CN113046243A

CN113046243A - Culture device

Info

Publication number: CN113046243A
Application number: CN202110341842.3A
Authority: CN
Inventors: 王玄
Original assignee: Shanghai Ruiyu Biotech Co Ltd
Current assignee: Shanghai Ruiyu Biotech Co Ltd
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2021-06-29

Abstract

The embodiment of the application discloses a culture device. The culture device comprises a substrate, the substrate surface comprising at least two culture areas for adhering a culture; the culture region interior surface has a submicron to nanometer scale structured structure such that the culture region interior has a greater adhesion affinity for the culture than the culture region exterior.

Description

Culture device

Technical Field

The application relates to the technical field of biological culture, in particular to a culture device.

Background

With the continuous development of modern society, the in vitro culture technology of organisms (such as cells, bacteria, viruses, etc.) is receiving more and more attention. Taking cells as an example, cell culture is an important and common technique in cell biology research methods, and a large number of cells can be obtained through cell culture, and signal transduction, anabolism, growth and proliferation of cells and the like of the cells can be researched by the cell culture.

Disclosure of Invention

One of the embodiments of the present application provides a culture device comprising a substrate, the substrate surface comprising at least two culture areas for adhering a culture; the culture region interior surface has a submicron to nanometer scale structured structure such that the culture region interior has a greater adhesion affinity for the culture than the culture region exterior.

In some embodiments, the sub-micron to nano-scale structured structure includes a plurality of micro-nano monomers arranged in an array, the micro-nano monomers including micro-nano columns, micro-nano tubes, micro-nano cones, and/or micro-nano walls.

In some embodiments, the micro-nano monomer has a diameter or maximum width of less than 1 μm.

In some embodiments, the head of the micro-nano monomer is mushroom-shaped.

In some embodiments, the interior of the culture region is etched such that the interior surface of the culture region has a submicron to nanometer scale regular structure.

In some embodiments, the etching process includes a soft etching technique, laser etching, plasma etching, electron beam etching, and/or chemical etching.

In some embodiments, the material of the substrate is a hydrophilic material.

In some embodiments, the inside of the culture region is subjected to a modification treatment, the hydrophilicity of the inside of the culture region after the modification treatment is stronger than that of the outside of the culture region, and the modification treatment includes plasma treatment, ray irradiation treatment and/or corona treatment.

In some embodiments, the material of the substrate is a hydrophobic material.

In some embodiments, the interior of the culture region is subjected to a modification treatment, the modified interior of the culture region has hydrophilicity, and the modification treatment comprises plasma treatment, ray irradiation treatment and/or corona treatment.

In some embodiments, the material of the substrate comprises at least one of: glass, quartz, silicon, mica, PS, PMMA, PSU, PC, PP, PE, PETG, LDPE, HDPE, PET, PVDF, PTFE, PEG, PEO, PPG, PLA, PGA, PLGA, PDMS, PVA, COC, COP, PMP, styrene/butadiene copolymer, styrene/acrylonitrile copolymer, cellulose acetate, cellulose nitrate, hydroxyethyl methacrylate, polyethersulfone, diallyl diethylene glycol polymer or nylon 66.

In some embodiments, the substrate surface is modified with a hydrophilic species or group comprising at least one of: collagen, fibronectin, laminin, polylysine, gelatin, hyaluronic acid, chitosan, RGD polypeptide, DNA, lysine, metallic gold, hydroxyl, carboxyl, carbonyl, amino, sulfydryl, sulfonic group, phosphate group, quaternary ammonium group, ether bond, carboxylic ester, amide group or block polyether.

In some embodiments, the substrate surface is modified with a hydrophobic substance or group comprising at least one of: albumin, a hydrocarbon group containing a double bond, a polyoxypropylene group, a long-chain perfluoroalkyl group, a polysiloxane group, or a hydrocarbon group containing an aryl, ester, ether, amine, or amide group.

In some embodiments, the interior of the culture region is modified with a hydrophilic substance or group.

In some embodiments, the exterior of the culture region is modified with a hydrophobic substance or group.

In some embodiments, the interior of the culture region is more hydrophilic than the exterior of the culture region.

Drawings

The present description will be further described by way of exemplary embodiments, which will be described in detail by way of the accompanying drawings. These embodiments are not intended to be limiting, and in these embodiments like numerals are used to indicate like structures, wherein:

FIG. 1 is a schematic view of a culture device according to some embodiments of the present application;

FIG. 2 is a schematic side view of a culture device according to some embodiments of the present application;

FIG. 3 is a schematic view of the structured structure of the interior surface of a culture region according to some embodiments of the present application;

FIG. 4 is a schematic view of the structure of the interior surface of a culture section according to yet another embodiment of the present application;

FIG. 5 is a schematic view of the structure of the inner surface of the culture section according to still another embodiment of the present application.

