WO2023132710A1 - Appareil d'immobilisation de cellules capable de collecter des cellules - Google Patents

Appareil d'immobilisation de cellules capable de collecter des cellules Download PDF

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Publication number
WO2023132710A1
WO2023132710A1 PCT/KR2023/000342 KR2023000342W WO2023132710A1 WO 2023132710 A1 WO2023132710 A1 WO 2023132710A1 KR 2023000342 W KR2023000342 W KR 2023000342W WO 2023132710 A1 WO2023132710 A1 WO 2023132710A1
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WO
WIPO (PCT)
Prior art keywords
glass
gel
curing chamber
cell
gel curing
Prior art date
Application number
PCT/KR2023/000342
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English (en)
Korean (ko)
Inventor
김승훈
강효정
배가은
노혜란
이정민
Original Assignee
주식회사 씨티셀즈
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from KR1020230001566A external-priority patent/KR20230107136A/ko
Application filed by 주식회사 씨티셀즈 filed Critical 주식회사 씨티셀즈
Publication of WO2023132710A1 publication Critical patent/WO2023132710A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • the present invention relates to a cell immobilization device capable of cell picking, and more particularly, by centrifugation in a state in which glass and gel curing chambers are fixed by a fixing unit, cell separation can be accurately and easily separated without loss of cells.
  • a cell immobilization device capable of cell picking, and more particularly, by centrifugation in a state in which glass and gel curing chambers are fixed by a fixing unit, cell separation can be accurately and easily separated without loss of cells.
  • it is possible to access the hydrogel with the gel curing chamber separated from the glass, making it possible to use cell picking equipment for immunostaining or subsequent analysis after immunostaining. it's about
  • Immunocytochemistry (ICC) analysis is an experimental technique that uses the property of antibodies to specifically bind to antigens to study and confirm the presence of specific substances in tissues or cells.
  • a method of attaching cells to glass using a cell centrifuge may be used.
  • cells can be attached to glass using a cytospin.
  • this device there is a risk that approximately 40% of all cells may be lost in the process of centrifugation.
  • the chamber has a state attached to the glass, and in this state, cell picking can be performed, but due to the wall structure of the chamber, it is difficult to pick cells for later analysis. can have
  • cells of a new configuration can minimize cell loss during centrifugation of the device, uniformly and precisely perform the curing process for cell staining, and facilitate easy access to cells and smooth cell picking.
  • immobilization devices There is a demand for the development of immobilization devices.
  • the present invention is a biomedical technology development (R&D) task of the National Research Foundation of Korea under the Ministry of Science and ICT, which is a mass production-based target cell separation device production and operation performance verification, and inertia for isolating fetal cells from pregnant women's blood for non-invasive early prenatal diagnosis. It is the result of a part of the centrifugal microfluidic technology development project.
  • centrifugation is performed in a state in which the glass and gel curing chamber are fixed by the fixing part, so that cell separation can be accurately and easily separated without loss of cells, and the gel curing chamber is separated from the glass.
  • a cell immobilization device capable of cell picking that allows access to a hydrogel and facilitates the use of a cell picking device for immunostaining or subsequent analysis after immunostaining.
  • An apparatus for immobilizing cells capable of picking cells includes a gel curing chamber having a hollow portion and having glass for curing a hydrogel disposed on a bottom surface open downward; a gel thickness formation unit that is retractable into the hollow portion of the gel curing chamber and limits exposure of the hydrogel placed on the glass in a sol state to ultraviolet rays by a set thickness within the gel curing chamber; and a fixing unit fixing the glass to the gel curing chamber.
  • the fixing part, the base member on which the glass is fixed to the upper surface and the gel curing chamber is disposed on the glass; and an intermediate member having a first through hole corresponding to the shape of the gel curing chamber, passing through the gel curing chamber through the first through hole, and disposed above the base member. and a fixing member fixing a position of the glass with respect to the gel curing chamber by fixing the intermediate member to the base member.
  • the fixing member is formed protruding in a circular band shape partially cut with the glass interposed therebetween from the upper surface of the base member, the outer surface of which is formed with a threaded portion; and rotatably coupled to the thread of the threaded portion passing through the second through hole of the intermediate member to correspond to the shape of the threaded portion, thereby fixing the intermediate member with respect to the base member, thereby forming the gel.
  • a fastening portion for fixing the position of the glass relative to the curing chamber may be included.
  • the upper surface of the base member may be provided with an insertion groove into which the glass is inserted.
  • a sealing portion interposed between the gel curing chamber and the glass may further include a sealing portion that closely adheres between the bottom of the gel curing chamber and the glass.
