimethylarsine (DMSO) / 95% complete growth medium.

培養基：Iscove之改質杜爾貝科氏培養基(ATCC® Catalog No.30-2005)+10%胎牛血清(Hyclone,Cat# SH30071.2)經組合以產生完全生長培養基。當在培育週期期間灌注時，在將完全生長培養基引入至 微流體器件中之前使用空氣中之5%二氧化碳來連續調節完全生長培養基。 Media : Iscove's modified Dulbecco's medium (ATCC® Catalog No. 30-2005) + 10% fetal bovine serum (Hyclone, Cat# SH30071.2) were combined to produce a complete growth medium. When perfused during the incubation period, the complete growth medium was continuously adjusted using 5% carbon dioxide in air prior to introduction of the complete growth medium into the microfluidic device.

預充溶液：含有0.1% Pluronic® F127(Life Technologies® Cat# P6866)之完全生長培養基 Pre-filled solution: Complete growth medium containing 0.1% Pluronic® F127 (Life Technologies® Cat# P6866)

系統及微流體器件：由Berkeley Lights公司製造。系統包含至少一流量控制器、溫度控制器、流體培養基調節及泵抽組件、用於光啟動DEP構形之光源、微流體器件、安裝台及一攝影機。用於此實驗中之微流體器件之生長室具有約1.4×10⁵立方微米之一體積。流動通道之橫截面積係約4×10³平方微米。微流體器件具有8個通道。 System and microfluidic devices: manufactured by Berkeley Lights. The system includes at least one flow controller, temperature controller, fluid medium conditioning and pumping assembly, light source for light activated DEP configuration, microfluidic device, mounting station, and a camera. For growing chamber microfluidic device of this experiment, the one having a volume of from about 1.4 × ¹⁰⁵ cubic microns. The cross-sectional area of the flow channel is about 4 x 10 ³ square microns. The microfluidic device has 8 channels.

培養準備：將微流體器件裝載至系統上且使用具有15psi之100%二氧化碳沖洗微流體器件達5分鐘。在二氧化碳沖洗之後，依5微升/秒即時灌注預充溶液通過微流體器件達8分鐘。接著，使完全生長培養基依5微升/秒流動通過微流體器件達5分鐘。 Culture Preparation: The microfluidic device was loaded onto the system and the microfluidic device was rinsed with 100% carbon dioxide at 15 psi for 5 minutes. Immediately after 5 liters per second, the prefilled solution was passed through the microfluidic device for 8 minutes after the carbon dioxide rinse. Next, the complete growth medium was flowed through the microfluidic device for 5 minutes at 5 microliters per second.

培養條件：使微流體器件之溫度維持於37℃處。在培養實驗之整個週期中依0.001微升/秒之一恆定速率灌注培養基。 Culture conditions: The temperature of the microfluidic device was maintained at 37 °C. The medium was perfused at a constant rate of 0.001 microliter/second throughout the entire culture period.

使用重力來使一單一K562細胞裝載至微流體器件之一生長室中。展示裝載細胞之後之t=0小時處之生長室之一照片(參閱圖10A)。箭頭1002指向生長室中之單一細胞之位置。 Gravity is used to load a single K562 cell into a growth chamber of a microfluidic device. A photograph of one of the growth chambers at t = 0 hours after loading the cells is shown (see Figure 10A). Arrow 1002 points to the location of a single cell in the growth chamber.

在16小時培養完成之後，將細胞擴增至2個細胞之一群體，如該時間點所拍攝之一照片中所展示(參閱圖10B)。箭頭1004指向生長室中之兩個細胞之位置。 After completion of the 16 hour culture, the cells were expanded to one of 2 cells as shown in one of the photographs taken at this time point (see Figure 10B). Arrow 1004 points to the location of the two cells in the growth chamber.

在34小時培養完成之後，將細胞群體增加至總共四個細胞，如圖10C之照片中所展示。箭頭1006及1008指向定位於生長室內之兩個細胞之兩個群組之各者。 After the 34 hour culture was completed, the cell population was increased to a total of four cells as shown in the photograph of Figure 10C. Arrows 1006 and 1008 point to each of two groups of two cells positioned within the growth chamber.

在54小時培養完成之後，將K562細胞群體增加至總共八個細胞，如圖10D之照片中所展示。箭頭1010及1012指向定位於生長室內 之細胞群組之任一側處之細胞。 After 54 hours of culture was completed, the K562 cell population was increased to a total of eight cells, as shown in the photograph of Figure 10D. Arrows 1010 and 1012 point to the growth chamber Cells at either side of the cell group.

在70小時培養完成之後，將K562細胞群體增加至總共16個細胞，如圖10E之照片中所展示。箭頭1014、1016及1018指向該群組之細胞。將K562之一選殖擴增群體提供於微流體器件之生長室中。 After 70 hours of culture was completed, the K562 cell population was increased to a total of 16 cells, as shown in the photograph of Figure 10E. Arrows 1014, 1016, and 1018 point to cells of the cohort. A colony-amplified population of K562 is provided in a growth chamber of the microfluidic device.

實例2. 一OKT3融合瘤細胞之培養及生長Example 2. Culture and growth of an OKT3 fusion tumor cell

材料：OKT3細胞(一鼠科骨髓性融合瘤細胞系)獲得自ATCC(ATCC® Cat.# CRL-8001^TM)。細胞經提供為一懸浮細胞系。藉由播種約1×10⁵個活細胞/mL至約2×10⁵個活細胞/mL且在37℃處使用空氣中之5%二氧化碳(作為氣體環境)來培育而維持培養。使細胞每隔2天至3天***。計數OKT3細胞數目及存活性且將細胞密度調整至5×10⁵/ml以裝載至微流體器件。 Material: OKT3 cells (a murine myeloid hybridoma cell line) obtained from ATCC (ATCC® Cat # CRL-8001 TM.). The cells are provided as a suspension cell line. The culture was maintained by sowing about 1 x 10 ⁵ viable cells/mL to about 2 x 10 ⁵ viable cells/mL and culturing at 37 ° C using 5% carbon dioxide in air (as a gaseous environment). The cells are allowed to divide every 2 to 3 days. The number and viability of OKT3 cells were counted and the cell density was adjusted to 5 x 10 ⁵ /ml to be loaded into the microfluidic device.

培養基：500mL Iscove之改質杜爾貝科氏培養基(ATCC® Catalog No.30-2005)、200mL胎牛血清(ATCC® Cat.#30-2020)及1mL青黴素-鏈黴素(Life Technologies® Cat.# 15140-122)經組合以產生培養基。透過一0.22μm過濾器而過濾完全培養基且在使用之前將完全培養基儲存於4℃之避光處。 Medium: 500 mL Iscove's modified Dulbecco's medium (ATCC® Catalog No. 30-2005), 200 mL fetal bovine serum (ATCC® Cat. #30-2020) and 1 mL penicillin-streptomycin (Life Technologies® Cat) .# 15140-122) Combined to produce a medium. The complete medium was filtered through a 0.22 [mu]m filter and the complete medium was stored at 4 °C in the dark before use.

當在培育週期期間灌注時，在將培養基引入至微流體器件中之前使用空氣中之5%二氧化碳連續調節培養基。 When perfused during the incubation period, the medium was continuously conditioned using 5% carbon dioxide in air prior to introduction of the medium into the microfluidic device.

預充溶液：含有0.1% Pluronic® F127(Life Technologies® Cat# P6866)之培養基。 Pre-filled solution: Medium containing 0.1% Pluronic® F127 (Life Technologies® Cat# P6866).

系統及微流體器件：由Berkeley Lights公司製造。系統包含至少一流量控制器、溫度控制器、流體培養基調節及泵抽組件、用於光啟動DEP構形之光源及投射器、微流體器件、安裝台及一攝影機。用於此實驗中之微流體器件之生長室具有約1.5×10⁶立方微米之一體積。一流動通道之橫截面積係8×10³平方微米，且總共六個通道存在於微流體器件上。 System and microfluidic devices: manufactured by Berkeley Lights. The system includes at least one flow controller, temperature controller, fluid media conditioning and pumping assembly, light source and projector for light-activated DEP configuration, microfluidic device, mounting station, and a camera. A growth chamber of a microfluidic device of this experiment, one having a volume of about 1.5 × 10 ⁶ cubic microns. The cross-sectional area of a flow channel is 8 x 10 ³ square microns and a total of six channels are present on the microfluidic device.