In the figure, 100 is a culture apparatus, 110 is a substrate, 120 is a culture region, 122 is a micro-nano monomer, 130 is a non-culture region, 140 is an etching treatment surface, and 150 is a polishing treatment surface.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

On the contrary, this application is intended to cover any alternatives, modifications, equivalents, and alternatives that may be included within the spirit and scope of the application as defined by the appended claims. Furthermore, in the following detailed description of the present application, certain specific details are set forth in order to provide a better understanding of the present application. It will be apparent to one skilled in the art that the present application may be practiced without these specific details.

The embodiment of the application relates to a culture device. The culture device can be used for in vitro culture of organisms (such as cells, bacteria, viruses and the like). Taking cell culture as an example, the culture that can be cultured in the culture device can include, but is not limited to, one or more of 2D adherent cells, suspension divided cells, 3D cell clusters, organoids, ex vivo living tissues, ex vivo living organs, non-ex vivo tissues, non-ex vivo organs, cell and microcarrier complexes, cell and scaffold complexes, and the like. Wherein, the scaffold can be gel material, tissue engineering porous scaffold, etc.

In some embodiments, the culture device can be used for three-dimensional culture of cells. Three-dimensional cell culture may refer to culturing cells to be cultured in a carrier (or scaffold) that may provide a three-dimensional environment in which the cells to be cultured grow, maintaining the cells to be cultured in a state of growing in the three-dimensional environment, and finally obtaining a three-dimensional cell culture product (e.g., organoids, tissue-like, etc.). In some embodiments, matrigel (e.g., temperature sensitive gel) can serve as a carrier (or scaffold) for three-dimensional cell culture. In some embodiments, the cells to be cultured may be mixed in a liquid matrigel prior to inoculating the culture, the liquid matrigel may undergo a phase transition under conditions that transition from a liquid state to a solid state (or gel state), and the solid state (or gel state) matrigel may provide a three-dimensional environment for the growth of the cells to be cultured. In some embodiments, the matrigel may be a temperature sensitive gel that may undergo a phase transition with a change in temperature. The temperature sensitive gel may include, but is not limited to, poly N-isopropylacrylamide (PNIPAM), block copolymers of poly N-isopropylacrylamide and polyethylene glycol (PNIPAM-PEG), polyethylene glycol (PEG), block copolymers of polylactic-co-glycolic acid (PEG-PLGA), PLGA-PEG-PLGA triblock polymers, and the like. In some embodiments, a culture may be understood as a matrigel mixed with cells to be cultured. In some alternative embodiments, the culture device may be used for two-dimensional culture of cells.

In some embodiments, the culture device may comprise one or more culture chambers. A culture chamber may refer to a device or structure that provides space for the cells to be cultured to survive, grow, and multiply. In some embodiments, the culture chamber may comprise a petri dish, a flask, a plate, or a column, among others. The substrate may be understood as the part of the culture chamber that is in contact with the culture (e.g. the bottom wall of the culture chamber).

An important step in the cultivation process (in the case of cell cultures) is the inoculation of the culture, i.e. the addition of the culture to the culture chamber. In some embodiments, the seeding of the cell culture may be by manual seeding. For example, the operator inoculates the culture on the substrate one by one (or row by row with a row needle) using a pipette. The manual one-by-one inoculation mode has low inoculation efficiency and slow speed, and may influence the inoculated culture and further influence the product quality of cell culture. For example, when the culture is matrigel mixed with cells to be cultured, because the phase transition temperature range of the matrigel is narrow (for example, the temperature-sensitive gel is liquefied at 4 ℃ and gradually coagulates at more than 10 ℃), if the temperature is improperly controlled, the culture may be already coagulated without being inoculated, thereby not only reducing the efficiency of cell culture, but also possibly causing the product of cell culture not to meet the culture requirements.

Some embodiments of the present disclosure provide a culture apparatus, wherein a patterned array that is alternately changed is designed on a surface of a substrate, and when a culture is inoculated, a liquid culture (e.g., matrigel mixed with cells to be cultured) can automatically stay in a culture region with a relatively high adhesion affinity (e.g., a relatively high roughness, a relatively high hydrophilicity, a relatively low hydrophobicity, a regular structure with a submicron to nanometer level, etc.), so that a plurality of spots can be automatically formed by one-time liquid feeding. The culture device can effectively improve the inoculation speed of the culture, and improve the efficiency of cell culture and the quality of cell culture products.

