Cell culture device, manufacturing method therefor and culture method thereof

Abstract

Provided are a cell culture device, a manufacturing method therefor and a culture method thereof. The cell culture device comprises a composite culture layer, the composite culture layer comprising: a culture through-hole layer, at least one vertically penetrating culture through hole being distributed in the culture through-hole layer; and an air-permeable microporous layer, the air-permeable microporous layer comprising at least one air-permeable portion, the surface of each air-permeable portion being provided with at least one micropore, and the material of the air-permeable portion being capable of allowing oxygen to pass through, wherein the air-permeable portions are in one-to-one correspondence with the culture through holes, and each air-permeable portion covers a port at one end of a corresponding culture through hole, so as to define, together with the corresponding culture through hole, a culture chamber, the micropores being located inside the culture chambers.

Description

Cell culture apparatus and its preparation and culture method

[0001] Related applications

[0002] This application claims priority to Chinese patent application filed on July 16, 2024, with application number 202410954219.9, entitled "Cell Culture Apparatus and Preparation and Culture Method Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biotechnology, and more specifically, to a cell culture device and its preparation and culture methods. Background Technology

[0004] Organoids are in vitro culture systems that combine small molecule regulation of stem cell development with 3D culture technology. They can better simulate some key morphological and genetic characteristics of corresponding organs. They have not only been successful in constructing various types of tissues and organs, but have also shown great application potential in organ development, precision medicine, regenerative medicine, drug screening, gene editing, disease modeling and other fields.

[0005] Traditional methods mostly use gel embedding to construct organoids, which takes 2 to 4 weeks to culture and the success rate is difficult to guarantee. Furthermore, the cells in the gel assemble randomly, making it impossible to obtain organoids with uniform size and cell composition.

[0006] Improving the efficiency and stability of organoid construction is a key challenge in organoid construction. Summary of the Invention

[0007] The primary objective of this application is to provide a cell culture apparatus, including a composite culture layer, the composite culture layer comprising:

[0008] A culture perforated layer is formed, wherein at least one culture perforated layer is distributed throughout the layer.

[0009] The breathable microporous layer includes at least one breathable part, and each breathable part has at least one micropore on its surface, and the material of the breathable part allows oxygen to pass through.

[0010] The ventilation section and the culture well correspond one-to-one. Each ventilation section covers one end of the corresponding culture well, so as to define a culture chamber for containing cells together with the corresponding culture well, and the micropores are located inside the culture chamber.

[0011] In some embodiments, the cross-section of the micropore is selected from at least one of circles and polygons.

[0012] In some embodiments, the longitudinal section of the micropore is selected from at least one of cylindrical, conical, and elliptical shapes.

[0013] In some implementations, when the cross-section of the micropore is a regular polygon, the diameter of the inscribed circle of the regular polygon is 40 μm to 1000 μm.

[0014] In some embodiments, the depth of the micropores is less than the thickness of the microporous permeable pore layer; optionally, the depth of the micropores is 40 μm to 1000 μm.

[0015] In some embodiments, the wall thickness between the micropores is 1 / 3 to 2 / 3 of the micropore depth.

[0016] In some embodiments, the material of the breathable microporous layer or breathable part is an elastomer.

[0017] Optionally, the material of the breathable microporous layer or breathable part includes one or both of polydimethylsiloxane and 4-methylpentene.

[0018] Optionally, the thickness of the breathable microporous layer is 0.5 mm to 5 mm.

[0019] Optionally, the oxygen permeability of the breathable microporous layer is 3.74 × 10⁻⁶. -13 mol / (cm 2 .s)~3.74×10 -12 mol / (cm 2 .s).

[0020] In some embodiments, the cell culture apparatus further includes:

[0021] A cover plate layer, which is stacked with the culture well layer to cover the end of each culture well away from the air-permeable micropore layer; and / or

[0022] The breathable perforated layer is stacked with the breathable microporous layer, and the breathable perforated layer has multiple breathable perforations corresponding to multiple breathable parts.

[0023] Optionally, the thickness of the cultured porous layer is 10 mm to 14 mm;

[0024] Optionally, the thickness of the breathable perforated layer is 0.5 mm to 5 mm.

[0025] In some embodiments, the cell culture apparatus further includes a connector for securing at least two of the culture perforation layer, the air-permeable micropore layer, and the air-permeable pore layer together.

[0026] Optionally, the fixed connection is selected from at least one of welding, riveting, bonding, threaded connection, pin connection, elastic deformation connection, locking connection and plug connection.

[0027] Optionally, the material used to cultivate the porous layer or the breathable porous layer is selected from at least one of stainless steel, titanium, zirconium oxide, alumina, polyethylene, polypropylene, polymethyl methacrylate, polycarbonate, polyurethane, polyester sulfone, polyetherketone, polysulfide, polyphenylene sulfide, polyimide, polytetrafluoroethylene, silicon, zirconium oxide / alumina composite, carbon fiber composite, glass fiber composite, zirconium oxide / titanium oxide composite, aluminum oxide, zirconium oxide / alumina / titanium oxide composite, silicon carbide, boron nitride, silicon nitride, silicon carbide, silicon oxide, cerium oxide, barium zirconate, zirconium oxide / aluminum nitride composite, alumina / aluminum nitride composite, aluminum gallium selenide, niobium oxide, titanium oxide, zinc oxide, magnesium oxide, calcium phosphate, zirconium oxide / niobium oxide composite, and zirconium oxide / magnesium oxide composite.

[0028] A second objective of this application is to provide a method for preparing the above-mentioned cell culture device, comprising:

[0029] A culture well layer is provided, wherein at least one culture well is distributed in the culture well layer that runs vertically through the culture well.

[0030] A breathable microporous layer is provided, the breathable microporous layer including a breathable part made of an oxygen-permeable material, and at least one micropore is formed on the surface of the breathable part;

[0031] A composite culture layer is formed by combining a breathable microporous layer and a culture through-hole layer, such that the breathable part covers the port of one end of the corresponding culture through-hole, and together with the corresponding culture through-hole, defines the culture chamber, and multiple micropores are located inside the corresponding culture chamber.

[0032] In some embodiments, the preparation steps of the breathable portion include:

[0033] Customize a target mold with a specific pattern according to the morphology of multiple micropores;

[0034] A mixture of oxygen-permeable material and curing agent is poured onto one side of the target mold that has a specific pattern.

[0035] The mixture on the surface of the target polydimethylsiloxane is cured and peeled off to form a breathable microporous layer with at least one micropore, which covers the portion of the culture pore as a breathable part.