培養準備：將微流體器件裝載至系統上且使用具有15psi之100%二氧化碳來沖洗微流體器件達5分鐘。在二氧化碳沖洗之後，依8微升/秒即時灌注預充溶液通過微流體器件，直至灌注2.5mL之一總體積通過微流體器件。接著，使培養基依8微升/秒流動通過微流體器件，直至灌注總共1mL之培養基通過微流體器件。圖11A之照片中展示引入細胞之前預備微流體器件。一列之四個生長室沿照片之底部延伸。 Culture Preparation: The microfluidic device was loaded onto the system and the microfluidic device was rinsed using 100% carbon dioxide with 15 psi for 5 minutes. After the carbon dioxide rinse, the pre-filled solution was immediately infused through the microfluidic device at 8 microliters per second until one of the total volume of 2.5 mL was perfused through the microfluidic device. Next, the medium was allowed to flow through the microfluidic device at 8 [mu]l/sec until a total of 1 mL of medium was perfused through the microfluidic device. The microfluidic device was prepared prior to introduction of cells in the photograph of Figure 11A. A row of four growth chambers extends along the bottom of the photo.

培養條件：使微流體器件之溫度維持於37℃處。在培養實驗之整個週期中使用一可變灌注方法來灌注培養基，該可變灌注方法包含依0.01微升/秒之一初始4小時灌注週期，接著為依8微升/秒之一短時高速灌注(約3秒)，接著為約小於1分鐘之一短灌注停止週期。在整個培養實驗中持續進行包含交替灌注速率及一停止之此循環。 Culture conditions: The temperature of the microfluidic device was maintained at 37 °C. The medium was perfused using a variable perfusion method throughout the entire period of the culture experiment, the variable perfusion method comprising an initial 4 hour perfusion cycle of 0.01 microliters per second followed by a short-term high speed of 8 microliters per second Perfusion (about 3 seconds) followed by a short perfusion stop period of about less than 1 minute. This cycle including alternating perfusion rates and one stop was continued throughout the culture experiment.

藉由重力而將一單一OKT3細胞引入至生長室中。圖11B中展示時間t=0處之具有一個細胞之生長室之一照片，其中箭頭1102指向左邊第二室，且尤其指向室內之單一細胞，其中駐留細胞之區域進一步由圓圈圈出。 A single OKT3 cell was introduced into the growth chamber by gravity. A photograph of one of the growth chambers with one cell at time t=0 is shown in Figure 11B, with arrow 1102 pointing to the second chamber to the left, and in particular to a single cell in the chamber, wherein the region of the resident cell is further circled by a circle.

圖12A至圖12C展示培養實驗中之隨後時間點處之微流體器件之照片且演示形成一選殖群體之細胞擴增。圖12A之照片拍攝於一天培養完成時且箭頭1202指向左邊第二室(單一OKT3細胞之引入點)中之一群組之約4個細胞。圖12B係在2天培養完成之後拍攝之一照片且箭頭1204指向左邊第二室中之一進一步增殖細胞群體。圖12C係在3天培養完成之後拍攝之一照片，且箭頭1206展示由培養單一OKT3細胞引起之眾多擴增OKT3細胞。 Figures 12A-12C show photographs of microfluidic devices at subsequent time points in a culture experiment and demonstrate cell expansion of a selected population. The photograph of Figure 12A was taken at approximately 4 cells in one of the groups in the second chamber (introduction point of single OKT3 cells) at the completion of one day of culture and arrow 1202. Figure 12B is a photograph of a further proliferating cell population taken after one day of culture completion and one of the arrows 1204 pointing to the left second chamber. Figure 12C is a photograph taken after 3 days of culture completion, and arrow 1206 shows numerous amplified OKT3 cells caused by culture of a single OKT3 cell.

圖13A至圖13C展示在三天培養完成之後(即，在圖12C之時間點之後)之微流體器件之照片，且演示使用由光電鑷子產生之一介電泳力來輸出擴增OKT3細胞之一選集。圖13A中將引發介電泳力之光圖案(即，箭頭1302所指向之一光阱)展示為圍繞細胞之一白框。由光學 致動之介電泳力使細胞自生長室之底部朝向流動通道移動。圖13B之照片展示擴增OKT3細胞進一步移動至流動區域中。仍將細胞陷留於光阱中且迫使細胞與光阱一起移動(箭頭1304)。圖13C之照片展示在將擴增細胞完全移動至流動區域中之後釋放擴增細胞(箭頭1306)。此等細胞藉由使用光學致動之DEP力、重力或流體流動而自微流體器件輸出此等細胞用於進一步研究或擴增。 Figures 13A-13C show photographs of microfluidic devices after three days of culture completion (i.e., after the time point of Figure 12C), and demonstrate the use of one of the dielectrophoretic forces generated by photoelectric tweezers to output an amplification of one of the OKT3 cells. . The light pattern that initiates the dielectrophoretic force (i.e., one of the light traps pointed by arrow 1302) is shown in Figure 13A as a white frame around one of the cells. By optics The actuated dielectrophoretic force moves the cells from the bottom of the growth chamber toward the flow channel. The photograph of Figure 13B shows that the expanded OKT3 cells are further moved into the flow region. The cells are still trapped in the light trap and the cells are forced to move with the light trap (arrow 1304). The photograph of Figure 13C shows the release of expanded cells after complete expansion of the expanded cells into the flow region (arrow 1306). These cells output such cells from the microfluidic device for further investigation or amplification by using optically actuated DEP forces, gravity or fluid flow.

此實驗演示藉由使用本文中所描述之器件及方法而提供之選擇性、精確度及靈活性。 This experiment demonstrates the selectivity, precision, and flexibility provided by using the devices and methods described herein.

實例3. 使用一無血清培養基來移除貼附細胞以調節一微流體器件之表面Example 3. Using a serum-free medium to remove adherent cells to modulate the surface of a microfluidic device

系統及微流體器件：如同實例1，使用具有約7×10⁵立方微米之一體積之生長室。 Systems and microfluidic device: as in Example 1, one having a volume of about 7 × 10 ⁵ cubic microns growth chamber.

預充方案：使250微升之100%二氧化碳依12微升/秒之一速率流入。此後，使含有0.1% Pluronic® F27(Life Technologies® Cat# P6866)之250微升之PBS依12微升/秒流入。預充之最後步驟包含：使250微升之PBS依12微升/秒流入。接著，引入培養基。 Pre-filling scheme : 250 μl of 100% carbon dioxide flows in at a rate of 12 μl/sec. Thereafter, 250 μl of PBS containing 0.1% Pluronic® F27 (Life Technologies® Cat# P6866) was allowed to flow in at 12 μl/sec. The final step of pre-filling consisted of allowing 250 microliters of PBS to flow in at 12 microliters per second. Next, the medium is introduced.

灌注方案：灌注方法係下列兩種方法之任一者： Perfusion protocol : The perfusion method is either of the following two methods:

1.依0.01微升/秒灌注2小時；依2微升/秒灌注64秒；且重複。 1. Perfusion for 2 hours at 0.01 microliters per second; perfusion for 6 seconds at 2 microliters per second; and repeated.

2.依0.02微升/秒灌注100秒；停止流動500秒；依2微升/秒灌注64秒；且重複。 2. Perfusion for 100 seconds at 0.02 microliters per second; stop flow for 500 seconds; perfusion for 6 seconds at 2 microliters per second; and repeat.

培養基：無血清培養基(ThermoFisher Scientific,Cat.No.12045-096)。 Medium : serum-free medium (ThermoFisher Scientific, Cat. No. 12045-096).