FIG. 1 is a schematic view of a culture device according to some embodiments of the present application; FIG. 2 is a schematic side view of a culture device according to some embodiments of the present application. The culture apparatus as claimed in the present application will be described below with reference to FIGS. 1 to 2. It is noted that the following description is for illustrative purposes only and is not intended to limit the scope of the present application.

As shown in fig. 1-2, the culture device 100 may include a substrate 110, the surface of the substrate 110 including at least two culture regions 120, the culture regions 120 may be used for adhering a culture (e.g., matrigel mixed with cells to be cultured). In some embodiments, the culture region 120 may be a regular shape. For example, the culture region 120 can be circular, square, triangular, oval, and the like. In some embodiments, the culture region 120 can also be irregularly shaped. In some embodiments, the culture region 120 can be circular, which can increase the contact area between the inoculated culture and the medium (e.g., the culture fluid) and increase the nutrient uptake rate.

In some embodiments, each culture region 120 can be the same shape and size, such that multiple batches of the same or nearly the same volume of culture can be quickly inoculated during cell culture. In practice, the volume of culture added may be determined by the desired spot size and the number of culture areas 120 in the culture device. After the liquid culture is added to the culture apparatus 100, the culture can be substantially uniformly adhered to each culture region 120 by shaking or tilting the culture apparatus. After the liquid culture becomes a solid state (or gel state), a culture medium may be added to the culture apparatus. The culture medium can be a nutrient solution for the growth and propagation of the culture, and can be prepared by combining different nutrient substances.

In some embodiments, the plurality of culture regions 120 may be arranged randomly or according to a certain rule. For example, the plurality of culture regions 120 may be arranged at equal intervals. In some embodiments, the size and spacing of the culture regions may be defined in order to ensure a more uniform and rapid uptake of the culture medium by the cells to be cultured. In some embodiments, the diameter of the circular culture area may be set between 0.1mm and 50 mm. Further, the diameter of the circular culture section may be set to be between 0.2mm and 10 mm. Further, the diameter of the circular culture section may be set between 1mm and 5 mm. In some embodiments, the spacing between any two culture regions can be greater than the diameter of the culture regions. In some embodiments, the spacing between any two culture regions can be greater than 1.5 times the diameter of the culture regions.

In some embodiments, the roughness inside the culture region may be different from the roughness outside the culture region, such that the adhesion affinity for the culture inside the culture region is greater than outside the culture region, thereby allowing for better adhesion of the culture inside the culture region 120.

In some embodiments, where the substrate surface is hydrophilic, the roughness of the interior of the culture region 120 can be greater than the roughness of the exterior of the culture region 120 (e.g., the non-culture region 130). In some embodiments, the substrate surface having hydrophilicity may include: the substrate material is one or more of a hydrophilic material, a modified treatment of part or all of the surface of the substrate, a modification of part or all of the surface of the substrate with a hydrophilic substance or group (hydrophilic modification), and the like. By making the roughness inside the culture region larger than outside the culture region on the substrate of the hydrophilic surface, the adhesion affinity to the culture inside the culture region can be made larger than outside the culture region, thereby making the culture inside the culture region adhere better. And during the culturing process, the drop of culture adhered inside the culture section 120 will take a relatively flat shape. The flat droplets, after solidifying to a gel state, facilitate better penetration of the external medium (e.g., broth) into the interior.

In some embodiments, the roughness inside the incubation area 120 may be greater than the roughness outside the incubation area 120 in a variety of ways. In some embodiments, the interior of the incubation area 120 can be polished to make the roughness inside the incubation area greater than the roughness outside the incubation area. For example, the inside of the culture region may be ground by sandpaper, pumice, or the like of a certain roughness so that the roughness inside the culture region is greater than the roughness outside the culture region. As shown in FIG. 2, the interior of the sanded culture region 120 may be shown as a sanded surface 150. The sanded surface 150 is an irregular rough surface (or is a non-regular structure). The differentiation of the roughness inside and outside the culture area is realized through a polishing mode, and the method has the effects of simple and rapid operation, low cost and the like. In some alternative embodiments, the roughness inside the incubation area may be larger than the roughness outside the incubation area by grinding (or polishing) the outside of the incubation area. In some embodiments, the interior of the incubation area may be etched such that the roughness of the interior of the incubation area is greater than the roughness of the exterior of the incubation area. In some embodiments, the etching process may include, but is not limited to, one or a combination of soft etching techniques, laser etching, plasma etching, e-beam etching, chemical etching, and the like. As shown in fig. 2, the interior of etched culture region 120 can be shown as etched surface 140. The etch process surface 140 is a regular rough surface. In some embodiments, the etch processing surface 140 may include a plurality of micro-scale or even nano-scale pillars. The method realizes the differentiation of the roughness inside and outside the culture area by an etching mode, and has the effects of good roughness controllability, high processing efficiency, good stability, good consistency among the culture areas and the like.