[0036] Optionally, the preparation steps of the mixture of oxygen-permeable material and curing agent include:

[0037] The thickness of the permeable microporous layer is determined according to the target oxygen permeability.

[0038] The amount of oxygen-permeable material and curing agent is determined based on the thickness of the air-permeable microporous layer, wherein the mass ratio of oxygen-permeable material to curing agent is (5~20):1.

[0039] Optionally, the oxygen-permeable material is selected from one or two of polydimethylsiloxane-4-methylpentene.

[0040] Optionally, the method further includes: providing a breathable perforated layer, wherein the breathable perforated layer and the breathable microporous layer are stacked together, and the breathable perforated layer is provided with breathable perforations corresponding to the breathable portion; and / or

[0041] A cover plate layer is provided, which is stacked with the culture perforation layer to cover the port of the culture chamber away from the vent; and / or

[0042] At least two of the culture through-hole layer, the air-permeable micropore layer, the air-permeable through-hole layer and the cover plate layer are fixedly connected using connectors;

[0043] Optionally, the fixed connection is selected from at least one of welding, riveting, bonding, threaded connection, pin connection, elastic deformation connection, locking connection and plug connection.

[0044] The third objective of this application is to provide a cell culture method, which uses the above-mentioned cell culture device to culture target cells.

[0045] In some implementations, the method satisfies at least one of the following conditions (1) to (4):

[0046] (1) The cell culture time is 1 to 2 days;

[0047] (2) Target cells include stem cells;

[0048] (3) The target cells are selected from any one of iPS induced stem cells, healthy tissue stem cells, organoid passaged digestive cells and tumor tissue stem cells;

[0049] (4) The target cells are cultured to obtain organoids or multicellular clusters. Optionally, the organoids include any one of intestinal organoids, liver organoids and pancreatic organoids.

[0050] In some implementations, the cell culture step includes:

[0051] The target cells and biogel reagent are mixed to prepare a cell suspension, and the cell suspension is injected into the culture chamber of a cell culture plate.

[0052] The cell suspension in the culture chamber was centrifuged, and the centrifuged cell suspension was then used for cell culture.

[0053] Optionally, the biogel reagent includes at least one of matrix gel, methacrylamide gelatin, and methacrylamide hyaluronic acid gelatin.

[0054] Optionally, the mass fraction of the matrix gel in the biogel reagent is 10% to 50%.

[0055] Optionally, the centrifugation speed is 300 rpm to 1000 rpm, and the centrifugation time is 30 s to 120 s.

[0056] Optionally, the cell density of the cell suspension is 1 x 10⁻⁶. 6 / mL~1x10 8 / mL.

[0057] Optionally, the size of the organoid shown is 100 μm to 400 μm. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in the specific embodiments or related technologies of this application, the drawings used in the description of the specific embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0059] Figure 1 is a cross-sectional schematic diagram of different micropores provided in an embodiment of this application.

[0060] Figure 2 is a schematic diagram of the longitudinal section of different micropores provided in an embodiment of this application.

[0061] Figures 3A to 3B are schematic diagrams of the structures of different cell culture devices provided in Embodiment 1 of this application. Figure 3A shows a 6-well cell culture device, Figure 3B shows a 24-well cell culture device, Figure 3C shows a 56-well cell culture device, and Figure 3D shows a 160-well cell culture device.

[0062] Figure 4 is a schematic diagram of the structure of micropore arrays of different sizes provided in Embodiment 1 of this application. In Figure 4, A represents a micropore array with a diameter of 126 μm, B represents a micropore array with a diameter of 200 μm, and C represents a micropore array with a diameter of 326 μm.

[0063] Figure 5 is a comparative schematic diagram of organoids cultured in different oxygen environment cell culture devices provided in Embodiment 2 of this application.

[0064] Figure 6 shows the statistical results of the size change trend of organoids cultured in different oxygen environment cell culture devices provided in Embodiment 2 of this application.

[0065] Figures 7A to 7C are light microscope images of the cell culture devices with different oxygen environments provided in Embodiment 2 of this application on the fifth day.

[0066] Figure 8 shows the statistical results of organoid size on the fifth day of cell culture using different oxygen environment cell culture devices provided in Embodiment 2 of this application.

[0067] Figure 9 is a technical roadmap for constructing multi-cell clusters under different in vitro environments provided in Embodiment 3 of this application.

[0068] Figures 10A and 10B show the detection results of intestinal cell barrier function expression provided in Example 3 of this application. Detailed Implementation

[0069] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0071] When using the terms “comprising,” “having,” and “including” as described herein, the intention is to cover non-exclusive inclusion, where another component may be added unless explicitly qualified terms such as “only,” “consisting of,” etc. are used. Unless otherwise stated, singular terms may include plural forms and should not be construed as having a quantity of one.

[0072] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0073] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0074] A first aspect of this application provides a cell culture apparatus, including a composite culture layer, the composite culture layer comprising:

[0075] A culture perforated layer is formed, wherein at least one culture perforated layer is distributed throughout the layer.

[0076] The breathable microporous layer includes at least one breathable part, and each breathable part has at least one micropore on its surface, and the material of the breathable part allows oxygen to pass through.

[0077] The ventilation section and the culture through hole are in a one-to-one correspondence. Each ventilation section covers one end of the corresponding culture through hole, so as to define the culture chamber together with the corresponding culture through hole, and the micropores are located inside the culture chamber.

[0078] In the above-described embodiments, this application creatively incorporates a breathable microporous layer inside the culture chamber. While constructing a high-oxygen culture environment, the micropores facilitate the orderly aggregation and assembly of cells, thereby significantly increasing the size of organoids while constructing uniformly sized organoids. Furthermore, it improves the construction efficiency of organoids, requiring only 1-2 days to complete the construction, which is a significant improvement compared to the 2-4 weeks required by traditional methods. Moreover, the breathable microporous layer can regulate the oxygen environment of cells to meet the oxygen consumption requirements of different types of organs.

[0079] In some implementations, to create multiple oxygen-permeable micropores at the bottom of the culture chamber, the cell culture device includes a permeable micropore layer. The material of the permeable micropore layer allows oxygen to pass through. The permeable micropore layer is stacked with a culture pore layer, covering the bottom of each culture pore to define the corresponding culture chamber. The portion of the permeable micropore layer covering the culture pore constitutes a permeable section. Micropores on the surface of the permeable section are located inside the corresponding culture chamber. During cell culture in the culture chamber, cells can be deposited in the micropores on the surface of the permeable section. The micropores allow for the orderly aggregation and assembly of cells, thereby constructing organoids of uniform size, significantly increasing organoid size and improving organoid construction efficiency.