系統及微流體器件：在培養之後自一微流體器件之流動通道移除貼附細胞之能力演示為：在36℃處於具有一調節培養基添加物(B-27®補充劑)(2% v/v)之一無血清培養基中預培育貼附細胞(其可為(例如)購自AddexBio,Cat.No.C000605之JIMT1細胞)達30分鐘。在預培 育之後，將貼附細胞引入至流動通道，停止流動，且培育貼附細胞達2小時至約24小時之一週期。在化驗結束之後，依5微升/秒之一速率引入無血清培養基之流動。約750微升之流量(其表示約150倍微流體器件體積)通過微流體器件，全部貼附JIMT1細胞自流動通道輸出而離開微流體器件。此實驗展示：可含有補充組分(諸如市售B27)之無血清培養基可防止併入貼附報導體細胞之一化驗之過程期間之黏著，且容許貼附細胞自微流體器件輸出。 System and microfluidic devices : The ability to remove adherent cells from a flow channel of a microfluidic device after incubation is demonstrated as having a conditioning medium supplement (B-27® supplement) at 36 °C (2% v/ v) Pre-incubation of adherent cells (which may be, for example, JIMT1 cells purchased from AddexBio, Cat. No. C000605) in serum-free medium for 30 minutes. After pre-incubation, the attached cells are introduced into the flow channel, flow is stopped, and the attached cells are incubated for a period of from 2 hours to about 24 hours. After the end of the assay, the flow of serum-free medium was introduced at a rate of 5 microliters per second. A flow rate of about 750 microliters (which represents about 150 times the volume of the microfluidic device) exits the microfluidic device through the microfluidic device, all attached to the JITT1 cells from the flow channel output. This experiment demonstrates that serum-free media, which may contain supplemental components, such as commercially available B27, prevents adhesion during the process of incorporation into one of the labeled conductor cells and allows the attached cells to be output from the microfluidic device.

實例4. 使用一調節混合物來移除貼附細胞以調節一微流體器件之表面Example 4. Using a conditioning mixture to remove adherent cells to modulate the surface of a microfluidic device

貼附細胞：如同上述實例3。 Attached cells: as in Example 3 above.

培養基：具有添加組分(其包含(但不限於)FBS(購自ThermoFisher Scientific,Cat.No.16000-036)及青黴素-鏈黴素(ThermoFisherScientific Cat.No.15140-163))之一無血清培養基(ThermoFisher Scientific,Cat.No.12045-076)。 Medium: serum-free with one of the added components including, but not limited to, FBS (purchased from ThermoFisher Scientific, Cat. No. 16000-036) and penicillin-streptomycin (ThermoFisher Scientific Cat. No. 15140-163) Medium (ThermoFisher Scientific, Cat. No. 12045-076).

調節混合物：細胞鬆弛素B(Sigma Aldrich,Catalog No.C2743-200UL)、DNaseI(New England Biosciences Cat No.M0303S)及RGD三肽(Santa Cruz Biotechnology Cat No.sc-201176)。 The mixture was adjusted: cytochalasin B (Sigma Aldrich, Catalog No. C2743-200 UL), DNase I (New England Biosciences Cat No. M0303S), and RGD tripeptide (Santa Cruz Biotechnology Cat No. sc-201176).

貼附細胞製備：使用調節混合物來改質培養基以具有下列之一最終濃度：4微莫耳細胞鬆弛素B、0.1單位/微莫耳DNaseI及1毫莫耳RGD三肽。在將貼附細胞輸入至微流體器件之前於36℃處培育貼附細胞達30分鐘。 Attachment Cell Preparation: The conditioning medium was used to modify the medium to have one of the following final concentrations: 4 micromolar cytochalasin B, 0.1 unit/micromolar DNase I and 1 millimolar RGD tripeptide. The attached cells were incubated at 36 ° C for 30 minutes before the attached cells were input to the microfluidic device.

系統及微流體器件。如同上文，使用具有約7×10⁵立方微米之一體積之生長室。 System and microfluidic devices. As above, a growth chamber having a volume of about 7 x 10 ⁵ cubic microns is used.

在培養之後自一微流體器件之流動通道移除貼附細胞(例如JIMT1細胞)之能力演示為：預培育使用一調節混合物來預培育之貼附細胞群體。值得注意地，調節混合物之使用容許將含血清培養基(諸 如用於此實例中之培養基)用於微流體環境內，同時仍給予貼附細胞之移除。 The ability to remove adherent cells (e.g., JIMT1 cells) from a flow channel of a microfluidic device after incubation is demonstrated as pre-incubation of a population of adherent cells pre-incubated with a conditioning mixture. Notably, the use of a conditioning mixture allows the use of serum-containing media ( The medium as used in this example was used in a microfluidic environment while still removing the adherent cells.

將預培育貼附細胞引入至微流體器件之流動通道中，且培育貼附細胞達2小時至約24小時之一週期。在化驗結束之後，依5微升/秒之一速率引入培養基之流動。約750微升之流量(其表示約150倍微流體器件體積)通過微流體器件，全部貼附細胞自流動通道輸出而離開微流體器件。此實驗展示：調節混合物可防止黏著且容許輸出貼附細胞。 The pre-incubated patch cells are introduced into the flow channel of the microfluidic device and the adherent cells are incubated for a period of from 2 hours to about 24 hours. After the end of the assay, the flow of medium was introduced at a rate of 5 microliters per second. A flow rate of about 750 microliters (which represents about 150 times the volume of the microfluidic device) passes through the microfluidic device, and all of the attached cells exit the flow channel and exit the microfluidic device. This experiment shows that adjusting the mixture prevents adhesion and allows the output to attach to the cells.

實例5. 具有調節表面之微流體器件之製備Example 5. Preparation of a microfluidic device with an conditioned surface

針對全部製備：微流體器件：如同上述實例1，由Berkeley Lights公司製造，且被直接使用。無論何種情況，具有圖案化聚矽氧(PPS)之矽基板及ITO/玻璃基板係在合成調節表面之前於一Nordson Asymtek電漿清洗器中清洗(100W電力、50s)之氧氣電漿。 For all preparation: microfluidic device: As in Example 1 above, manufactured by Berkeley Lights, Inc., and used directly. In either case, the patterned polyfluorene oxide (PPS) substrate and the ITO/glass substrate were cleaned (100 W power, 50 s) of oxygen plasma in a Nordson Asymtek plasma cleaner prior to synthesizing the conditioning surface.

A.全氟烷基矽烷氧基調節表面A. Perfluoroalkyl decyloxy adjusting surface

材料：十七氟-1,1,2,2-四氫十二烷基三甲氧基矽烷獲得自Gelest(Cat.No.SIH5841.5)且被直接使用。直接使用MgSO₄．7H₂O(Acros)。 Materials: Heptafluoro-1,1,2,2-tetrahydrododecyltrimethoxydecane was obtained from Gelest (Cat. No. SIH5841.5) and used directly. Use MgSO ₄ directly. 7H ₂ O (Acros).

製備之方法：藉由使經組裝之微流體器件在降低壓力下於升高溫度處暴露於十七氟-1,1,2,2-四氫十二烷基三甲氧基矽烷及水蒸汽而化學地改質經組裝之微流體器件。添加300微升十七氟-1,1,2,2-四氫十二烷基三甲氧基矽烷及0.5克MgSO₄．7H₂O(水源)以分離在一清潔乾燥6"玻璃真空乾燥器之底部中之鋁舟。將微流體器件支撐於矽烷試劑及水合鹽(水源)上方之一多孔板上。在室溫處將乾燥器泵抽至750mTorr且密封乾燥器。接著，將乾燥劑放置至一110℃爐中達24小時。接著，具有全氟烷基調節表面之微流體器件自乾燥器移除且被使用。 Method of preparation: by exposing the assembled microfluidic device to heptafluoro-1,1,2,2-tetrahydrododecyltrimethoxynonane and water vapor at elevated temperature under reduced pressure The assembled microfluidic device is chemically modified. 300 μl of heptafluoro-1,1,2,2-tetrahydrododecyltrimethoxydecane and 0.5 g of MgSO _{4 were added} . 7H ₂ O (water source) to separate the aluminum boat in the bottom of a clean and dry 6" glass vacuum dryer. Support the microfluidic device on a perforated plate above the decane reagent and hydrated salt (water source). The dryer was pumped to 750 mTorr and the dryer was sealed. The desiccant was then placed in a furnace at 110 ° C for 24 hours. Next, the microfluidic device with perfluoroalkyl conditioned surface was removed from the dryer and used .