  • an azide-based coating material may be coated on the glass.
  • the gel thickness forming unit may be provided with a hydrophobic material for separation from the hydrogel.
  • the gel curing chamber may be provided in a black series.
  • a stepped member is provided at the lower end of the inner wall of the gel curing chamber to be stepped toward the center of the gel curing chamber, and the gel thickness forming unit so that the bottom surface of the gel thickness forming unit is spaced apart from the upper surface of the glass.
  • a holding member caught on the stepped member may be provided at a lower end of the outer wall.
  • the formed length of the stepped member is greater than the formed length of the holding member, so that the lower end of the gel thickness forming part is spaced apart from the glass by 10 micrometers to 200 micrometers, so that the hydrogel can be formed in the spaced space.
  • centrifugation is performed in a state where the glass and gel curing chamber are fixed by the fixing part, so that cell separation can be accurately and easily separated without loss of cells, and the gel curing chamber is separated from the glass.
  • this state access to the hydrogel is possible, which facilitates the use of cell picking equipment for immunostaining or subsequent analysis after immunostaining.
  • FIG. 1 is an exploded perspective view of a cell immobilization device capable of picking cells according to an embodiment of the present invention.
  • FIG. 2 is a cross-sectional view of a coupled state of the cell immobilization device of FIG. 1 .
  • FIG. 3 is a view schematically illustrating a subsequent process after removing the gel curing chamber from the cell immobilization device of FIG. 2 .
  • Figure 4 is a view schematically showing the coupling structure of the gel curing chamber and the gel thickness forming portion shown in Figure 1;
  • Figure 5 is a three-dimensional perspective view showing the coupling structure of Figure 4.
  • FIG. 1 is an exploded perspective view of a cell immobilization device capable of cell picking according to an embodiment of the present invention
  • FIG. 2 is a cross-sectional view of a coupled state of the cell immobilization device of FIG. 1
  • FIG. 3 is a cell immobilization device of FIG. It is a diagram schematically showing the subsequent process after removing the gel curing chamber from the device.
  • a cell fixation device 100 capable of cell picking according to an embodiment of the present invention includes a gel curing chamber 110, a gel thickness forming unit 120, a fixing unit 150 ) may be included.
  • the gel curing chamber 110 of this embodiment may have a columnar shape having a hollow portion as a whole.
  • it may have a rectangular column shape as a whole, the center portion of which is penetrated in the height direction, and may be provided in a rectangular column shape by the four chamber walls 111.
  • the shape of the gel curing chamber 110 is not limited to the above-mentioned case, and the imaging range of the device, such as the hydrogel shape, the concentration of cells in the suspension (cfu/ml) and a fluorescence microscope for observing the stained cells.
  • the shape of the chamber 110 may be provided in various ways to suit the resolution, for example, it may be a cylindrical shape having a hollow portion, and in this case, the glass may be circular.
  • Such a gel curing chamber 110 in the prior art, for example, if made of a polymethylsiloxane (PDMS) material, in the case of this embodiment, it may be made of a polycarbonate (PC) material, but the mass of the gel curing chamber 110 Any material suitable for production can be applied without limitation.
  • PDMS polymethylsiloxane
  • PC polycarbonate
  • the gel curing chamber 110 of this embodiment is polyimide (polyimide), polyamide (polyamide), polyamic acid (polyamicacid), polyethylene (poly ethylene (PE), polyetherimide (polyetherimide), polyaramid ( polyaramide), poly-r-benzylglumate, poly-p-phenylene terephthalamide, polyacrylonitrile (PAN), polyethylene terephthalate ( polyethyleneterephthalate (PET), nylon 6 (nylon 6), polyurethane (PU), polystyrene (PS), polycarbonate (PC), polyvinylchloride (PVC), polyvinylalcohol; PVA), polyvinylidene fluoride (PVdF), DNA, PLA / PLGA (polyactic acid), polyether ether ketone (PEEK) and polyethylene oxide (polyethyleneoxide; PEO) selected from polymers consisting of It may be provided with a copolymer material formed by a crosslinking reaction of one
  • the gel curing chamber 110 may be provided in black, including black, to reduce fluorescence during fluorescence imaging. However, it is not limited thereto.
  • the glass 130 of this embodiment may be provided in a rectangular plate shape having an area larger than the left and right widths of the gel curing chamber 110 .
  • the curing process of the hydrogel 101 (see FIG. 3) on the glass 130 can be reliably performed.