[0080] In some implementations, the number of wells in the culture vias is 6, 24, 56, 88, 160, or 384.

[0081] In some implementation schemes, the culture vias are square holes with a side length of 3.5 mm to 8.5 mm.

[0082] In some implementation schemes, the thickness of the cultured porous layer is 10 mm to 14 mm.

[0083] In some specific implementations, the cultured porous layer has a length of 128 mm, a width of 84.5 mm, and a height of 12 mm.

[0084] In some embodiments, the material used to cultivate the through-pore layer is selected from at least one of stainless steel, titanium, zirconium oxide, alumina, polyethylene, polypropylene, polymethyl methacrylate, polycarbonate, polyurethane, polyester sulfone, polyetherketone, polysulfide, polyphenylene sulfide, polyimide, polytetrafluoroethylene, silicon, zirconium oxide / alumina composite, carbon fiber composite, glass fiber composite, zirconium oxide / titanium oxide composite, aluminum oxide, zirconium oxide / alumina / titanium oxide composite, silicon carbide, boron nitride, silicon nitride, silicon carbide, silicon oxide, cerium oxide, barium zirconate, zirconium oxide / aluminum nitride composite, alumina / aluminum nitride composite, aluminum gallium selenide, niobium oxide, titanium oxide, zinc oxide, magnesium oxide, calcium phosphate, zirconium oxide / niobium oxide composite, and zirconium oxide / magnesium oxide composite.

[0085] In some implementation schemes, the thickness of the breathable microporous layer is 0.5 mm to 5 mm. By adjusting the thickness of the breathable microporous layer, the oxygen permeability of the layer can be controlled, thereby regulating the oxygen environment of the cultured cells and providing a more favorable oxygen environment for cell growth. For example, the thickness of the breathable microporous layer is 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, and 5 mm.

[0086] In some specific implementations, the breathable microporous layer has a length of 128 mm and a width of 84.5 mm.

[0087] In some specific embodiments, the material of the breathable microporous layer or breathable portion is selected from at least one of polydimethylsiloxane, polymethyl methacrylate, amorphous polycarbonate, and polyethylene. These materials possess certain oxygen permeability and formability, allowing them to be assembled with the culture porous layer to provide a high-oxygen environment for cells, while simultaneously creating micropores to accommodate cells and facilitate orderly aggregation and assembly. For example, polydimethylsiloxane (PDMS), the most commonly used material in soft lithography, possesses soft and flexible properties, transparency to ultraviolet and visible light, and oxygen permeability, making it suitable for preparing the breathable portion or breathable microporous layer of this application for cell culture.

[0088] Specifically, PDMS and curing agent are mixed in proportion, and the curing temperature is 20℃~150℃, for example, around 85℃, to quickly cure and obtain an elastic rubber body. This body is used as a breathable microporous layer or breathable part and is assembled with a culture through-hole layer to cover one end of the culture through-hole, thus defining the corresponding culture chamber for cell culture.

[0089] Generally speaking, the oxygen permeability of a standard 1mm thick PDMS film without special treatment is approximately 1.87 × 10⁻⁶. -12 mol / (cm 2.s), and the thickness is inversely proportional to the oxygen permeability.

[0090] In some implementations, when the thickness of the breathable microporous layer is 0.5 mm to 5 mm, the oxygen permeability can be 3.74 × 10⁻⁶. -13 mol / (cm 2 .s)~3.74×10 -12 mol / (cm 2 Gas permeability (GTR) refers to the total amount of gas that permeates through two parallel planes of a plastic film per unit time and per unit area under test conditions. The SI unit for GTR is mol / (cm²). 2 .s).

[0091] In some embodiments, the cross-section of the micropore is selected from at least one of circles and polygons. For example, the cross-section of the micropore is selected from at least one of the various shapes shown in FIG1.

[0092] In some embodiments, the longitudinal section of the micropore is selected from at least one of cylindrical, conical and elliptical shapes. For example, the longitudinal section of the micropore is selected from at least one of the various shapes shown in Figure 2.

[0093] In some implementations, when the cross-section of the micropore is a regular polygon, the diameter of the inscribed circle of the regular polygon is 40μm to 1000μm. In some specific implementations, the diameter of the inscribed circle of the regular polygonal micropore can be 46μm, 55μm, 76μm, 126μm, 200μm, 326μm, or 500μm.

[0094] In some implementations, the depth of the micropores is less than the thickness of the microporous permeable pore layer to accommodate cells; in some specific implementations, the depth of the micropores is 40 μm to 1000 μm.

[0095] In some implementations, the wall thickness between the micropores is 1 / 3 to 2 / 3 of the micropore depth.

[0096] In some specific implementations, when the cross-section of the micropore is a regular hexagon, the longitudinal section is cylindrical, the inscribed circle diameter of each regular hexagonal micropore is 126μm, the depth is 133μm, and the wall thickness between micropores is 50μm.

[0097] In some specific implementation schemes, the number of micropores is N. It is understood that N is a natural number.

[0098] In some implementations, the cell culture plate further includes a cover plate layer, and the cover plate layer and the culture well layer are stacked to cover the end of the culture wells away from the ventilated part.

[0099] In some specific implementation schemes, the outer contour of the cover plate layer is 130mm long, 86.5mm wide, and 6mm high.

[0100] In some implementations, the cell culture plate also includes:

[0101] The air-permeable pore layer is stacked with the air-permeable micropore layer. The air-permeable pore layer is provided with air-permeable pores corresponding to the air-permeable parts on the air-permeable micropore layer, which is used to ensure the air permeability of the culture chamber.

[0102] In some specific implementations, the outer contour dimensions of the breathable perforated layer are 128 mm in length, 84.5 mm in width, and 0.5 mm to 5 mm in thickness.

[0103] In some specific implementation schemes, the material of the breathable perforated layer is a rigid material.

[0104] In some specific embodiments, the material of the breathable perforated layer is selected from at least one of stainless steel, titanium, zirconium oxide, alumina, polyethylene, polypropylene, polymethyl methacrylate, polycarbonate, polyurethane, polyester sulfone, polyetherketone, polysulfide, polyphenylene sulfide, polyimide, polytetrafluoroethylene, silicon, zirconium oxide / alumina composite material, carbon fiber composite material, glass fiber composite material, zirconium oxide / titanium oxide composite material, aluminum oxide, zirconium oxide / alumina / titanium oxide composite material, silicon carbide, boron nitride, silicon nitride, silicon carbide, silicon oxide, cerium oxide, barium zirconate, zirconium oxide / aluminum nitride composite material, alumina / aluminum nitride composite material, aluminum gallium selenide, niobium oxide, titanium oxide, zinc oxide, magnesium oxide, calcium phosphate, zirconium oxide / niobium oxide composite material, and zirconium oxide / magnesium oxide composite material.