在一些實驗中，在將微流體器件安裝至印刷電路板之前化學地改質微流體器件。 In some experiments, the microfluidic device was chemically upgraded prior to mounting the microfluidic device to the printed circuit board.

B.聚葡萄糖調節表面B. Polydextrose regulating surface

材料：藉由使用疊氮化鈉來置換溴化物部分而由11-溴十一烷基三甲氧基矽烷(Gelest)合成11-疊氮十一烷基三甲氧基矽烷。在一典型反應中，將4.00克11-溴十一烷基三甲氧基矽烷(Gelest)添加至含有2.00克疊氮化鈉(Sigma-Aldrich)之一60微升無水二甲基甲醯胺(DMF)(Acros)溶液中。在氮氣下於室溫處攪拌溶液達24小時。接著，過濾溶液，且使用乾燥戊烷(Acros)來提取濾液。粗製11-疊氮十一烷基三甲氧基矽烷產物藉由旋轉蒸發而濃縮且藉由兩次連續真空蒸餾而純化。 Materials : 11-azidoundecyltrimethoxydecane was synthesized from 11-bromoundecyltrimethoxydecane (Gelest) by replacing the bromide moiety with sodium azide. In a typical reaction, 4.00 g of 11-bromoundecyltrimethoxydecane (Gelest) was added to 60 μl of anhydrous dimethylformamide containing one of 2.00 g of sodium azide (Sigma-Aldrich). DMF) (Acros) solution. The solution was stirred at room temperature under nitrogen for 24 hours. Next, the solution was filtered, and the filtrate was extracted using dry pentane (Acros). The crude 11-azidoundecyltrimethoxydecane product was concentrated by rotary evaporation and purified by two successive vacuum distillations.

二苯并環辛炔(DBCO)改質聚葡萄糖(MW：約3000Da)購自Nanocs且被直接使用。 Dibenzocyclooctyne (DBCO) modified polydextrose (MW: about 3000 Da) was purchased from Nanocs and used directly.

準備方法：引入一表面改質配位基。藉由使經組裝微流體器件之表面在降低壓力下於升高溫度處暴露於11-疊氮十一烷基三甲氧基矽烷及水蒸汽而化學地改質經組裝微流體器件之表面。添加300微升11-疊氮十一烷基三甲氧基矽烷及0.5克MgSO₄．7H₂O(水源)以分離一清潔乾燥6"玻璃真空乾燥器之底部中之鋁舟。將微流體器件支撐於矽烷及水合鹽(水源)上方之一多孔板上。在室溫處將乾燥器泵抽至750mTorr且密封乾燥器。接著，將乾燥器放置至一110℃爐中達24小時。接著，自乾燥器移除具有表面改質配位基(11-疊氮十一烷基矽烷氧基部分)之微流體晶片。在一些實驗中，在將微流體器件安裝至印刷電路板之前化地學改質微流體器件。 Preparation method: Introducing a surface modification ligand. The surface of the assembled microfluidic device is chemically modified by exposing the surface of the assembled microfluidic device to 11-azidodecyltrimethoxydecane and water vapor at elevated temperatures under reduced pressure. Add 300 μl of 11-azidoundecyltrimethoxydecane and 0.5 g of MgSO ₄ . 7H ₂ O (water source) to separate the aluminum boat in the bottom of a clean and dry 6" glass vacuum dryer. Support the microfluidic device on a perforated plate above decane and hydrated salt (water source). The dryer was pumped to 750 mTorr and the dryer was sealed. Next, the dryer was placed in a furnace at 110 ° C for 24 hours. Then, the surface modified ligand (11-azidodecyl group) was removed from the dryer. Microfluidic wafers of the decyloxy moiety. In some experiments, the modified microfluidic device was modified prior to mounting the microfluidic device to the printed circuit board.

聚葡萄糖調節表面之引入：藉由在氣相沈積之後使含有166微莫耳DBCO聚葡萄糖之一至少250微升水溶液流動通過具有表面改質疊氮配位基之微流體器件而使疊氮基封端之微流體器件表面與DBCO聚葡萄糖反應。允許反應在室溫處進行達至少1小時。接著，藉由使至少250微升DI水流動通過晶片而沖洗晶片。 Introduction of a polydextrose-regulating surface: an azide group is obtained by flowing at least 250 microliters of an aqueous solution containing one of 166 micromoles of DBCO polydextrose through a microfluidic device having a surface-modified azide ligand after vapor deposition The surface of the capped microfluidic device reacts with DBCO polydextrose. The reaction is allowed to proceed at room temperature for at least 1 hour. The wafer is then rinsed by flowing at least 250 microliters of DI water through the wafer.

C.聚乙二醇(PEG)調節表面C. Polyethylene glycol (PEG) conditioning surface

材料：如上文般合成11-疊氮十一烷基三甲氧基矽烷。炔基改質PEG(MW：約5000Da)購自JenKem且被直接使用。抗壞血酸鈉及五水合硫酸銅購自Sigma-Aldrich且被直接使用(THPTA(三(3-羥丙基***基甲基)胺)銅催化點擊試劑(Glen Research))。 Materials: 11-azidodecyltrimethoxydecane was synthesized as above. Alkynyl modified PEG (MW: about 5000 Da) was purchased from JenKem and used directly. Sodium ascorbate and copper sulfate pentahydrate were purchased from Sigma-Aldrich and used directly (THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) copper catalyzed click reagent (Glen Research)).

準備之方法：引入一表面改質配位基。如上文般製備具有11-疊氮十一烷基矽烷氧基表面改質配位基之微流體晶片。 Preparation method: Introducing a surface modification ligand. A microfluidic wafer having a surface modification ligand of 11-azidodecyldecyloxy group was prepared as above.

PEG調節表面之引入：藉由使含有333微莫耳炔基改質PEG、500微莫耳硫酸銅、500微莫耳THPTA配位基及5毫莫耳抗壞血酸鈉之一至少250微升水溶液流動通過具有11-疊氮十一烷基矽烷氧基表面改質配位基之微流體器件而使微流體器件之疊氮基封端之表面與炔基改質PEG反應。允許反應在室溫處進行達至少1小時。接著，藉由使至少250微升去離子水流動通過器件而沖洗具有一PEG調節表面之微流體器件。 Introduction of PEG-regulating surface: by flowing at least 250 microliters of aqueous solution containing one of 333 micromolar alkyne modified PEG, 500 micromolar copper sulfate, 500 micromolar THPTA ligand, and 5 millimoles of sodium ascorbate The azide-terminated surface of the microfluidic device is reacted with an alkynyl-modified PEG by a microfluidic device having a 11-azidodecyldecyloxy surface modifying ligand. The reaction is allowed to proceed at room temperature for at least 1 hour. The microfluidic device having a PEG conditioning surface is then rinsed by flowing at least 250 microliters of deionized water through the device.

D.具有至表面之膦酸酯鍵聯基團之烷基改質表面D. an alkyl modified surface having a phosphonate linkage group to the surface

材料：膦酸十八酯購自Sigma Aldrich且被直接使用。丙酮及乙醇購自Sigma Aldrich。 Materials: Hexadesphosphonate was purchased from Sigma Aldrich and used directly. Acetone and ethanol were purchased from Sigma Aldrich.