  • An azide-based coating material may be coated on the glass 130 to improve adhesion between cells and hydrogel.
  • the type of coating material is not limited thereto, and any coating material capable of enhancing the adhesion of cells and hydrogel to the glass 130 can be applied, such as Poly-L-Lysin.
  • a sealing unit 140 may be provided between the gel curing chamber 110 and the glass 130 . Airtightness between the gel curing chamber 110 and the glass 130 may be maximized through the sealing unit 140 .
  • the sealing unit 140 has a rectangular band shape corresponding to the rectangular shape of the gel curing chamber 110, and seals when the gel curing chamber 110 is fixed to the glass 130 by the fixing unit 150 to be described later.
  • the space between them can be sealed by the unit 140, and through this, leakage of liquid during centrifugation of the cell immobilization device 100 of the present embodiment can be prevented, and imaging interference can occur because the bond is not applied. can prevent
  • a groove into which the sealing part 140 can be inserted may be provided on the bottom surface of the gel curing chamber 110, and the thickness of the sealing part 140 may be equal to or slightly greater than the depth of the groove.
  • the sealing unit 140 may be made of a material having elasticity or a material having adhesive strength.
  • a material having adhesive strength a tape whose adhesive strength is reduced when exposed to ultraviolet rays can be used. Through this, when the adhesive strength of the sealing portion 140 is weakened by irradiating ultraviolet rays to the side of the glass 130, the gel curing chamber 110 is separated. can do.
  • the sealing unit 140 may be provided with, for example, a gasket. However, it is not limited thereto.
  • the gel thickness forming unit 120 of this embodiment is drawn into the hollow portion of the gel curing chamber 110 and placed on the glass 130 in a sol state.
  • the gel As the gel is exposed to UV rays only as much as the set thickness, it may have a shape that is open upward and the lower end is blocked.
  • liquid hydrogel is injected onto the glass 130 in a state where the gel curing chamber 110 and the glass 130 are firmly coupled by the fixing unit 150, and the gel thickness forming unit 120 After pressurization, the hydrogel is cured. At this time, the hydrogel is cured to a certain thickness by the separation space (110S, see FIG. 4) between the bottom surface of the gel thickness forming unit 120 and the upper surface of the glass 130 can
  • the hydrogel can be formed to a desired thickness, particularly into an ultra-thin film.
  • the bottom part of the gel thickness forming part 120 of this embodiment is a part in direct contact with the hydrogel, it may be provided with a hydrophobic material.
  • the hydrogel has hydrophilicity, when the gel thickness forming portion 120 is made of a hydrophilic material, the separation of the bottom portion of the gel thickness forming portion 120 from the hydrogel may not be performed well.
  • the gel thickness forming part 120 of this embodiment is made of a hydrophobic material.
  • the side portion of the gel thickness forming unit 120 has a passage hole 125 for passing the hydrogel rising between the side portion and the inner wall of the gel curing chamber 110 when the hydrogel is pressed with the bottom portion. ) is formed, and the hydrogel flows out of the gel curing chamber 110 by inducing the hydrogel to move into the gel thickness forming part 120 through the passage hole 125 while pressing the gel thickness forming part 120. can prevent it from happening.
  • the process of picking the cells in the hydrogel must proceed.
  • the wall structure of the chamber conventionally has limitations in that it is not easy to enter and manipulate the cell picking device. there was.
  • the gel curing process can be uniform and accurate, and the gel curing chamber 110 can be removed from the glass 130 to facilitate access to the cells thereafter.
  • a structure is required, which can be implemented by the fixing unit 150 of this embodiment.
  • the fixing part 150 can firmly maintain the coupling of the gel curing chamber 110 to the glass 130 during centrifugation, and also removes the gel from the glass 130 during cell picking. By separating the curing chamber 110, a desired operation can be performed smoothly and accurately.
  • the fixing part 150 of this embodiment may fix the position of the glass 130 with respect to the gel curing chamber 110 while the glass 130 is placed on the upper surface.
  • the position of the gel curing chamber 110 relative to the glass 130 is firmly fixed so as to reliably prevent loss of cells during centrifugation of the device.
  • the fixing part 150 includes a base member 151 on which a glass 130 is fixed to an upper surface and a gel curing chamber 110 is disposed on the glass 130, and a gel curing chamber A first through hole 156 corresponding to the cross-sectional shape of (110) is formed, and through the first through hole 156, the intermediate member 155 is placed on top of the base member 151 through the gel curing chamber 110. ), and a fixing member 160 for fixing the position of the glass 130 with respect to the gel curing chamber 110 by fixing the intermediate member 155 with respect to the base member 151.