[0105] In some implementations, the cell culture plate also includes a connector for securing at least two of the culture well layer, the aeration micropore layer, the aeration well layer, and the cover plate layer.

[0106] In some specific implementations, the fixing connection includes detachable fixing connections and non-detachable fixing connections. It is understood that the fixing connection methods for the culture perforated layer, the aeration microporous layer, the aeration perforated layer, and the cover plate layer in this application are not limited, as long as the fixing connection can be achieved and the cell culture effect of this application is not affected.

[0107] Specifically, non-removable fixed connections include at least one of welding, riveting, and bonding; detachable fixed connections include at least one of threaded connections, pin connections, elastic deformation connections, locking connections, and plug connections.

[0108] In some specific implementation schemes, when the fixed connection is a threaded connection, in order to realize the threaded connection between the culture through-hole layer, the air-permeable micropore layer and the air-permeable through-hole layer, a number of first connection holes are also provided on the culture through-hole layer, and the number of first connection holes are dispersedly arranged on the culture through-hole layer;

[0109] The breathable microporous layer is provided with second connecting holes corresponding to each of the first connecting holes;

[0110] The breathable perforated layer is provided with a third connecting hole corresponding to each second connecting hole;

[0111] Bolts are used to pass through the first, second, and third connecting holes, and nuts are used to fix the culture through-hole layer, the air-permeable micropore layer, and the air-permeable through-hole layer together.

[0112] In some specific implementations, the diameter of at least one of the first connecting hole, the second connecting hole, or the third connecting hole is 4 mm.

[0113] In some specific implementations, the number of at least one of the first connecting hole, the second connecting hole, or the third connecting hole is 12.

[0114] A second aspect of this application provides a method for preparing a cell culture plate as described above, comprising:

[0115] A culture well layer is provided, which has multiple culture wells that run vertically through it;

[0116] A breathable microporous layer is provided, the breathable microporous layer including a breathable part made of an oxygen-permeable material, and at least one micropore is formed on the surface of the breathable part;

[0117] A composite culture layer is formed by combining a culture through-hole layer and a breathable micropore layer. The breathable part covers one end of the corresponding culture through-hole, so as to define the corresponding culture chamber together with the corresponding culture through-hole, and multiple micropores are located inside the corresponding culture chamber.

[0118] It is understandable that when combined with the culture perforated layer, each air-permeable part can be connected to each culture perforated layer separately at intervals, thereby combining with the culture perforated layer, or it can be combined with the culture perforated layer as a whole.

[0119] In some implementations, the preparation steps of the breathable part include:

[0120] Customize a target mold with a specific pattern according to the morphology of multiple micropores;

[0121] A mixture of oxygen-permeable material and curing agent is poured onto one side of the target mold that has a specific pattern.

[0122] The mixture on the surface of the target mold is cured and peeled off to form a breathable microporous layer with multiple micropores on the surface. The breathable microporous layer serves as a breathable part for the part covering the culture through holes.

[0123] Specifically, after quantitatively mixing oxygen-permeable materials and curing agents, the quantitative mixture is poured onto the target mold, dried to form an elastomer, and the elastomer is peeled off for cutting, cleaning and drying, PLASMA surface treatment, surface hydrophobic treatment liquid immersion and secondary cleaning to obtain an air-permeable microporous layer with multiple micropores on the surface.

[0124] In some specific implementation schemes, the drying temperature in the above steps is 70℃~80℃, and the drying time is greater than 12h.

[0125] In some specific implementation schemes, anhydrous ethanol is used for cleaning in the above steps.

[0126] In some specific implementations, the PLASMA surface treatment time in the above steps is 10s to 30s. PLASMA is an oxygen plasma surface treatment that hydrophilizes the surface of the breathable microporous layer, facilitating subsequent hydrophobic treatment.

[0127] In some specific implementation schemes, the soaking time in the above steps is 2 hours to 12 hours. For example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, and 12 hours.

[0128] In some specific implementations, a second washing with PBS buffer is used in the above steps;

[0129] In some specific implementations, the oxygen-permeable material is PDMS or PMP. Polydimethylsiloxane (PDMS) is an organosilicon material with many remarkable properties, such as good thermal stability, biocompatibility, corrosion resistance, flexibility, low cost, ease of use, chemical inertness, proliferative properties, and air permeability.

[0130] PMP (poly-4-methyl-1-pentene) is a highly crystalline polyolefin material with high thermal stability, chemical resistance, and excellent gas permeability.

[0131] When selecting PMP material, it should be noted that PMP is a thermoplastic material. It is solid at high temperatures and liquid at room temperature. Therefore, when preparing a breathable microporous layer using PMP material, it is necessary to heat the PMP material above its melting point, pour it onto the mold, and then cool and solidify it.

[0132] When selecting PDMS material, in some implementation schemes, in order to control the oxygen permeability of the venting section, the preparation steps of the mixture of oxygen-permeable material and curing agent include:

[0133] The thickness of the permeable microporous layer is determined according to the target oxygen permeability.

[0134] The amount of oxygen-permeable material and curing agent is determined based on the thickness of the air-permeable microporous layer, wherein the mass ratio of oxygen-permeable material to curing agent is (5-20):1. For example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1.

[0135] In some implementations, the method further includes: providing a breathable perforated layer, wherein the breathable perforated layer and the breathable microporous layer are stacked together, and the breathable perforated layer is provided with breathable perforations corresponding to the breathable portion.

[0136] In some implementations, the method further includes: providing a cover layer, which is stacked with the culture well layer to cover the end of the culture well away from the vent.

[0137] In some implementations, the method further includes using connectors to fix at least two of the culture perforation layer, the air permeable micropore layer, the air permeable pore layer, and the cover plate layer.

[0138] Specifically, the fixed connection method of at least two of the cultivation through-hole layer, air-permeable microporous layer, air-permeable through-hole layer and cover plate layer has been described in detail above and will not be repeated here.

[0139] In some specific implementation schemes, in order to achieve threaded connections between the orifice plates, the method further includes:

[0140] A plurality of first connecting holes are dispersedly arranged on the culture through-hole layer, a second connecting hole is arranged on the air-permeable microporous layer at a position corresponding to the first connecting hole, and a third connecting hole is arranged on the air-permeable through-hole layer at a position corresponding to the second connecting hole, so as to insert bolts to fix at least two of the culture through-hole layer, the air-permeable microporous layer, the air-permeable through-hole layer and the cover plate layer.