In some embodiments, the substrate surface has hydrophobicity, the roughness of the interior of the culture region is less than the roughness of the exterior of the culture region, and the exterior surface of the culture region is a non-regular structure. In some embodiments, the substrate surface being hydrophobic may include: the substrate material is a hydrophobic material, and a part or the whole surface of the substrate is modified by one or more of hydrophobic substances or groups (hydrophobic modification). By making the roughness inside the culture region smaller than the outside of the culture region on the substrate of the hydrophobic surface and making the surface outside the culture region be a non-regular structure, the adhesion affinity of the inside of the culture region to the culture can be made larger than that of the outside of the culture region, thereby making the culture inside the culture region better adhere. And during the cultivation, the drop of the culture attached to the inside of the cultivation region 120 will be in an approximately round and spherical shape. The spherical droplets help provide a more three-dimensional growth space for the culture after solidification to a gel state, thereby facilitating the culture of complex structures (e.g., organoids). In some embodiments, the exterior of the incubation area may be polished to make the roughness inside the incubation area less than the roughness outside the incubation area. In some alternative embodiments, the roughness of the inside of the incubation area may also be made smaller than the roughness of the outside of the incubation area by grinding (or polishing) the inside of the incubation area.

In some embodiments, the interior surface of the culture region can have a submicron to nanometer scale regular structure such that the interior of the culture region has a greater adhesion affinity for the culture than the exterior of the culture region, thereby allowing for better adhesion of the culture within the culture region 120. In some embodiments, the structured structure on the submicron to nanometer scale may include a plurality of micro-nano monomers arranged in an array. The micro-nano monomer can comprise one or more of a micro-nano column, a micro-nano tube, a micro-nano cone, a micro-nano wall and the like. In some embodiments, the micro-nano monomers may be arranged in a circular array, a rectangular array, or the like. In some embodiments, the distances between each two micro-nano monomers may be the same or different. In some embodiments, when the culture region interior surface has a submicron to nanometer scale regular structure, the culture region exterior surface can be non-regular (i.e., not have a regular structure).

FIG. 3 is a schematic view of a structured structure of the interior surface of a culture region according to some embodiments of the present application. FIG. 4 is a schematic view of the structure of the inner surface of the culture section according to still another embodiment of the present application. FIG. 5 is a schematic view of the structure of the inner surface of the culture section according to still another embodiment of the present application. As shown in fig. 3-5, the inner surface of the culture region 120 may have a submicron-to-nanometer scale regular structure, and the submicron-to-nanometer scale regular structure may include a plurality of micro-nano monomers 122 arranged in an array. As shown in fig. 3, the micro-nano monomer 122 may include micro-nano pillars. As shown in fig. 4, the micro-nano monomer 122 may include a micro-nano cone. In some embodiments, as shown in fig. 5, the micro-nano monomer 122 may be a micro-nano column, and a head of the micro-nano monomer 122 may be mushroom-shaped. The head of the micro-nano monomer is arranged to be mushroom-shaped, so that the adhesion affinity of a regular structure from submicron to nanometer level to a culture is stronger. In some embodiments, the head of the micro-nano monomer 122 may also have other shapes (e.g., spherical, planar, wedge-shaped, etc.).

In some embodiments, the micro-nano monomer has a diameter or maximum width of less than 1 μm. Further, the diameter or the maximum width of the micro-nano monomer can be 1-500 nm. Further, the diameter or the maximum width of the micro-nano monomer can be 50-200 nm. In some alternative embodiments, the diameter or maximum width of the micro-nano-monomer may be greater than or equal to 1 μm. For example, the diameter or the maximum width of the micro-nano monomer can be 1 to 100 μm. In some embodiments, the micro-nano monomer has a height of less than 5 μm. Further, the height of the micro-nano monomer can be 5-1000 nm. Further, the height of the micro-nano monomer can be 200-500 nm. In some embodiments, the ratio of the height of the micro-nano monomer to the diameter or the maximum width thereof may be 2:1 to 10: 1. In some embodiments, the distance between any two micro-nano monomers may be greater than the diameter or the maximum width of the micro-nano monomers. In some embodiments, the distance between any two micro-nano monomers may be greater than 1.5 times the diameter or the maximum width of the micro-nano monomers. In some embodiments, the distance between two adjacent micro-nano monomers may be 1 to 3 times the diameter or the maximum width of the micro-nano monomer.

