Cell culturing vessel, method for producing same, and method for culturing cells

Abstract

This cell culturing vessel has a base material having an oxygen permeation part, and a coating layer disposed on a surface of the base material on a side where cells are to be disposed. The oxygen permeation part is made of a resin and has an oxygen permeability coefficient of 200-2400 cm3×mm / (m2×24h×atm). The bubble contact angle of the coating layer in water is 30-100°.<sp / > <sp / >

Description

Cell culture vessel, method for manufacturing the same, and method for culturing cells

[0001] The present invention relates to a cell culture vessel, a method for producing the same, and a method for culturing cells.

[0002] Methods for improving cell culture efficiency by using oxygen-permeable cell culture vessels as cell culture vessels for proliferation, growth, storage, and transportation of cells have been investigated. For example, cell culture vessels are known in which an oxygen-permeable resin sheet or film is attached to the top or bottom surface of a cylindrical, dish-shaped, or other substrate (see, for example, Patent Document 1).

[0003] Furthermore, the surface of a cell culture vessel to which cells are attached may be coated with a culture substrate such as laminin (see, for example, Patent Document 2).

[0004] Japanese Patent Application Laid-Open No. 2016-111975

[0005] According to the findings of the present inventors, cell adhesion may be low in cell culture vessels with increased oxygen permeability as described in Patent Document 1. Coating the culture substrate as described in Patent Document 2 is expected to increase cell adhesion, but in cell culture vessels with increased oxygen permeability, coating the culture substrate did not increase cell adhesion as much as expected.

[0006] The present invention has been made in consideration of the above-mentioned problems of the conventional technology, and aims to provide a cell culture vessel that is oxygen permeable and has improved cell adhesion after coating a culture substrate, a method for manufacturing the cell culture vessel, and a method for culturing cells using the cell culture vessel.

[0007] One aspect of the present invention for solving the above problems relates to the cell culture vessels of the following [1] to

[16] . [1] A cell culture vessel comprising: a substrate having an oxygen permeable portion; and a coating layer disposed on the surface of the substrate on which cells are disposed, wherein the oxygen permeable portion is made of resin and has an oxygen permeability coefficient of 200 cm ３ × mm / (m ２ ×24h×atm) or more 2400cm３ ×mm / (m ２× 24h × atm) or less, and the bubble contact angle of the coating layer in water is 30° or more and 100° or less. [2] The cell culture vessel according to [1], wherein a difference between the bubble contact angle of the coating layer in water and the bubble contact angle of the oxygen permeable portion in water is 35° or more and 75° or less. [3] The cell culture vessel according to claim 1, wherein the water contact angle of the coating layer is 40° or more and 90° or less. [4] The cell culture vessel according to claim 1, wherein the coating layer contains a polyalkoxyalkyl (meth)acrylate and / or a polyhydroxyalkyl (meth)acrylate. [5] The polyalkoxyalkyl (meth)acrylate is selected from the group consisting of polymethoxymethyl acrylate, poly(2-methoxyethyl acrylate), polymethoxypropyl acrylate, polymethoxybutyl acrylate, polyethoxymethyl acrylate, polyethoxyethyl acrylate, polyethoxypropyl acrylate, polyethoxybutyl acrylate, polypropoxymethyl acrylate, polypropoxyethyl acrylate, polypropoxypropyl acrylate, polypropoxybutyl acrylate, polybutoxymethyl acrylate, polybutoxyethyl acrylate, polybutoxypropyl acrylate, polybutoxybutyl acrylate, polymethoxy 5. The cell culture vessel according to claim 4, further comprising at least one compound selected from the group consisting of dimethyl methacrylate, polymethoxyethyl methacrylate, polymethoxypropyl methacrylate, polymethoxybutyl methacrylate, polyethoxymethyl methacrylate, polyethoxyethyl methacrylate, ethoxypropyl methacrylate, polyethoxybutyl methacrylate, polypropoxymethyl methacrylate, polypropoxyethyl methacrylate, polypropoxypropyl methacrylate, polypropoxybutyl methacrylate, polybutoxymethyl methacrylate, polybutoxyethyl methacrylate, polybutoxypropyl methacrylate, and polybutoxybutyl methacrylate.[6] The cell culture vessel according to [4] or [5], wherein the polyalkoxyalkyl(meth)acrylate and / or polyhydroxyalkyl(meth)acrylate has a number average molecular weight Mn of 10,000 or more and 500,000 or less. [7] The cell culture vessel according to any one of [1] to [6], wherein the coating layer contains gelatin. [8] The coating layer has a deposition amount of 0.1 μg / cm. ２ 100 μg / cm or more ２ The cell culture vessel according to any one of [1] to [7], wherein the base material contains at least one resin selected from the group consisting of polystyrene, polyethylene, polypropylene, 4-methyl-1-pentene (co)polymers, and cyclic olefin (co)polymers.

[10] The cell culture vessel according to any one of [1] to [9], wherein the oxygen permeable portion has an air bubble contact angle of 100° or more in water.