[0141] A third aspect of this application provides a cell culture method, comprising culturing target cells using the aforementioned cell culture apparatus, orderly guiding cell aggregation and assembly, and increasing cell growth rate, thereby significantly increasing the size of organoids while improving the uniformity of organoid size.

[0142] In some implementation schemes, the target cells include stem cells.

[0143] In some implementation schemes, the target cells are selected from any one of iPS-induced stem cells, healthy tissue stem cells, organoid passaged digested cells, and tumor tissue stem cells. Accordingly, the target cells are injected into the culture chamber of a cell culture device for cell culture to obtain organoids or multicellular clusters.

[0144] In some specific implementations, organoids include any one of intestinal organoids, liver organoids, and pancreatic organoids.

[0145] Organoids are three-dimensional cellular structures cultured in vitro that can mimic certain functions and structural features of real organs. Organoids are typically induced to differentiate from stem cells or pluripotent stem cells (such as induced pluripotent stem cells, iPSCs) under specific culture conditions. These cells can self-assemble into three-dimensional structures with specific tissue types under specific conditions and can mimic the functions of the corresponding organs to a certain extent.

[0146] In some implementations, the organoids are 100 μm to 400 μm in size. For example, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, and 400 μm.

[0147] The cell culture device of this application can further significantly increase the size of organoids by improving the uniformity of organoid size.

[0148] In some implementation plans, the cell culture time is 1 to 2 days.

[0149] In some implementation schemes, the cell culture steps include:

[0150] The target cells and biogel reagent are mixed to prepare a target cell suspension, and the target cell suspension is injected into the culture chamber of the cell culture device;

[0151] The target cell suspension in the culture chamber is centrifuged, and the centrifuged target cell suspension is then used for cell culture.

[0152] In some specific implementations, the biogel reagent includes at least one of matrix gelatin, methacrylamide gelatin, and methacrylamide hyaluronic acid gelatin. Matrix gelatin, also known as the extracellular matrix (ECM), is a complex three-dimensional structure existing in the intercellular space surrounding cells, mainly composed of various molecules such as proteins, polysaccharides, and minerals. Protein components include collagen, fibronectin, and calcium lactate phosphate, while polysaccharide components include alginate and hyaluronic acid. Matrix gelatin plays multiple important roles in vivo, including maintaining tissue structure, cell attachment, signal transduction, and cell migration.

[0153] In some specific implementations, the mass fraction of the matrix gel in the biogel reagent is 10% to 50%.

[0154] In some specific implementations, the centrifugation speed is 300 rpm to 1000 rpm, and the centrifugation time is 30 s to 120 s. For example, the centrifugation speed is 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, or 1000 rpm, and the centrifugation time is 30 s, 40 s, 50 s, 60 s, 70 s, 80 s, 90 s, 100 s, 110 s, or 120 s.

[0155] The centrifugation step is used to precipitate cells at the bottom of the culture chamber, which in turn allows the cells to be deposited in multiple micropores on the surface of the vent. This enables the directional aggregation and assembly of cells using the multiple micropores on the surface of the vent, thus constructing cell clusters with specific aggregation structures.

[0156] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0157] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0158] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0159] Example 1

[0160] (1) Structural features of cell culture device

[0161] The cell culture apparatus includes a cover plate layer, a culture well layer, a permeable microporous layer, a permeable air well layer, and fixing screws. The top cover has an outer contour of 130 mm in length, 86.5 mm in width, and 6 mm in height, with a wall thickness of 1 mm. The culture well layer has a length of 128 mm, a width of 84.5 mm, and a height of 12 mm. The culture wells in the culture well layer are 3.5 mm squares.

[0162] To prevent liquid leakage and improve the sealing between the culture through-hole layer and the air-permeable microporous layer, a certain space is left between every two culture through-holes for screw fixing. Threaded holes are distributed around the first plate, totaling 12, with a thread diameter of 4mm.

[0163] The permeable microporous layer is 128 mm long, 84.5 mm wide, and 1 mm thick. The part of the permeable microporous layer covering the culture wells is the permeable part, which has a micropore array. The micropores are hexagonal, with an inscribed circle diameter of 126 μm and a depth of 133 μm. The wall thickness between the micropores is 50 μm.

[0164] The outer dimensions of the ventilated perforated layer are 128mm in length, 84.5mm in width, and 2mm in thickness. The ventilated perforated layer has certain ventilation holes, the size and position of which are consistent with the dimensions and positions of the ventilation section and the threaded holes, for gas exchange and bolt connection fixation.

[0165] (2) Material selection and preparation method: The material for the culture through-hole layer is PMMA acrylic, the material for the air-permeable microporous layer is PDMS, and the material for the air-permeable through-hole layer is stainless steel. The cover plate layer, culture through-hole layer, and air-permeable through-hole layer are machined using a CNC milling machine. The air-permeable microporous layer is machined using soft photolithography.

[0166] (3) Preparation of cell culture apparatus:

[0167] Step 1: Prepare a breathable microporous layer;

[0168] The mold for the breathable microporous layer can be constructed using photolithography to customize the mold. The specific steps are as follows:

[0169] 1) Cleaning: Rinse the silicon wafer with deionized water and dry it with nitrogen gas;

[0170] 2) Pre-baking: Bake the silicon wafer at 95℃ for about 30 minutes;

[0171] 3) Spin coating: Using a spin coater to spin-coat photoresist onto a silicon wafer;

[0172] 4) Pre-baking: Select baking time and parameters according to the performance parameters of different photoresists;

[0173] 5) Exposure: Select the exposure time and light intensity according to the performance parameters of different photoresists. The shape of the customized photomask can determine the exposure pattern.

[0174] 6) Post-baking: Select baking time and parameters according to the performance parameters of different photoresists;

[0175] 7) Development: Rinse alternately with developer and anhydrous ethanol until the structure is intact;

[0176] 8) Hardening the film: Bake at 150℃ for 30 minutes;

[0177] 9) Etching: A specific pattern on a silicon wafer is etched using a reactive ion etching system to obtain a mold, including but not limited to etching with CHF3 gas. For example, to obtain a hexagonal micro-hole array, a mold with a hexagonal prism array needs to be constructed using photolithography, and the hexagonal array photoresist needs to be UV exposed on the silicon wafer. When a positive photoresist is selected, the specific pattern is a hexagonal mesh array; when a negative photoresist is selected, the specific pattern is a hexagonal array.