  • the base member 151 of this embodiment is provided in a rectangular plate shape, as shown in FIGS. 1 and 2, and an insertion groove corresponding to the shape of the glass 130 on its upper surface. 152 is provided so that the glass 130 can be inserted and maintained in position.
  • the four vertex areas of the base member 151 are provided with support portions 153 protruding upward, so that the four vertex portions of the intermediate member 155 to be described later can be supported thereon.
  • the glass 130 is placed on the base member 151, the gel curing chamber 110 is placed thereon so that the sealing part 140 is positioned therebetween, and then the first through hole 156 formed in the intermediate member 155 By penetrating the gel curing chamber 110 through the middle member 155 can be raised on the base member 151.
  • the outer surface of the four side portions of the gel curing chamber 110 may be provided with a right-triangle-shaped penetrating member 115 that grows outward as it goes downward, and the middle member 155
  • One through hole 156 may be formed to correspond to a cross section in a horizontal direction of the gel curing chamber 110 in which the through member 115 is provided.
  • the penetrating member 115 of the gel curing chamber 110 in the lower part of the first through hole 156 Since the size (horizontal length) of the gel curing chamber 110 is larger than the size of the first through hole 156, it is possible to prevent the gel curing chamber 110 from falling upward through the first through hole 156. That is, the position of the gel curing chamber 110 can be maintained by the intermediate member 155 .
  • the intermediate member 155 since the position of the intermediate member 155 with respect to the base member 151 is not fixed, the intermediate member 155 must be fixed with respect to the base member 151, which is achieved by the fixing member 160 of the present embodiment. It can be done.
  • the fixing member 160 of this embodiment is formed protruding in a circular band shape partially cut from the upper surface of the base member 151 with the glass 130 in between, and on the outer surface
  • the threaded portion 161 penetrated through the threaded portion 161 where the screw thread 162 is formed and the second through hole 157 of the intermediate member 155 penetrated to correspond to the shape of the threaded portion 161.
  • It may include a fastening portion 165 that is rotatably coupled to the screw thread.
  • the fixing member 160 of this embodiment can fix or unfix the intermediate member 155 with respect to the base member 151 by tightening and releasing the tightening operation using the screw coupling principle.
  • the intermediate member 155 has a second through hole 157 formed in a cut circular shape, through which the threaded portion 161 of the fixing member 160 provided in the base member 151 is formed. Penetrating through the intermediate member 155, its upper end may be positioned above the intermediate member 155.
  • the fastening part 165 having a nut structure When the circular fastening part 165 having a nut structure is coupled to the threaded part 161 and then rotated, the fastening part 165 is in close contact with the intermediate member 155 in the direction of the screw coupling principle, and the intermediate member 155
  • the base member 151 may be fixed in a state of being in close contact with the base member 151 .
  • the gel curing chamber 110 which receives force from the intermediate member 155, can be strongly adhered to the glass 130, so that subsequent processes such as centrifugation can be performed accurately and smoothly.
  • the fastening part 165 is released by the fixing part 150, that is, from the threaded part 161, and the intermediate member 155 is placed on the base.
  • the fixation of the gel curing chamber 110 to the glass 130 can be released, and thus the hydrogel 101 can be accessed to facilitate cell 102 picking. there is.
  • single cell picking can be accurately performed by the cell picking device 200, or as shown in (c) of FIG. 3, by the standard equipment 300.
  • Immunofluorescent staining of cells can be performed by an automated process.
  • immunofluorescence staining of cells may be performed before or after separation of the gel curing chamber 110 .
  • the fixing structure of the fixing part 150 due to the fixing structure of the fixing part 150, cell loss can be surely prevented during centrifugation of the device, and the gel curing process can be performed uniformly and accurately, and then the fixing part 150 is fixed. After separating the gel curing chamber 110 from the glass 130 due to release, cell picking from the hydrogel can be smoothly and accurately performed.
  • FIG. 4 is a schematic view of a coupling structure of a gel curing chamber and a gel thickness forming unit shown in FIG. 1
  • FIG. 5 is a three-dimensional perspective view of the coupling structure of FIG. 4 .
  • a stepped member 117 is provided at the lower end of the inner wall of the gel curing chamber 110 of this embodiment in a stepwise manner in the center direction of the gel curing chamber 110, and the gel thickness forming portion 120 A holding member 127 caught on the stepped member 117 may be provided at the lower end of the outer wall, and through this, the bottom surface of the gel thickness forming part 120 is spaced apart from the glass 130, thereby creating a space for hydrogel formation ( 110S) can be formed.