[11] The cell culture vessel according to any one of [1] to

[10] , wherein the oxygen permeable portion contains at least one resin selected from the group consisting of polyethylene, polypropylene, polyethylene terephthalate, and 4-methyl-1-pentene (co)polymers.

[12] The cell culture vessel according to any one of [1] to

[11] , wherein the base material has a frame having through holes and a resin sheet arranged on the top or bottom surface of the through holes.

[13] The cell culture vessel according to

[12] , wherein the oxygen permeable portion is formed in the resin sheet.

[14] The cell culture vessel according to

[12] or

[13] , wherein the resin sheet has a thickness of 10 μm or more and 500 μm or less.

[15] The cell culture vessel according to any one of

[12] to

[14] , wherein the resin sheet is formed of a thermoplastic resin different from that of the base material.

[16] The cell culture vessel according to

[12] to

[14] , wherein the sterility assurance level (SAL) of the medical device measured in accordance with BS EN556-1:2001 is 10 －２ The cell culture vessel according to any one of [1] to

[15] , which is:

[0008] One aspect of the present invention for solving the above problems relates to the following methods for manufacturing a cell culture vessel

[17] to

[20] .

[17] A method for manufacturing a cell culture vessel, comprising the steps of: preparing a substrate having an oxygen permeable portion; and applying a coating material to the surface of the substrate on which cells are cultured, drying the applied coating material, and disposing a coating layer, wherein the oxygen permeable portion is made of resin and has an oxygen permeability coefficient of 200 cm ３ × mm / (m ２ ×24h×atm) or more 2400cm ３ × mm / (m ２ × 24h × atm) or less, and an air bubble contact angle in water of the disposed coating layer is 30° or more and 100° or less.

[18] A method for manufacturing a cell culture vessel according to

[17] , comprising the steps of: packaging the cell culture vessel after disposing the coating layer; and sterilizing the packaged cell culture vessel.

[19] A method for manufacturing a cell culture vessel according to

[17] or

[18] , wherein the coating layer contains polyalkoxyalkyl (meth)acrylate and / or polyhydroxyalkyl (meth)acrylate.

[20] A method for manufacturing a cell culture vessel according to any of

[17] to

[19] , wherein the coating material contains gelatin and a solvent.

[0009] One aspect of the present invention for solving the above-mentioned problems relates to a method for culturing cells according to the following item

[21] :

[21] The method for culturing cells comprises the steps of: preparing a cell culture vessel according to any one of items [1] to

[16] ; forming a culture substrate layer containing a natural polymer selected from the group consisting of collagen, laminin, laminin fragments, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan on the surface of the coating layer; and culturing cells in the cell culture vessel.

[0010] According to the present invention, there are provided a cell culture vessel that has oxygen permeability and enhances cell adhesion after coating a culture substrate, a method for manufacturing the cell culture vessel, and a method for culturing cells using the cell culture vessel.

[0011] FIG. 1 is a perspective view of a cell culture vessel. FIG. 2 is a cross-sectional view of a portion of the cell culture vessel. FIG. 3A is a bottom perspective view of another cell culture vessel, and FIG. 3B is a top perspective view of the cell culture vessel. FIG. 4 is a cross-sectional view of a portion of the cell culture vessel. FIGS. 5A to 5C are micrographs obtained when cell adhesiveness was evaluated in Experiment 1 of the Example. FIG. 5A is a micrograph of a cell culture vessel evaluated as ⊚, FIG. 5B is a micrograph of a cell culture vessel evaluated as O, and FIG. 5C is a micrograph of a cell culture vessel evaluated as ×. FIGS. 6A to 6C are micrographs obtained when cell adhesiveness was evaluated in Experiment 2 of the Example. FIG. 3A is a micrograph of a cell culture vessel evaluated as O, FIG. 3B is a micrograph of a cell culture vessel evaluated as △, and FIG. 3C is a micrograph of a cell culture vessel evaluated as ×.

[0012] 1. Cell Culture Vessel The first embodiment of the present invention relates to a cell culture vessel. The cell culture vessel can be used for culturing cells, etc. In this specification, culturing cells means growing and proliferating the cells, or maintaining them in a living state.

[0013] Fig. 1 is a perspective view of a cell culture vessel 100 for culturing cells. Fig. 2 is a cross-sectional view of a portion of the cell culture vessel 100.

[0014] The cell culture vessel 100 is used, for example, for culturing cells derived from humans. Such a cell culture vessel 100 has a plurality of storage sections 210 (see FIG. 2 ) for culturing cells. The cell culture vessel 100 is housed in the culture space of a culture device (e.g., an incubator) with a drug (culture medium) and cells to be cultured (hereinafter referred to as "target cells") stored in the storage sections 210. Note that the cell culture vessel 100 does not need to be used while housed in the culture device. The cell culture vessel 100 may be used in various situations depending on the target cells.