[0178] Mix PDMS and curing agent at a concentration of 10:1. Calculate the required thickness of the base film to be cast based on the preset oxygen permeability. Calculate the amount of PDMS and curing agent to be used. Cast the quantitative mixture of PDMS and curing agent onto the photolithographically etched silicon wafer and cure it in an oven at 70-80℃ for more than 12 hours.

[0179] Step 2: Peel off the breathable microporous layer, cut along the reserved outline, and drill holes along the reserved bolt connection positions.

[0180] Step 3: After cleaning and drying with anhydrous ethanol, treat the surface with PLASMA for 15 seconds.

[0181] Step 4: Soak the substrate in the prepared surface hydrophobic treatment solution for 3 hours.

[0182] Step 5: Place the cover plate layer, culture perforated layer, and air perforated layer in a clean bench for ultraviolet sterilization for 3 hours.

[0183] Step 6: Wash the bottom membrane with PBS at least 3 times.

[0184] Step 7: Assemble the cover plate layer, culture well layer, air-permeable micropore layer, and air-permeable pore layer, and fix them with bolts to obtain the cell culture device.

[0185] Examples of cell culture apparatuses with different numbers of wells are shown in Figures 3A-D. Figure 3A shows a 6-well cell culture apparatus, Figure 3B shows a 24-well cell culture apparatus, Figure 3C shows a 56-well cell culture apparatus, and Figure 3D shows a 160-well cell culture apparatus. In Figures 3A-D, "1" represents the cover plate layer, "2" represents the culture well layer, "3" represents the aeration microporous layer, and "4" represents the aeration through-pore layer.

[0186] Taking a 160-well cell culture device as an example, the array of micropores of different sizes on the surface of the aeration section of the cell culture device is shown in Figures 4A to 4C. In Figure 4, A represents a micropore array with a diameter of 126 μm, B represents a micropore array with a diameter of 200 μm, and C represents a micropore array with a diameter of 326 μm.

[0187] (4) Use of cell culture equipment

[0188] Taking the culture of small intestinal organoids as an example, the culture method includes the following steps:

[0189] Step 1: Prepare a 50% matrix gel using culture medium, and resuspend the dispersed small intestinal organoid cell suspension to achieve a cell density of 1 x 102. 7 / mL.

[0190] Step 2: Place a 24 μL drop of cell suspension in the center of the culture chamber of the cell culture device;

[0191] Step 3: Centrifuge at 4℃, 500 rpm, for 1 minute;

[0192] Step 4: After cross-linking in a 37℃ cell culture incubator for 30 min, add 120 μL of culture medium;

[0193] Step 5: Add 20 μL of culture medium every 1 to 2 days.

[0194] Example 2: Organoid Culture under Different Oxygen Environments

[0195] The cell culture effects of traditional gel embedding culture, hypoxic microplate culture, and high oxygen permeability microplate culture were compared. For both traditional gel embedding culture and hypoxic microplate culture, Corning 96-well cell culture plates (catalog number 3599) were used.

[0196] 1. Traditional gel embedding culture methods are as follows:

[0197] Small intestinal organoids were digested into single-cell suspensions using pancreatic enzymes;

[0198] Centrifuge and resuspend the single cells in 50% matrix gel;

[0199] Add 24 μL of cell suspension to one well of a 96-well plate, and repeat 3 times.

[0200] Centrifuge at 4℃, 500 rpm, for 1 min;

[0201] Place the 96-well plate in a 37°C cell culture incubator and crosslink for 30 min;

[0202] Supplement with 120 μL of complete organoid culture medium and change the medium daily.

[0203] 2. The low-oxygen microplate culture method is as follows:

[0204] First, a low-oxygen microporous plate with a micropore array at the bottom is prepared:

[0205] The prepared PDMS substrate with microporous array was punched with 6mm holes using a puncher to prepare a PDMS film with a diameter of 6mm and microporous array.

[0206] The PDMS film with microporous array was cleaned and dried with anhydrous ethanol and then surface-treated with PLASMA for 15 seconds.

[0207] Soak in the prepared surface hydrophobic treatment solution for 3 hours;

[0208] Wash with PBS at least 3 times;

[0209] A PDMS film with a micropore array was placed in the culture wells of a 96-well cell culture plate manufactured by Corning Incorporated (product number 3599).

[0210] Small intestinal organoids were digested into single-cell suspensions using pancreatic enzymes;

[0211] Centrifuge and resuspend the single cells in 50% matrix gel;

[0212] Add 24 μL of cell suspension to one well of a 96-well plate, and repeat 3 times.

[0213] Centrifuge at 4℃, centrifuge speed at 500 rpm, and centrifuge time at 1 min;

[0214] Place the 96-well plate in a 37°C cell culture incubator and crosslink for 30 min;

[0215] Supplement with 120 μL of complete organoid culture medium and change the medium daily.

[0216] 3. The high-oxygen microplate culture method is as follows:

[0217] Cell culture was performed using a 96-well cell culture apparatus prepared according to Example 1, and the steps included:

[0218] Prepare a 50% matrix gel using culture medium, and resuspend the dispersed small intestinal organoid cell suspension to achieve a cell density of 1 x 10⁻⁶ cells / mL. 7 / mL;

[0219] Place a 24 μL drop of cell suspension in the center of the culture chamber of the cell culture apparatus;

[0220] Centrifuge at 4℃, centrifuge speed at 500 rpm, and centrifuge time at 1 min.

[0221] After cross-linking in a 37°C cell culture incubator for 30 min, add 120 μL of culture medium and change the medium daily.

[0222] After organoids were implanted in different ways, photographs were taken daily to observe and the size of the organoids was recorded. The specific photographic results are shown in Figure 5. The main difference between the hypoxic microplate culture method and the hyperxic microplate culture method is that the hypoxic microplate culture method involves cutting a PDMS breathable microporous layer with a microporous array into 6mm discs and placing them in the culture wells of an existing cell culture plate. The culture wells are a one-piece structure, and the bottom of the culture well itself is not oxygen permeable. In contrast, the hyperxic microplate culture method is based on the cell culture device prepared in Example 1. In this device, a PDMS bottom membrane with a microporous array is used as the bottom of the culture well, thus the bottom of the culture well has high oxygen permeability.

[0223] The results showed that, 48 hours after inoculation, the organoids formed by the traditional culture mode and the two culture methods that added PDMS microwell array to the traditional culture mode were significantly smaller than the organoids formed by the organoid culture process of the cell culture device in Example 1. The relevant statistical results are shown in Figure 6.