In some embodiments, the interior of the culture region can be etched such that the interior surface of the culture region has a regular structure on the submicron to nanometer scale. The etching process may include one or a combination of soft etching techniques, laser etching, plasma etching, electron beam etching, chemical etching, and the like. In some embodiments, the engineered sub-micron to nano-scale structured structures may be attached to the substrate surface by gluing, clamping, or the like to form the culture region.

In some embodiments, the roughness inside the culture region can be different from the roughness outside the culture region, and the culture region interior surface has a submicron to nanometer scale regular structure, such that the adhesion affinity to the culture inside the culture region is greater than outside the culture region, thereby resulting in better adhesion of the culture inside the culture region 120. In some embodiments, the substrate surface has a hydrophilic property, the roughness inside the culture region is greater than the roughness outside the culture region, and the culture region inner surface has a submicron to nanometer scale regular structure. By using roughness differences in combination with the submicron to nanometer scale regular structures, the adhesion affinity to the culture inside the culture region can be further increased. In some embodiments, the substrate surface has hydrophobic properties, the roughness inside the culture region is greater than the roughness outside the culture region, and the culture region interior surface has a submicron to nanometer scale regular structure.

In some embodiments, the material of the substrate 110 may be a hydrophilic material. For example, the material of the substrate may include, but is not limited to, a combination of one or more of the following: glass, quartz, silicon, mica, PS (polystyrene), PMMA (polymethyl methacrylate/organic glass), PSU (polysulfone), PC (polycarbonate), PP (polypropylene), PE (polyethylene), PETG (polyethylene terephthalate-1, 4-cyclohexanedimethanol ester, a product obtained by Polycondensation of Terephthalic Acid (PTA), Ethylene Glycol (EG) and 1, 4-Cyclohexanedimethanol (CHDM) by an ester exchange method), LDPE (low density polyethylene), HDPE (high density polyethylene), PET (polyethylene terephthalate), PVDF (polyvinylidene fluoride), PTFE (polytetrafluoroethylene), PEG (polyethylene glycol), PEO (polyethylene oxide), PPG (polytrimethylene glutarate), PPO (poly 2, 6-dimethyl-1, 4-phenylene oxide), PLA (polylactic acid/polylactide), PGA (polyglutamic acid), PLGA (polylactic-co-glycolic acid), PDMS (polydimethylsiloxane), PVA (polyvinyl alcohol), COC (cyclic olefin copolymer), COP (cyclic olefin), PMP (poly-4-methyl-1-pentene), styrene/butadiene copolymer, styrene/acrylonitrile copolymer, cellulose acetate, cellulose nitrate, hydroxyethyl methacrylate, polyethersulfone, diallyl diethylene glycol polymer, nylon 66, or the like. In some embodiments, the base material itself may be hydrophilic. In some embodiments, the substrate material may be made hydrophilic by a specific processing method and processing technology. By using a substrate of hydrophilic material, both inside the culture region 120 and outside the culture region 120 can be made hydrophilic, and during the culture, the drop of culture adhering inside the culture region 120 will take a relatively flat shape. The flat droplets, after solidifying to a gel state, facilitate better penetration of the external medium (e.g., broth) into the interior. In some embodiments, the substrate 110 may be a transparent material (e.g., glass, etc.) to facilitate viewing of the culture.

In some embodiments, the interior of the culture region 120 may be modified based on the hydrophilic material of the substrate 110, and the modified interior of the culture region 120 is more hydrophilic than the exterior of the culture region 120. In some embodiments, the modification treatment may include a combination of one or more of plasma treatment, radiation treatment, corona treatment, and the like. For example only, in the modification treatment, the non-culture region 130 of the substrate 110 may be shielded by a shield (e.g., a mask, a baffle, etc.), and then the culture region 120 may be modified by a plasma generator or an ultraviolet lamp. In some embodiments, the greater hydrophilicity inside the culture region 120 than outside the culture region 120 may be understood as: the hydrophilic-lipophilic balance (HLB) value inside the culture region 120 is greater than outside the culture region 120, or the water contact angle inside the culture region 120 is less than outside the culture region 120. In some embodiments, the substrate 110 (e.g., inside the culture region 120) may be first treated for surface roughness and then modified; or the substrate 110 may be modified first and then subjected to surface roughness treatment; alternatively, the substrate 110 may be simultaneously subjected to the modification treatment and the surface roughness treatment. In some embodiments, the inside of the culture region may be processed (e.g., etched) to make the surface of the inside of the culture region have a regular structure in a submicron to nanometer scale, and then the inside of the culture region may be modified; or the interior of the culture region may be modified and then processed (e.g., etched) to make the interior surface of the culture region have a regular structure in the submicron to nanometer level. The hydrophilicity inside the culture region 120 is made stronger than that outside the culture region 120 by the modification treatment, and the adhesion affinity to the liquid droplet inside the culture region 120 can be made stronger.