  • the hooking part provided at the upper end of the gel thickness forming part has a structure that is caught on the upper wall of the chamber, and at this time, the bottom surface of the gel thickness forming part is provided so that it can be spaced apart from the glass.
  • the lower part of the gel curing chamber 110 in which this space is formed and the lower part of the gel thickness forming part 120 are provided to minimize thickness deviation.
  • the occurrence of tolerance according to can be minimized.
  • the formation length (L1) of the stepped member 117 is greater than the formation length (L2) of the holding member 127, so that the bottom surface of the gel thickness formation part 120 is 10 micrometers to 200 micrometers with respect to the glass 130.
  • a hydrogel may be formed in the spaced space 110S in a micrometer range, preferably 100 micrometers apart.
  • the formation length (L1) of the stepped member 117 of the gel curing chamber 110 is 1000 micrometers and the formation length (L2) of the holding member 127 is 900 micrometers, these
  • the bottom surface of the gel thickness forming unit 120 may be spaced apart from the glass 130 by the difference, that is, by 100 micrometers, and accordingly, a hydrogel having a uniform thickness may be formed.
  • centrifugation is performed in a state where the glass 130 and the gel curing chamber 110 are fixed by the fixing part 150, and cell separation can be accurately and easily separated without loss of cells.
  • access to the hydrogel is possible while the gel curing chamber 110 is separated from the glass 130 by the fixing part 150, so it is easy to use cell picking equipment for immunostaining or subsequent analysis after immunostaining. can do

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Abstract

Appareil d'immobilisation de cellules capable de collecter des cellules, selon un mode de réalisation de la présente invention, pouvant comprendre : une chambre de durcissement de gel qui comprend une partie creuse et dans laquelle du verre destiné au durcissement d'un hydrogel est disposé sur une surface inférieure qui est ouverte vers le bas ; une partie de formation d'épaisseur de gel qui peut être rétractée dans la partie creuse de la chambre de durcissement de gel, et maintient l'hydrogel placé dans un état de sol sur le verre dans les limites de la chambre de durcissement de gel pour qu'il soit exposé à des rayons ultraviolets jusqu'à une épaisseur définie ; et une partie d'immobilisation destinée à immobiliser le verre par rapport à la chambre de durcissement de gel.
PCT/KR2023/000342 2022-01-07 2023-01-07 Appareil d'immobilisation de cellules capable de collecter des cellules WO2023132710A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20220002465 2022-01-07
KR10-2022-0002465 2022-01-07
KR1020230001566A KR20230107136A (ko) 2022-01-07 2023-01-05 세포 피킹이 가능한 세포 고정화 장치
KR10-2023-0001566 2023-01-05

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WO2023132710A1 true WO2023132710A1 (fr) 2023-07-13

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140116997A (ko) * 2013-03-25 2014-10-07 테고사이언스 (주) 세포배양장치 및 이를 이용한 세포배양방법
US20190070291A1 (en) * 2000-05-19 2019-03-07 Genentech, Inc. GENE DETECTION ASSAY FOR IMPROVING THE LIKELIHOOD OF AN EFFECTIVE RESPONSE TO AN ErbB ANTAGONIST CANCER THERAPY
US20190106667A1 (en) * 2017-10-10 2019-04-11 Cypre, Inc. Methods and devices for cell culture
US20210023273A1 (en) * 2018-03-12 2021-01-28 Centre D'etude Des Cellules Souches Method and device for preparing an implant obtained from a culture of stem cells
KR20210119051A (ko) * 2020-03-24 2021-10-05 재단법인대구경북과학기술원 무손실 세포 염색 방법 및 장치

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190070291A1 (en) * 2000-05-19 2019-03-07 Genentech, Inc. GENE DETECTION ASSAY FOR IMPROVING THE LIKELIHOOD OF AN EFFECTIVE RESPONSE TO AN ErbB ANTAGONIST CANCER THERAPY
KR20140116997A (ko) * 2013-03-25 2014-10-07 테고사이언스 (주) 세포배양장치 및 이를 이용한 세포배양방법
US20190106667A1 (en) * 2017-10-10 2019-04-11 Cypre, Inc. Methods and devices for cell culture
US20210023273A1 (en) * 2018-03-12 2021-01-28 Centre D'etude Des Cellules Souches Method and device for preparing an implant obtained from a culture of stem cells
KR20210119051A (ko) * 2020-03-24 2021-10-05 재단법인대구경북과학기술원 무손실 세포 염색 방법 및 장치

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