製備之方法：使微流體器件之表面在35℃處暴露於一10毫莫耳之膦酸十八酯無水乙醇溶液中達48小時。在沈積之後使用乙醇及DI水來充分沖洗具有經由膦酸酯鍵聯基團而附接之烷基調節表面之所得微流體器件。 Method of preparation: The surface of the microfluidic device was exposed to a 10 millimolar solution of octadecylphosphonic acid in absolute ethanol at 35 ° C for 48 hours. The resulting microfluidic device having an alkyl modulating surface attached via a phosphonate linkage group is thoroughly rinsed with ethanol and DI water after deposition.

實例6. 一調節微流體表面上之T淋巴細胞之培養及輸出Example 6. One regulation of the culture and output of T lymphocytes on the surface of microfluidics

材料：CD3+細胞，其來自AllCells公司且依1珠粒/1細胞之一速率與抗CD3/抗CD28磁珠(Dynabeads®,Thermofisher Scientific,Cat.No.11453D)混合。在37℃處於一5% CO₂培育箱中之於相同於培養實驗本身之培養基中培育混合物達5小時。在培育之後，使T細胞/珠粒 混合物再懸浮而供使用。 Materials: CD3+ cells from AllCells Inc. were mixed with anti-CD3/anti-CD28 magnetic beads (Dynabeads®, Thermofisher Scientific, Cat. No. 11453D) at a rate of 1 bead/1 cell. The mixture was incubated for 5 hours at 37 ° C in a 5% CO ₂ incubator in the same medium as the culture experiment itself. After incubation, the T cell/bead mixture is resuspended for use.

培養基：RPMI-1640(GIBCO®，ThermoFisher Scientific,Cat.No.11875-127)、10% FBS、2%人類AB血清(50U/ml IL2；R&D系統) Medium: RPMI-1640 (GIBCO®, ThermoFisher Scientific, Cat. No. 11875-127), 10% FBS, 2% human AB serum (50 U/ml IL2; R&D system)

預充程序：如同上述實例3。 Precharge program: Like Example 3 above.

灌注方案：如同上述實例3。 Perfusion protocol: as in Example 3 above.

系統及微流體器件：如同上述實例3。生長室具有約7×10⁵立方微米之一體積。 System and microfluidic device: as in Example 3 above. The growth chamber has a volume of about 7 x 10 ⁵ cubic microns.

調節表面：微流體器件具有如上文所描述般製備之一共價鍵聯聚葡萄糖調節表面。 Conditioning Surface: The microfluidic device has one of the covalently bonded polydextrose conditioning surfaces prepared as described above.

藉由使再懸浮液流動通過一流體入口且進入微流體通道而將(T細胞+珠粒)懸浮液引入至微流體器件中。藉由使晶片傾斜且允許重將動T細胞/珠粒拉至生長室中而停止流動且使T細胞/珠粒隨機裝載至生長室中。 The (T cell + bead) suspension is introduced into the microfluidic device by flowing the resuspension through a fluid inlet and into the microfluidic channel. Flow is stopped and T cells/beads are randomly loaded into the growth chamber by tilting the wafer and allowing heavy T cells/beads to be pulled into the growth chamber.

在將T細胞/珠粒裝載至生長室之後，灌注培養基通過奈流體晶片之微流體通道達4天之一時間週期。圖14A展示微流體器件之生長室之聚葡萄糖調節表面上之T細胞之生長。相對於一類似微流體器件(資料未展示)之一非調節表面而改良聚葡萄糖調節表面上之T細胞之生長。 After loading the T cells/beads into the growth chamber, the perfusion medium passes through the microfluidic channel of the nanofluidic wafer for one of four days. Figure 14A shows the growth of T cells on the polydextrose modulating surface of a growth chamber of a microfluidic device. The growth of T cells on the polydextrose modulating surface is improved relative to a non-regulating surface of a similar microfluidic device (not shown).

接著，藉由重力(例如，使微流體器件傾斜)而使T細胞自生長室移除。圖14B展示在一20分鐘週期結束時自生長室移除之程度(其演示使擴增T細胞輸出至流動通道中之卓越能力)，其相較於自一類似微流體器件之一非調節表面移除T細胞之程度而改良。接著，使T細胞自微流體器件(圖中未展示)移除。 The T cells are then removed from the growth chamber by gravity (eg, tilting the microfluidic device). Figure 14B shows the extent of removal from the growth chamber at the end of a 20 minute period (which demonstrates the superior ability to export expanded T cells into the flow channel) as compared to a non-regulated surface from a similar microfluidic device Improved by removing the extent of T cells. Next, the T cells are removed from the microfluidic device (not shown).

本文中所展示之實例係例示性的且絕不限制[實施方式]中所描述之方法及裝置之範疇。 The examples shown herein are illustrative and in no way limit the scope of the methods and apparatus described in the [embodiments].

900‧‧‧微流體器件 900‧‧‧Microfluidic devices

902‧‧‧封閉區域 902‧‧‧closed area

904‧‧‧第一介電泳(DEP)基板 904‧‧‧First Dielectrophoresis (DEP) Substrate

906‧‧‧第二介電泳(DEP)基板 906‧‧‧Second Dielectrophoresis (DEP) Substrate

910‧‧‧內表面 910‧‧‧ inner surface

912‧‧‧內表面 912‧‧‧ inner surface

914‧‧‧矽烷氧基鍵聯基團 914‧‧‧ 矽 alkoxy linkage group

916‧‧‧調節表面 916‧‧‧Adjustment surface

Claims (90)