[0224] The size of organoids formed by each culture method on the fifth day was statistically analyzed in the light micrographs, as shown in Figures 7A-7C. The statistical results are shown in Figure 8. According to the statistical results shown in Figure 8, the organoids formed by the traditional organoid culture method are relatively small, and the size of the organoids varies greatly. When a PDMS microwell array is added to the traditional well plate, the size difference of the formed organoids is reduced, improving the uniformity of the organoids to some extent. When the culture process based on the cell culture device of Example 1 is used, in addition to a significant improvement in uniformity compared to the traditional well plate, the size of the organoids formed under high oxygen conditions is further significantly increased compared to the traditional culture method with the addition of the PDMS microwell array.

[0225] Example 3: In vitro construction of multi-cell clusters

[0226] The intestine was chosen as a representative organ with multicellular composition. Multicellular clusters were constructed under a hyperoxia environment. The construction routes of multicellular models under different oxygen permeability conditions are shown in Figure 9. Using the Caco-2 clonal colon adenocarcinoma cell line, which has a structure and function similar to differentiated small intestinal epithelial cells, primary human umbilical vein endothelial cells (HUVEC), and the 3T3 fibroblast cell line, cell clusters were constructed in a 3D culture method in a microwell array. This was compared with the cluster construction method in ordinary commercial culture plates, resulting in a tissue model with better in vitro functionality.

[0227] Two experimental groups were set up: one with a ratio of intestinal epithelial cells, vascular endothelial cells and fibroblasts of 4.5:4.5:1, and the other with a ratio of intestinal epithelial cells, vascular endothelial cells and fibroblasts of 4:4:2. A control group was set up with clusters of the same cell components in ordinary oxygen-free cell culture plates.

[0228] The colon adenocarcinoma cell line Caco-2 (purchased from ATCC (American Type Culture Collection), HTB-37) was cultured in high-glucose DMEM medium (purchased from Gibco, 11960044) supplemented with 10% (v / v) fetal bovine serum (purchased from Gibco) and 1% (v / v) penicillin and streptomycin (purchased from Gibco). Cells were passaged in culture flasks when they reached 80% confluence, with a passage ratio of 1:3.

[0229] Primary human umbilical vein endothelial cells (Huvec, purchased from Zhongqiao Xinzhou, DFSC-EC-01) were cultured in endothelial cell-specific medium (Zhongqiao Xinzhou, ZQ-1304), and 3T3 cells (purchased from ATCC, CRL-1658) were cultured in high-glucose DMEM medium (purchased from Gibco, 11960044), supplemented with 10% (v / v) fetal bovine serum (FBS, Gibco) and 1% (v / v) penicillin and streptomycin (Gibco). Cells were passaged when the confluence in the culture flask reached 80%, with a passage ratio of 1:3.

[0230] 1. The method for low-oxygen microplate culture is as follows:

[0231] Multi-cell co-culture clusters were constructed using the hypoxic microplate culture method described in Example 2. The steps included:

[0232] Caco-2, 3T3, and Huvec were digested with trypsin to form single-cell suspensions, centrifuged, and counted.

[0233] Cell suspensions containing multiple cell types were prepared by resuspending the cells in a special culture medium for endothelial cells according to a predetermined cell combination ratio.

[0234] Add 25 μL of cell suspension to one well of a 96-well plate, and repeat for 3 replicates.

[0235] Centrifuge at room temperature at a speed of 500 rpm for 1 minute.

[0236] Supplement with 120 μL of complete organoid culture medium.

[0237] 2. The high-oxygen microplate culture method is as follows:

[0238] Multi-cell co-culture clusters were constructed using the hyperoxia microplate culture method described in Example 2. The steps included:

[0239] Caco-2, 3T3, and Huvec were digested with trypsin to form single-cell suspensions, centrifuged, and counted.

[0240] Cell suspensions containing multiple cell types were prepared by resuspending the cells in a special culture medium for endothelial cells according to a predetermined cell combination ratio.

[0241] Add 25 μL of cell suspension to one culture chamber of a 96-well cell culture apparatus, and repeat 3 times.

[0242] Centrifuge at room temperature at a speed of 500 rpm for 1 minute.

[0243] Supplement with 120 μL of complete organoid culture medium.

[0244] After two days of culture, DAPI was selected as the stain for nuclear localization; E-cadherin was selected as the characteristic protein of Caco-2 cell barrier function, and the E-cadherin protein on the cell membrane was stained using immunofluorescence staining; CD31 was selected as the specific antibody for Huvec cells, and Huvec cells were stained; phalloidin was used to specifically stain the cytoskeleton of all cells. The immunofluorescence staining results are shown in Figure 10A, where green represents E-cadherin protein on the membrane, blue represents the cell nucleus, red represents endothelial cells, and purple represents the cytoskeleton. The expression level of E-cadherin was quantified using ImageJ image processing software, and the fluorescence intensity of E-cadherin was normalized to the fluorescence intensity of DAPI. The statistical results are shown in Figure 10B.

[0245] The statistical results in Figure 10B show that the in vitro constructed intestinal barrier with multicellular components has better barrier function under culture conditions with higher oxygen permeability, indicating that the cell cluster construction method based on the cell culture device prepared in Practice 1 can obtain a more functional in vitro multicellular model.

[0246] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0247] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A cell culture device comprising a composite culture layer, the composite culture layer comprising: a culture through-hole layer, the culture through-hole layer being provided with at least one culture through-hole extending vertically therethrough; a gas-permeable microporous layer, the gas-permeable microporous layer comprising at least one gas-permeable portion, each gas-permeable portion being provided with at least one micropore on a surface thereof, and the material of the gas-permeable portion being permeable to oxygen; wherein the gas-permeable portion and the culture through-hole correspond to each other, each gas-permeable portion covers a port at one end of the corresponding culture through-hole to jointly define a culture chamber with the corresponding culture through-hole, and the micropore is located inside the culture chamber. 2.The cell culture device according to claim 1, wherein: a cross section of the micropore is selected from at least one of a circle and a polygon; and / or a longitudinal section of the micropore is selected from at least one of a column, a cone, and an ellipse.

3. The cell culture device of claim 1 or 2, wherein, a cross section of the micropore is a regular polygon, and a diameter of an inscribed circle of the regular polygon is 40 μm to 1000 μm.

4. The cell culture device according to any one of claims 1 to 3, wherein a depth of the micropore is less than a thickness of the gas-permeable microporous layer, and the depth of the micropore is 40 μm to 1000 μm.