In some embodiments, the material of the substrate 110 may be a hydrophobic material. For example, the material of the substrate may include, but is not limited to, a combination of one or more of the following: glass, quartz, silicon, mica, PS (polystyrene), PMMA (polymethyl methacrylate/organic glass), PSU (polysulfone), PC (polycarbonate), PP (polypropylene), PE (polyethylene), PETG (polyethylene terephthalate-1, 4-cyclohexanedimethanol ester, a product obtained by Polycondensation of Terephthalic Acid (PTA), Ethylene Glycol (EG) and 1, 4-Cyclohexanedimethanol (CHDM) by an ester exchange method), LDPE (low density polyethylene), HDPE (high density polyethylene), PET (polyethylene terephthalate), PVDF (polyvinylidene fluoride), PTFE (polytetrafluoroethylene), PEG (polyethylene glycol), PEO (polyethylene oxide), PPG (polytrimethylene glutarate), PPO (poly 2, 6-dimethyl-1, 4-phenylene oxide), PLA (polylactic acid/polylactide), PGA (polyglutamic acid), PLGA (polylactic-co-glycolic acid), PDMS (polydimethylsiloxane), PVA (polyvinyl alcohol), COC (cyclic olefin copolymer), COP (cyclic olefin), PMP (poly-4-methyl-1-pentene), styrene/butadiene copolymer, styrene/acrylonitrile copolymer, cellulose acetate, cellulose nitrate, hydroxyethyl methacrylate, polyethersulfone, diallyl diethylene glycol polymer, nylon 66, or the like. In some embodiments, the substrate material itself may be hydrophobic. In some embodiments, the substrate material may be made hydrophobic by a specific processing method and processing technology. By using a substrate made of hydrophobic material, both the inside of the culture region 120 and the outside of the culture region 120 can be made hydrophobic, and during the culture process, the culture liquid drops adhered to the inside of the culture region 120 will be in the shape of an approximately round sphere. The spherical droplets help provide a more three-dimensional growth space for the culture after solidification to a gel state, thereby facilitating the culture of complex structures (e.g., organoids).

In some embodiments, the interior of the culture region 120 may be modified based on the hydrophobic material of the substrate 110, and the interior of the culture region 120 after the modification has hydrophilic property. In some embodiments, the modification treatment may include a combination of one or more of plasma treatment, radiation treatment, corona treatment, and the like. The modification treatment makes the inside of the culture region 120 hydrophilic and the outside of the culture region 120 hydrophobic, enabling the liquid droplets to be more easily adhered inside the culture region 120. In some embodiments, the substrate 110 (e.g., inside the culture region 120) may be first treated for surface roughness and then modified; or the substrate 110 may be modified first and then subjected to surface roughness treatment; alternatively, the substrate 110 may be simultaneously subjected to the modification treatment and the surface roughness treatment. In some embodiments, the inside of the culture region may be processed (e.g., etched) to make the surface of the inside of the culture region have a regular structure in a submicron to nanometer scale, and then the inside of the culture region may be modified; or the interior of the culture region may be modified and then processed (e.g., etched) to make the interior surface of the culture region have a regular structure in the submicron to nanometer level.