一種用於培養一或多個生物細胞之微流體器件，其包括：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其包括一隔離區域及一連接區域，該隔離區域與該連接區域流體地連接且該連接區域包括至該流動區域之一近端開口，其中該至少一生長室進一步包括經調節以支援該微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 A microfluidic device for culturing one or more biological cells, comprising: a flow region configured to flow with one of a first fluid medium; and at least one growth chamber including an isolation region and a a connection region fluidly coupled to the connection region and the connection region includes a proximal opening to one of the flow regions, wherein the at least one growth chamber further includes an adjustment to support cell growth, storage in the microfluidic device At least one surface of activity, portability, or any combination thereof. 如請求項1之微流體器件，其中使用支援該微流體器件內之細胞可移植性之一或多個試劑來調節該至少一調節表面。 The microfluidic device of claim 1, wherein the at least one conditioning surface is adjusted using one or more reagents that support cell portability within the microfluidic device. 如請求項1或2之微流體器件，其中使用包括伸烷基醚部分之一聚合物來調節該至少一調節表面。 A microfluidic device according to claim 1 or 2, wherein the polymer comprising one of the alkylene ether moieties is used to adjust the at least one conditioning surface. 如前述請求項中任一項之微流體器件，其中使用包括糖類部分之一聚合物來調節該至少一調節表面。 A microfluidic device according to any of the preceding claims, wherein the at least one conditioning surface is adjusted using a polymer comprising one of the saccharide moieties. 如前述請求項中任一項之微流體器件，其中使用包括胺基酸部分之一聚合物來調節該至少一調節表面。 A microfluidic device according to any of the preceding claims, wherein a polymer comprising one of the amino acid moieties is used to adjust the at least one conditioning surface. 如前述請求項中任一項之微流體器件，其中使用包括羧酸部分、磺酸部分、核酸部分或膦酸部分之一聚合物來調節該微流體器件之該至少一調節表面。 A microfluidic device according to any of the preceding claims, wherein the at least one conditioning surface of the microfluidic device is adjusted using a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety or a phosphonic acid moiety. 如前述請求項中任一項之微流體器件，其中該至少一調節表面包括共價鍵聯至該微流體器件之一表面之一鍵聯基團，且其中該鍵聯基團鍵聯至經構形以支援該微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之一部分。 The microfluidic device of any of the preceding claims, wherein the at least one conditioning surface comprises a covalently bonded to one of the surface of the microfluidic device, and wherein the linking group is linked to The configuration is to support a portion of any combination of cell growth, viability, portability, or the like within the microfluidic device. 如請求項7之微流體器件，其中該鍵聯基團係一個矽烷氧基鍵聯 基團。 The microfluidic device of claim 7, wherein the linking group is a decyloxy linkage Group. 如請求項7或8之微流體器件，其中該至少一調節表面包括烷基或氟烷基部分。 The microfluidic device of claim 7 or 8, wherein the at least one conditioning surface comprises an alkyl or fluoroalkyl moiety. 如請求項9之微流體器件，其中該等烷基或氟烷基部分具有大於10個碳之一主鏈長度。 The microfluidic device of claim 9, wherein the alkyl or fluoroalkyl moiety has a backbone length of greater than 10 carbons. 如請求項7至10中任一項之微流體器件，其中該鍵聯基團經由一鍵聯子而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。 The microfluidic device of any one of claims 7 to 10, wherein the linking group is indirectly linked via a linkage to a conformation to support cell growth, viability, portability, or the like Combine this part. 如請求項11之微流體器件，其中該鍵聯子包括一個伸***基部分。 The microfluidic device of claim 11, wherein the linker comprises a triazole-based moiety. 如前述請求項中任一項之微流體器件，其中該至少一調節表面包括糖類部分。 The microfluidic device of any of the preceding claims, wherein the at least one conditioning surface comprises a carbohydrate moiety. 如前述請求項中任一項之微流體器件，其中該至少一調節表面包括伸烷基醚部分。 The microfluidic device of any of the preceding claims, wherein the at least one conditioning surface comprises an alkyl ether moiety. 如前述請求項中任一項之微流體器件，其中該至少一調節表面包括胺基酸部分。 The microfluidic device of any of the preceding claims, wherein the at least one conditioning surface comprises an amino acid moiety. 如請求項7至15中任一項之微流體器件，其中該至少一調節表面包括兩性離子。 The microfluidic device of any of claims 7 to 15, wherein the at least one conditioning surface comprises a zwitterion. 如前述請求項中任一項之微流體器件，其中該至少一調節表面包括至少一細胞黏著阻斷分子。 The microfluidic device of any of the preceding claims, wherein the at least one conditioning surface comprises at least one cell adhesion blocking molecule. 如請求項17之微流體器件，其中該至少一細胞黏著阻斷分子係一含RGD之肽。 The microfluidic device of claim 17, wherein the at least one cell adhesion blocking molecule is an RGD-containing peptide. 如請求項17或18之微流體器件，其中該至少一細胞黏著阻斷分子係多於一種類型之細胞黏著阻斷分子之一組合。 The microfluidic device of claim 17 or 18, wherein the at least one cell adhesion blocking molecule is more than one of a combination of one type of cell adhesion blocking molecule. 如前述請求項中任一項之微流體器件，其中該調節表面包括一可裂解部分。 A microfluidic device according to any of the preceding claims, wherein the conditioning surface comprises a cleavable portion. 如前述請求項中任一項之微流體器件，其中該微流體器件之該至少一調節表面包括哺乳動物血清之一或多個組分。 A microfluidic device according to any of the preceding claims, wherein the at least one conditioning surface of the microfluidic device comprises one or more components of mammalian serum. 如前述請求項中任一項之微流體器件，其中該微流體器件進一步包括具有一介電泳(DEP)構形之一基板。 A microfluidic device according to any of the preceding claims, wherein the microfluidic device further comprises a substrate having a dielectrophoresis (DEP) configuration. 如請求項22之微流體器件，其中光學地致動該DEP構形。 The microfluidic device of claim 22, wherein the DEP configuration is optically actuated. 如前述請求項中任一項之微流體器件，其中該至少一生長室包括經調節以支援一哺乳動物細胞之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 The microfluidic device of any of the preceding claims, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or the like, of any combination of mammalian cells. 如前述請求項中任一項之微流體器件，其中該至少一生長室包括經調節以支援一免疫細胞之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 The microfluidic device of any of the preceding claims, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or the like of any combination of immune cells. 如請求項25之微流體器件，其中該免疫細胞係一淋巴細胞或白血球。 The microfluidic device of claim 25, wherein the immune cell line is a lymphocyte or a white blood cell. 如請求項25之微流體器件，其中該免疫細胞係一B細胞、一T細胞、NK細胞、一巨噬細胞或一樹突細胞。 The microfluidic device of claim 25, wherein the immune cell is a B cell, a T cell, an NK cell, a macrophage or a dendritic cell. 如請求項1至24中任一項之微流體器件，其中該至少一生長室包括經調節以支援一貼附細胞之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 The microfluidic device of any one of claims 1 to 24, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or the like of any combination of attached cells . 如請求項1至24中任一項之微流體器件，其中該至少一生長室包括經調節以支援一融合瘤細胞之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 The microfluidic device of any one of claims 1 to 24, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of a fusion tumor cell . 如前述請求項中任一項之微流體器件，其中該至少一生長室包括經調節以支援一單一細胞及生物細胞之一對應選殖群落之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 A microfluidic device according to any of the preceding claims, wherein the at least one growth chamber comprises cells adapted to support cell growth, viability, portability or the like of a single cell and one of the biological cells of the selected colony. At least one surface of any combination. 一種用於在一微流體器件上培養一或多個生物細胞之系統，該系統包括： 一微流體器件，其包括：一流動區域，其經構形以含有一第一流體培養基之一流動；及至少一生長室，其中該生長室具有經調節以支援該微流體器件中之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 A system for culturing one or more biological cells on a microfluidic device, the system comprising: A microfluidic device comprising: a flow region configured to flow with one of a first fluid medium; and at least one growth chamber, wherein the growth chamber has an adjustment to support cell growth in the microfluidic device At least one surface of any combination of survivability, portability, or the like. 如請求項31之系統，其中該微流體器件係如請求項1至30中任一項之微流體器件。 