5. The cell culture device according to any one of claims 1 to 4, wherein a wall thickness between the micropores is 1 / 3 to 2 / 3 of the depth of the micropore. 6.The cell culture device according to any one of claims 1 to 5, wherein: the material of the gas-permeable microporous layer or the gas-permeable portion is an elastomer, and the elastomer has a good oxygen permeability. 7.The cell culture device according to any one of claims 1 to 6, wherein the material of the gas-permeable microporous layer or the gas-permeable portion comprises one or both of polydimethylsiloxane and 4-methylpentene. 8.The cell culture device according to any one of claims 1 to 7, wherein a thickness of the gas-permeable microporous layer is 0.5 mm to 5 mm.

9. The cell culture apparatus according to any one of claims 1 to 8, wherein the oxygen permeability of the permeable microporous layer or the permeable portion is 3.74 × 10⁻⁶. -13 mol / (cm 2 .s)~3.74×10 -12 mol / (cm 2 .s).

10. The cell culture device according to any one of claims 1 to 9, wherein, The cell culture device further comprises: a cover plate layer, the cover plate layer being stacked with the culture through-hole layer to cover one end of each culture through-hole away from the gas-permeable microporous layer.

11. The cell culture device according to any one of claims 1 to 10, wherein, The cell culture device further comprises a gas-permeable through-hole layer, the gas-permeable through-hole layer being stacked with the gas-permeable microporous layer, and the gas-permeable through-hole layer being provided with a gas-permeable through-hole corresponding to each gas-permeable portion; a thickness of the culture through-hole layer is 10 mm to 14 mm; a thickness of the gas-permeable through-hole layer is 0.5 mm to 5 mm.

12. The cell culture device of claim 11, wherein, The cell culture device further comprises a connecting member, the connecting member being used to fixedly connect at least two of the culture through-hole layer, the gas-permeable microporous layer, and the gas-permeable through-hole layer.

13. The cell culture device of claim 12, wherein, The fixed connection comprises one or more of welding, riveting, bonding, threaded connection, pin connection, elastic deformation connection, lock connection, and plug-in connection.

14. The cell culture device according to any one of claims 11 to 13, wherein, The material of the culture through-hole layer or the gas-permeable through-hole layer is selected from at least one of stainless steel, titanium, zirconium oxide, aluminum oxide, polyethylene, polypropylene, polymethyl methacrylate, polycarbonate, polyurethane, polyester sulfone, polyether ketone, polysulfide, polyphenylene sulfide, polyimide, polytetrafluoroethylene, silicon, zirconium oxide / aluminum oxide composite material, carbon fiber composite material, glass fiber composite material, zirconium oxide / titanium composite material, aluminum oxide, zirconium oxide / aluminum oxide / titanium oxide composite material, silicon carbide, boron nitride, silicon nitride, silicon carbide, silicon oxide, cerium oxide, barium zirconate, zirconium oxide / aluminum nitride composite material, aluminum oxide / aluminum nitride composite material, aluminum gallium selenide, niobium oxide, titanium oxide, zinc oxide, magnesium oxide, calcium phosphate, zirconium oxide / niobium oxide composite material, and zirconium oxide / magnesium oxide composite material.

15. A method of producing a cell culture device as claimed in any one of claims 1 to 14, wherein, Comprise: a culture through-hole layer is provided, and the culture through-hole layer is distributed with at least one culture through-hole penetrating up and down; a gas-permeable microporous layer is provided, and the gas-permeable microporous layer comprises a gas-permeable part prepared based on an oxygen-permeable material, and a surface of the gas-permeable part is provided with at least one micropore; the gas-permeable microporous layer and the culture through-hole layer are compounded to form a compound culture layer, so that the gas-permeable part covers a port at one end of a corresponding culture through-hole, and together with the corresponding culture through-hole, defines a culture chamber, and the micropore is located inside the corresponding culture chamber.

16. The method of manufacturing according to claim 15, wherein, The preparation step of the gas-permeable part comprises: a target mold with a specific pattern is customized according to the morphology of the micropore; the thickness of the gas-permeable microporous layer is determined according to the target oxygen permeation amount; the amount of the oxygen-permeable material and the curing agent is determined according to the thickness of the gas-permeable microporous layer, and the mass ratio of the oxygen-permeable material and the curing agent is (5-20):1; the oxygen-permeable material comprises one or both of polydimethylsiloxane and 4-methylpentene; the mixture on the surface of the target mold is cured and peeled off to form a gas-permeable microporous layer with at least one micropore, and the gas-permeable microporous layer covers part of each culture through-hole as a gas-permeable part.

17. The method of making according to claim 16, wherein, The method further comprises: a gas-permeable through-hole layer is provided, and the gas-permeable through-hole layer is arranged in layers with the gas-permeable microporous layer, and the gas-permeable through-hole layer is provided with a gas-permeable through-hole corresponding to the gas-permeable part; and / or a cover plate layer is provided, and the cover plate layer is arranged in layers with the culture through-hole layer to cover the culture chamber; and / or at least two of the culture through-hole layer, the gas-permeable microporous layer, the gas-permeable through-hole layer, and the cover plate layer are fixedly connected by using a connecting piece; the fixed connection is selected from at least one of welding, riveting, bonding, threaded connection, pin connection, elastic deformation connection, lock connection, and insertion.

18. A method of cell culturing, wherein, The cell culture device according to any one of claims 1-14 is used for cell culture of target cells.

19. The culturing method according to claim 18, wherein, The method satisfies at least one of the following conditions (1)-(4): (1) the time of the cell culture is 1-2 days; (2) the target cells comprise stem cells; (3) the target cells are selected from any one of iPS induced stem cells, healthy tissue stem cells, organoid passage digestion cells, and tumor tissue stem cells; (4) the target cells are obtained as organoids or multicellular clusters after cell culture, and the organoids include any one of intestinal organoids, liver organoids and pancreatic organoids.

20. The culturing method according to claim 19, wherein, The step of cell culture comprises: mixing the target cells and a biological gel reagent to prepare a cell suspension, and injecting the cell suspension into a culture cavity of a cell culture well plate; centrifuging the cell suspension in the culture cavity, and culturing the cells in the centrifuged cell suspension; the biological gel reagent includes at least one of Matrigel, methacrylated gelatin and methacrylated hyaluronic acid gelatin; the mass fraction of Matrigel in the biological gel reagent is 10% to 50%; the centrifuging speed is 300 rpm to 1000 rpm, and the centrifuging time is 30 s to 120 s; The cell density of the cell suspension is 1 x 10 6 / mL to 1 x 10 8 / mL. the size of the organoids is 100 μm to 400 μm.

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