In some embodiments, the substrate surface may be modified with hydrophilic species or groups. The hydrophilic species or group (hydrophilic group) may include, but is not limited to, a combination of one or more of the following: collagen, fibronectin, laminin, polylysine, gelatin, hyaluronic acid, chitosan, RGD polypeptide, DNA, lysine, metallic gold, hydroxyl, carboxyl, carbonyl, amino, sulfydryl, sulfonic group, phosphate group, quaternary ammonium group, ether bond, carboxylic ester, amide group or block polyether, etc. In some embodiments, hydrophilic species or groups (e.g., materials containing hydrophilic groups as described above) can be coated on the substrate surface. In some embodiments, a substance bearing hydrophilic groups may be used to react with the substrate surface to chemically modify the substrate surface. In some embodiments, the substrate material itself may be a hydrophilic material or a hydrophobic material. The substrate surface can be conveniently rendered hydrophilic by hydrophilic modification of the substrate surface. By using a substrate having a hydrophilic surface, both inside the culture region 120 and outside the culture region 120 can be made hydrophilic, and the liquid droplets adhering inside the culture region 120 will take a relatively flat shape during the culture. The flat droplets, after solidifying to a gel state, contribute to better penetration of the external culture medium into the interior. In some embodiments, the substrate 110 (e.g., inside the culture region 120) may be first surface roughened and then modified with hydrophilic substances or groups. In some embodiments, the interior of the culture region may be processed (e.g., etched) to make the surface of the interior of the culture region have a regular structure in the submicron to nanometer scale, and then the surface of the substrate 110 may be modified hydrophilically.

In some embodiments, the substrate surface may be modified with hydrophobic substances or groups (hydrophobic groups). The hydrophobic substance or group may include, but is not limited to, combinations of one or more of the following: albumin, a hydrocarbon group having a double bond, a polyoxypropylene group, a long-chain perfluoroalkyl group, a polysiloxane group, or a hydrocarbon group having an aryl group, an ester group, an ether group, an amine group, or an amide group. In some embodiments, a hydrophobic substance or group (e.g., a material containing a hydrophobic group as described above) may be coated on the substrate surface. In some embodiments, a substance with hydrophobic groups may be used to react with the substrate surface to chemically modify the substrate surface. In some embodiments, the substrate material itself may be a hydrophilic material or a hydrophobic material. The substrate surface can be made hydrophobic conveniently by hydrophobic modification of the substrate surface. By using a substrate having a hydrophobic surface, both the inside of the culture region 120 and the outside of the culture region 120 can be made hydrophobic, and during the culture, the liquid droplets adhered to the inside of the culture region 120 will be in the shape of an approximately round sphere. The spherical droplets help provide a more three-dimensional growth space for the culture after solidification to a gel state, thereby facilitating the cultivation of complex structures. In some embodiments, the substrate 110 (e.g., inside the culture region 120) may be first surface roughened and then modified with hydrophobic substances or groups. In some embodiments, the interior of the culture region may be processed (e.g., etched) to make the surface of the interior of the culture region have a regular structure in the submicron to nanometer scale, and then the surface of the substrate 110 may be hydrophobically modified.

In some embodiments, the interior of the culture region 120 can be modified with hydrophilic substances or groups. When the base material is a hydrophilic material, the hydrophilicity inside the culture region 120 can be made stronger than the hydrophilicity outside the culture region 120 by hydrophilically modifying the inside of the culture region, thereby making the adhesion affinity to the liquid droplets inside the culture region 120 stronger. When the base material is a hydrophobic material, the inside of the culture region 120 can be made hydrophilic by hydrophilic modification of the inside of the culture region, while the outside of the culture region 120 is made hydrophobic, so that liquid droplets are more easily adhered to the inside of the culture region 120. In some embodiments, the exterior of the culture region 120 can be modified with hydrophobic substances or groups. By performing hydrophilic modification inside the culture region and hydrophobic modification outside the culture region, the hydrophilicity and hydrophobicity of the substrate surface can be easily changed, while enabling the liquid droplets to be more easily adhered inside the culture region 120.

In some embodiments, the interior of the culture region can be more hydrophilic than the exterior of the culture region. In some embodiments, the hydrophilicity inside the culture region is greater than outside the culture region can be achieved in a variety of ways. For example, the inside and/or outside of the culture region may be treated by modification (such as plasma treatment, radiation treatment, and/or corona treatment) so that the inside of the culture region is more hydrophilic than the outside of the culture region. For another example, the interior of the culture region may be hydrophilically modified (e.g., with a hydrophilic substance or group) such that the interior of the culture region is more hydrophilic than the exterior of the culture region. For example, the inside of the culture region may be modified with a hydrophilic substance and then modified with a hydrophilic substance (e.g., modified with a hydrophilic substance or a group) so as to make it more hydrophilic than the outside of the culture region.

In some embodiments, there may be no height difference inside the culture region 120 and outside the culture region 120. In some embodiments, the interior of the culture region 120 can be higher than the exterior of the culture region 120. In some embodiments, the interior of the culture region 120 can be lower than the exterior of the culture region 120. For example, the interior of the culture region 120 can be lower by a certain height (e.g., 1um, 2um, 10um, etc.) than the exterior of the culture region 120. By setting the inside of the culture region 120 lower than the outside of the culture region 120, the culture can be made to adhere better in the culture region 120.