The system of claim 31, wherein the microfluidic device is the microfluidic device of any one of claims 1 to 30. 如請求項31或32之系統，其進一步包括經構形以灌注至少該第一流體培養基之一流量控制器。 The system of claim 31 or 32, further comprising a flow controller configured to perfuse at least one of the first fluid medium. 如請求項33之系統，其中該控制器經構形以非連續地灌注該至少第一流體培養基。 The system of claim 33, wherein the controller is configured to non-continuously perfuse the at least first fluid medium. 如請求項31至34中任一項之系統，其中該微流體器件進一步包括具有一介電泳(DEP)構形之一基板，該基板經構形以將一或多個生物細胞引入至該生長室中或將該一或多個生物細胞移出該生長室。 The system of any one of claims 31 to 34, wherein the microfluidic device further comprises a substrate having a dielectrophoresis (DEP) configuration, the substrate being configured to introduce one or more biological cells into the growth chamber Or moving the one or more biological cells out of the growth chamber. 如請求項35之系統，其中光學地致動該DEP構形。 The system of claim 35, wherein the DEP configuration is optically actuated. 如請求項31至36中任一項之系統，其進一步包括經構形以含有該第一流體培養基之一儲液器，其中該儲液器流體地連接至該微流體器件。 The system of any one of claims 31 to 36, further comprising a reservoir configured to contain the first fluid medium, wherein the reservoir is fluidly coupled to the microfluidic device. 如請求項37之系統，其中該儲液器經構形以由能夠使該第一流體培養基飽含溶解氣體分子之一氣體環境接觸。 The system of claim 37, wherein the reservoir is configured to be contacted by a gaseous environment capable of causing the first fluid medium to be saturated with one of the dissolved gas molecules. 如請求項31至38中任一項之系統，其進一步包括連接至該微流體器件之至少一入口之一感測器，其中該感測器經構形以偵測該第一流體培養基之一pH。 The system of any one of claims 31 to 38, further comprising a sensor coupled to at least one inlet of the microfluidic device, wherein the sensor is configured to detect one of the first fluid medium pH. 如請求項31至39中任一項之系統，其進一步包括連接至至少一 出口之一感測器，其中該感測器經構形以在該第一流體培養基離開該微流體器件時偵測該第一流體培養基之一pH。 The system of any one of claims 31 to 39, further comprising connecting to at least one One of the exit sensors, wherein the sensor is configured to detect a pH of the first fluid medium as the first fluid medium exits the microfluidic device. 如請求項39或40之系統，其中該感測器係一光學感測器。 The system of claim 39 or 40, wherein the sensor is an optical sensor. 如請求項31至41中任一項之系統，其進一步包括經構形以擷取該至少一生長室及其內所含之任何生物細胞之一影像之一偵測器。 The system of any one of claims 31 to 41, further comprising a detector configured to capture an image of the at least one growth chamber and any one of the biological cells contained therein. 一種組合物，其包括：一基板，其包括一介電泳(DEP)構形及一表面；及一調節表面，其共價鍵聯至該基板之該表面之氧化物部分。 A composition comprising: a substrate comprising a dielectrophoresis (DEP) configuration and a surface; and an conditioning surface covalently bonded to the oxide portion of the surface of the substrate. 如請求項43之組合物，其中該調節表面包括共價鍵聯至該表面之該等氧化物部分之一鍵聯基團，且其中該鍵聯基團進一步鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之一部分。 The composition of claim 43, wherein the conditioning surface comprises a linkage group covalently bonded to one of the oxide moieties of the surface, and wherein the linkage group is further linked to the conformation to support the cell Part of any combination of growth, viability, portability, or the like. 如請求項44之組合物，其中該鍵聯基團係一個矽烷氧基鍵聯基團。 The composition of claim 44, wherein the linking group is a decyloxy linking group. 如請求項44或45之組合物，其中該鍵聯基團間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。 The composition of claim 44 or 45, wherein the linking group is indirectly linked to the portion configured to support cell growth, viability, portability, or any combination thereof. 如請求項44至46中任一項之組合物，其中該鍵聯基團經由連接至一鍵聯子之一第一端而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。 The composition of any one of claims 44 to 46, wherein the linking group is indirectly bonded to the conformation to support cell growth, viability, and portability via attachment to one of the first ends of the linkage. This part of the sex or any combination thereof. 如請求項47之組合物，其中該鍵聯子進一步包括一直鏈部分，其中該直鏈部分之一主鏈包括選自包括矽原子、碳原子、氮原子、氧原子、硫原子及磷原子之群組之任何組合之1個至200個非氫原子。 The composition of claim 47, wherein the bond further comprises a straight chain moiety, wherein one of the linear chain backbones comprises a group selected from the group consisting of a germanium atom, a carbon atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a phosphorus atom. One to 200 non-hydrogen atoms of any combination of the groups. 如請求項47至48中任一項之組合物，其中該鍵聯子進一步包括 一個伸***基部分。 The composition of any one of claims 47 to 48, wherein the bond further comprises A triazole-based moiety. 如請求項43至49中任一項之組合物，其中經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分包括烷基、氟烷基、單糖或多糖、醇類、多元醇、伸烷基醚、聚電解質、胺基、羧酸、膦酸、磺酸陰離子、羧基甜菜鹼、磺基甜菜鹼、胺磺酸或胺基酸。 The composition of any one of claims 43 to 49, wherein the moiety configured to support cell growth, viability, portability, or any combination thereof, etc., comprises an alkyl group, a fluoroalkyl group, a monosaccharide or a polysaccharide. An alcohol, a polyhydric alcohol, an alkylene ether, a polyelectrolyte, an amine group, a carboxylic acid, a phosphonic acid, a sulfonic acid anion, a carboxybetaine, a sulfobetaine, an aminesulfonic acid or an amino acid. 如請求項43至50中任一項之組合物，其中該至少一調節表面包括胺基酸、烷基部分、全氟烷基部分、聚葡萄糖部分及/或伸烷基醚部分。 The composition of any one of claims 43 to 50, wherein the at least one conditioning surface comprises an amino acid, an alkyl moiety, a perfluoroalkyl moiety, a polydextrose moiety, and/or an alkylene ether moiety. 如請求項43至51中任一項之組合物，其中該調節表面進一步包括一或多個可裂解部分。 The composition of any one of claims 43 to 51, wherein the conditioning surface further comprises one or more cleavable moieties. 如請求項52之組合物，其中該可裂解部分經構形以容許破壞該調節表面，藉此促進該一或多個生物細胞在培養之後之可移植性。 The composition of claim 52, wherein the cleavable moiety is configured to permit disruption of the conditioning surface, thereby facilitating portability of the one or more biological cells after culture. 一種用於培養一生物細胞之套組，其包括：一微流體器件，其包括：一流動區域，其經構形以含有一第一流體培養基之一流動；及其中該至少一生長室進一步包括經調節以支援該微流體器件內之細胞生長、存活性、可移植性或其等之任何組合之至少一表面。 A kit for culturing a biological cell, comprising: a microfluidic device comprising: a flow region configured to flow with one of a first fluid medium; and wherein the at least one growth chamber further comprises At least one surface that is conditioned to support cell growth, viability, portability, or any combination thereof, within the microfluidic device. 如請求項54之套組，其中該微流體器件係如請求項1至30中任一項之微流體器件。 The kit of claim 54, wherein the microfluidic device is the microfluidic device of any one of claims 1 to 30. 如請求項54或55之套組，其中該微流體器件之該至少一調節表面包括烷基部分、氟烷基部分、單糖或多糖部分、醇類部分、多元醇部分、伸烷基醚部分、聚電解質部分、胺基部分、羧酸 部分、膦酸部分、磺酸酯部分、羧基甜菜鹼部分、磺基甜菜鹼部分、胺磺酸部分或胺基酸部分。 The kit of claim 54 or 55, wherein the at least one conditioning surface of the microfluidic device comprises an alkyl moiety, a fluoroalkyl moiety, a monosaccharide or polysaccharide moiety, an alcohol moiety, a polyol moiety, an alkyl ether moiety Polyelectrolyte moiety, amine moiety, carboxylic acid Part, phosphonic acid moiety, sulfonate moiety, carboxybetaine moiety, sulfobetaine moiety, amine sulfonic acid moiety or amino acid moiety. 如請求項54至56中任一項之套組，其中該微流體器件之該至少一調節表面包括下列之至少一者：糖類部分、伸烷基醚部分、烷基部分、氟烷基部分或胺基酸部分。 The kit of any one of claims 54 to 56, wherein the at least one conditioning surface of the microfluidic device comprises at least one of: a sugar moiety, an alkyl ether moiety, an alkyl moiety, a fluoroalkyl moiety or Amino acid moiety. 如請求項57之套組，其中該等烷基或氟烷基部分具有大於10個碳之一主鏈長度。 The kit of claim 57, wherein the alkyl or fluoroalkyl moiety has a backbone length of greater than 10 carbons. 如請求項54至58中任一項之套組，其中該至少一調節表面包括共價鍵聯至該微流體器件之一表面之一鍵聯基團，且其中該鍵聯基團進一步鍵聯至經構形以支援該微流體器件內之該一或多個生物細胞之生長、存活性、可移植性或其等之任何組合之一部分。 The kit of any one of claims 54 to 58, wherein the at least one conditioning surface comprises a covalently bonded to one of the surface of the microfluidic device, and wherein the linking group is further linked To a portion configured to support growth, viability, portability, or the like of the one or more biological cells within the microfluidic device. 如請求項59之套組，其中該鍵聯基團係一個矽烷氧基鍵聯基團。 A kit of claim 59, wherein the linkage group is a decyloxy linkage group. 如請求項59或60之套組，其中該鍵聯基團間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。 A kit of claim 59 or 60, wherein the linkage group is indirectly linked to the portion configured to support cell growth, viability, portability, or any combination thereof. 如請求項61之套組，其中該鍵聯基團經由一鍵聯子而間接鍵聯至經構形以支援細胞生長、存活性、可移植性或其等之任何組合之該部分。 A kit of claim 61, wherein the linking group is indirectly linked via a linkage to the portion configured to support cell growth, viability, portability, or any combination thereof. 如請求項54至62中任一項之套組，其進一步包括一表面調節試劑。 The kit of any of claims 54 to 62, further comprising a surface conditioning reagent. 如請求項63之套組，其中該表面調節試劑包括一聚合物，該聚合物包括下列之至少一者：伸烷基醚部分、羧酸部分、磺酸部分、膦酸部分、胺基酸部分、核酸部分或糖類部分。 The kit of claim 63, wherein the surface conditioning agent comprises a polymer comprising at least one of an alkyl ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, and an amino acid moiety , a nucleic acid moiety or a carbohydrate moiety. 如請求項63之套組，其中該表面調節試劑包括至少一細胞黏著 阻斷分子。 The kit of claim 63, wherein the surface conditioning reagent comprises at least one cell adhesion Block the molecule. 如請求項63之套組，其中該表面調節試劑包括哺乳動物血清之一或多個組分。 The kit of claim 63, wherein the surface conditioning agent comprises one or more components of a mammalian serum. 如請求項54至66中任一項之套組，其進一步包括一培養基添加物，該培養基添加物包括經構形以加強生長室之該至少一表面之該調節之一試劑。 The kit of any of claims 54 to 66, further comprising a medium supplement comprising one of the conditioning agents configured to enhance the at least one surface of the growth chamber. 如請求項54至67中任一項之套組，其中該調節表面包括一可裂解部分。 The kit of any of claims 54 to 67, wherein the conditioning surface comprises a cleavable portion. 如請求項68之套組，其中該套組進一步包括經構形以使該調節表面之該可裂解部***解之一試劑。 The kit of claim 68, wherein the kit further comprises a reagent configured to lyse the cleavable portion of the conditioning surface. 一種在一微流體器件中培養至少一生物細胞之方法，該微流體器件具有經構形以含有一第一流體培養基之一流動之一流動區域、及至少一生長室，該方法包括下列步驟：將該至少一生物細胞引入至該至少一生長室中，其中該至少一生長室經構形以具有經調節以支援細胞生長、存活性、可移植性或其等之任何組合之至少一表面；及在至少足以擴增該至少一生物細胞而產生生物細胞之一群落之一長時間週期內培育該至少一生物細胞。 A method of culturing at least one biological cell in a microfluidic device having a flow region configured to contain one of a first fluid medium flow, and at least one growth chamber, the method comprising the steps of: Introducing the at least one biological cell into the at least one growth chamber, wherein the at least one growth chamber is configured to have at least one surface conditioned to support cell growth, viability, portability, or any combination thereof; And cultivating the at least one biological cell for a long period of time at least one of the colonies that are at least sufficient to amplify the at least one biological cell to produce one of the biological cells. 如請求項70之方法，其中該微流體器件係如請求項1至30中任一項之微流體器件。 The method of claim 70, wherein the microfluidic device is the microfluidic device of any one of claims 1 to 30. 如請求項70至71中任一項之方法，其中該至少一調節表面包括共價鍵聯至該表面之一鍵聯基團，且其中該鍵聯基團進一步鍵聯至經構形以支援該微流體器件內之該一或多個生物細胞之細胞生長、存活性、可移植性或其等之任何組合之一部分。 The method of any one of clauses 70 to 71, wherein the at least one conditioning surface comprises covalently bonded to one of the bonding groups of the surface, and wherein the linking group is further bonded to the conformation to support Part of any combination of cell growth, viability, portability, or the like of the one or more biological cells within the microfluidic device. 如請求項70至72中任一項之方法，其中該至少一調節表面包括烷基或全氟烷基部分。 The method of any one of claims 70 to 72, wherein the at least one conditioning surface comprises an alkyl or perfluoroalkyl moiety. 如請求項70至72中任一項之方法，其中該至少一調節表面包括伸烷基醚部分或聚葡萄糖部分。 The method of any one of claims 70 to 72, wherein the at least one conditioning surface comprises an alkyl ether moiety or a polydextrose moiety. 如請求項70至74中任一項之方法，其進一步包括：調節該至少一生長室之至少一表面。 The method of any one of claims 70 to 74, further comprising: adjusting at least one surface of the at least one growth chamber. 如請求項75之方法，其中調節包括：使用包括一聚合物之一調節試劑來處理該至少一生長室之該至少一表面。 The method of claim 75, wherein the adjusting comprises: treating the at least one surface of the at least one growth chamber with a conditioning agent comprising a polymer. 如請求項75至76中任一項之方法，其中調節包括：使用哺乳動物血清之一或多個組分來處理該至少一生長室之至少一表面。 The method of any one of clauses 75 to 76, wherein the adjusting comprises: treating at least one surface of the at least one growth chamber with one or more components of the mammalian serum. 如請求項75至77中任一項之方法，其中調節包括：使用至少一細胞黏著阻斷分子來處理該至少一生長室之至少一表面。 The method of any one of clauses 75 to 77, wherein the adjusting comprises: treating at least one surface of the at least one growth chamber with at least one cell adhesion blocking molecule. 如請求項70至78中任一項之方法，其中將該至少一生物細胞引入至該至少一生長室中包括：使用具有足以移動該至少一生物細胞之力量之一介電泳(DEP)力。 The method of any one of claims 70 to 78, wherein introducing the at least one biological cell into the at least one growth chamber comprises using a dielectrophoresis (DEP) force having a force sufficient to move the at least one biological cell. 如請求項79之方法，其中光學地致動該DEP力。 The method of claim 79, wherein the DEP force is optically actuated. 如請求項70至80中任一項之方法，其進一步包括：在該培育步驟期間灌注該第一流體培養基，其中該第一流體培養基經由該微流體器件之至少一入口而引入且經由該微流體器件之至少一出口而輸出，其中在輸出之後，該第一流體培養基視情況包括來自第二流體培養基之組分。 The method of any one of claims 70 to 80, further comprising: injecting the first fluid medium during the incubating step, wherein the first fluid medium is introduced via the at least one inlet of the microfluidic device and via the micro The fluid device is outputted from at least one outlet, wherein after the output, the first fluid medium optionally includes components from the second fluid medium. 如請求項70至81中任一項之方法，其進一步包括下列步驟：在該培育步驟之後使該調節表面之一或多個可裂解部***解，藉此促進該一或多個生物細胞自該生長室或其隔離區域輸出而進入該流動區域。 The method of any one of claims 70 to 81, further comprising the step of lysing one or more cleavable moieties of the modulating surface after the culturing step, thereby promoting the one or more biological cells from the The growth chamber or its isolated area is output and enters the flow area. 如請求項70至82中任一項之方法，其進一步包括下列步驟：使一或多個生物細胞自該生長室或其之該隔離區域輸出而進入該流動區域。 The method of any one of claims 70 to 82, further comprising the step of outputting one or more biological cells from the growth chamber or the isolated region thereof into the flow region. 如請求項70至83中任一項之方法，其中該至少一生物細胞係一哺乳動物細胞。 The method of any one of claims 70 to 83, wherein the at least one biological cell line is a mammalian cell. 如請求項70至84中任一項之方法，其中該至少一生物細胞係一免疫細胞。 The method of any one of clauses 70 to 84, wherein the at least one biological cell line is an immune cell. 如請求項85之方法，其中該免疫細胞係一淋巴細胞或白血球。 The method of claim 85, wherein the immune cell line is a lymphocyte or a white blood cell. 如請求項85之方法，其中該免疫細胞係一B細胞、一T細胞、NK細胞、一巨噬細胞或一樹突細胞。 The method of claim 85, wherein the immune cell is a B cell, a T cell, an NK cell, a macrophage or a dendritic cell. 如請求項70至85中任一項之方法，其中該至少一生物細胞係一貼附細胞。 The method of any one of claims 70 to 85, wherein the at least one biological cell line attaches to the cell. 如請求項70至85中任一項之方法，其中該至少一生物細胞係一融合瘤細胞。 The method of any one of claims 70 to 85, wherein the at least one biological cell line is a fusion tumor cell. 如請求項70至89中任一項之方法，其中將該至少一生物細胞引入至該至少一生長室中包括：將一單一細胞引入至該生長室中，且其中藉由該培育步驟而產生之該生物細胞群落係一選殖群落。 The method of any one of claims 70 to 89, wherein introducing the at least one biological cell into the at least one growth chamber comprises: introducing a single cell into the growth chamber, and wherein the culturing step is generated The biological cell community is a colonization community.