It should be noted that the above description of the culture device is for illustrative purposes only and is not intended to limit the present application. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the principles of the culture device in the examples described herein. However, such modifications and variations are intended to be within the scope of the present application. For example, the substrate surface may be subjected to any combination of one or more of roughness treatment, processing to form a regular structure having a submicron to nanometer scale, modification treatment, hydrophilic modification, hydrophobic modification, and the like.

The beneficial effects that may be brought by the embodiments of the present application include, but are not limited to: (1) the culture can be inoculated quickly and in batches, and the culture efficiency is effectively improved; (2) the quality of cell culture products can be improved; (3) different culture liquid drop shapes can be formed to be suitable for different culture requirements; (4) the culture cost can be reduced. It is to be noted that different embodiments may produce different advantages, and in different embodiments, any one or combination of the above advantages may be produced, or any other advantages may be obtained.

The present invention is not intended to be limited to the particular embodiments shown and described, but is to be accorded the widest scope consistent with the principles and novel features herein disclosed.

Claims

1. A culture device comprising a substrate, wherein,

the substrate surface comprises at least two culture areas for adhering cultures;

the culture region interior surface has a submicron to nanometer scale structured structure such that the culture region interior has a greater adhesion affinity for the culture than the culture region exterior.

2. The culture apparatus of claim 1, wherein the structured structure at the submicron-to-nanometer level comprises a plurality of micro-nano monomers arranged in an array, the micro-nano monomers comprising micro-nano columns, micro-nano tubes, micro-nano cones, and/or micro-nano walls.

3. The culture device of claim 2, wherein the micro-nano monomer has a diameter or maximum width of less than 1 μm.

4. The culture device of claim 2, wherein the head of the micro-nano monomer is mushroom-shaped.

5. The culture device of claim 1, wherein the interior of the culture region is etched such that the interior surface of the culture region has a regular structure on the submicron to nanometer scale.

6. Culture device according to claim 5, wherein the etching treatment comprises soft lithography, laser lithography, plasma lithography, electron beam lithography and/or chemical lithography.

7. The culture device of claim 1, wherein the substrate is a hydrophilic material.

8. The culture device according to claim 7, wherein the inside of the culture region is subjected to a modification treatment, the inside of the culture region after the modification treatment is more hydrophilic than the outside of the culture region, and the modification treatment comprises a plasma treatment, a radiation irradiation treatment, and/or a corona treatment.

9. The culture device of claim 1, wherein the substrate is made of a hydrophobic material.

10. The culture device according to claim 9, wherein the inside of the culture region is subjected to a modification treatment, the modified inside of the culture region has hydrophilicity, and the modification treatment comprises plasma treatment, radiation treatment and/or corona treatment.

11. The culture device of claim 1, wherein the substrate material comprises at least one of: glass, quartz, silicon, mica, PS, PMMA, PSU, PC, PP, PE, PETG, LDPE, HDPE, PET, PVDF, PTFE, PEG, PEO, PPG, PLA, PGA, PLGA, PDMS, PVA, COC, COP, PMP, styrene/butadiene copolymer, styrene/acrylonitrile copolymer, cellulose acetate, cellulose nitrate, hydroxyethyl methacrylate, polyethersulfone, diallyl diethylene glycol polymer or nylon 66.

12. The culture device of claim 1, wherein the substrate surface is modified with a hydrophilic substance or group comprising at least one of: collagen, fibronectin, laminin, polylysine, gelatin, hyaluronic acid, chitosan, RGD polypeptide, DNA, lysine, metallic gold, hydroxyl, carboxyl, carbonyl, amino, sulfydryl, sulfonic group, phosphate group, quaternary ammonium group, ether bond, carboxylic ester, amide group or block polyether.

13. The culture device of claim 1, wherein the substrate surface is modified with a hydrophobic substance or group comprising at least one of: albumin, a hydrocarbon group containing a double bond, a polyoxypropylene group, a long-chain perfluoroalkyl group, a polysiloxane group, or a hydrocarbon group containing an aryl, ester, ether, amine, or amide group.

14. The culture device according to claim 1, wherein the inside of the culture region is modified with a hydrophilic substance or group.

15. The culture device of claim 14, wherein the exterior of the culture region is modified with a hydrophobic substance or group.

16. The culture device of claim 1, wherein the interior of the culture region is more hydrophilic than the exterior of the culture region.