[0015] The cell culture vessel 100 or at least one of the resin film 112 and the resin film 114 has a sterility assurance level (SAL) of 10 or more for medical devices measured in accordance with BS EN556-1:2001. －２Preferably, it is 10 or less. －３ More preferably, it is 10 －６ Here, SAL indicates the degree of sterility of the sterilization target after the sterilization process, and is expressed as the probability of microorganisms being expected to exist per unit number of the sterilization target after sterilization. SAL is 10 －ｎ The number of viable bacteria per sterilized item is expressed as 10 －ｎ n is preferably 2 or more, more preferably 3 or more, and even more preferably 6 or more.

[0016] The cell culture vessel 100 has a substrate 200 having an oxygen permeable portion, and a coating layer 300 arranged on the surface of the substrate 200 on the side where cells are arranged (the inner surface of the storage portion 210).

[0017] 1-1. Base Material 200 The base material 200 has a frame 220 and a resin sheet 230 .

[0018] The frame 220 has a frame portion 222 and a plurality of wells 224. The frame 220 is made of, for example, a thermoplastic resin and is a one-piece molded product produced by injection molding. Examples of thermoplastic resins that make up the frame 220 include polystyrene and polyolefin. Examples of the polyolefin include polyethylene, polypropylene, 4-methyl-1-pentene (co)polymers, and cyclic olefin (co)polymers. Of these, 4-methyl-1-pentene polymers are preferred from the viewpoint of increasing the gas permeability of the frame 220. Alternatively, 4-methyl-1-pentene polymers, polystyrene, polypropylene, and cyclic olefin polymers are preferred from the viewpoint of increasing the heat resistance of the frame 220. As the 4-methyl-1-pentene polymer, the 4-methyl-1-pentene polymer described as the material for the adhesion layer of the cell culture vessel sheet can be used in the same way, but a polymer different from the material for the adhesion layer may also be used.

[0019] The frame portion 222 is configured by a rectangular frame-shaped member. The well 224 is provided in a space surrounded by the frame portion 222. The well 224 is provided integrally with the frame portion 222.

[0020] The wells 224 are cylindrical through-holes that open upward. In this embodiment, the shape of the opening of the wells 224 (in other words, the outer shape in a plan view) is circular. The wells 224 are arranged side by side in the left-right direction and the front-rear direction. The number of wells 224 may be, for example, 6, 24, 96, or 384. Of course, the number of wells 224 may be other than 6, 24, 96, and 384.

[0021] Adjacent wells 224 are connected to each other by connecting portions 326. The left-end well 224, the right-end well 224, the front-end well 224, and the rear-end well 224 are connected to the inner circumferential surface of the frame portion 222 via connecting portions (not shown).

[0022] The resin sheet 230 is attached to the lower surface of the frame 220 via an adhesive layer 240. As a result, the resin sheet 230 closes the top or bottom surface of the cylindrical portion 310 (through-hole) and forms the top or bottom surface of the container 1. In this embodiment, the resin sheet 230 is made of a material with high oxygen permeability. As a result, the resin sheet 230 also functions as an oxygen permeable portion that supplies oxygen to the cells cultured inside the storage portion 210.

[0023] The resin sheet 230 is a transparent resin sheet with high oxygen permeability. Examples of such transparent resins include polyethylene, polypropylene, polyethylene terephthalate, and 4-methyl-1-pentene (co)polymers.

[0024] It is preferable that the resin used to make the resin sheet 230 be a different thermoplastic resin from the thermoplastic resin used to make the frame 220. For example, the thermoplastic resin used to make the frame 220 can be polystyrene or a cyclic olefin (co)polymer, and the resin used to make the resin sheet 230 can be a 4-methyl-1-pentene (co)polymer. Alternatively, the thermoplastic resin used to make the frame 220 can be a 4-methyl-1-pentene (co)polymer, and the resin used to make the resin sheet 230 can be a 4-methyl-1-pentene (co)polymer containing a different comonomer type or ratio. By using different thermoplastic resins as these materials, different properties can be imparted to the frame 220 and the resin sheet 230. For example, by imparting rigidity to the frame 220 and oxygen permeability to the resin sheet 230, a cell culture vessel 100 can be obtained that combines rigidity and oxygen permeability.

[0025] From the viewpoint of increasing the metabolic activity of the cells by making the inside of the storage section 210 aerobic, the resin sheet 230 has an oxygen permeability coefficient of 200 cm ３ × mm / (m ２ ×24hr×atm) or more 2400cm ３ × mm / (m ２ From the same viewpoint, it is preferable that the resin sheet 230 has an oxygen permeability of 4500 cm ３ / (m ２ ×24hr×atm) or more 120000cm ３ / (m ２ From the viewpoint of effectively increasing oxygen permeability, the resin sheet 230 is preferably a sheet-like member containing a polymer having a structural unit derived from 4-methyl-1-pentene.

[0026] The oxygen permeability coefficient is determined by a differential pressure gas permeability measurement method at a temperature of 23°C and a relative humidity of 0% [cm ３ / (m ２× 24h × atm)] is measured, and the oxygen permeability is divided by the thickness (mm) of the resin sheet 230. The device used for the measurement is not particularly limited as long as it uses a differential pressure gas permeability measurement method, but for example, a BT-3MT-C3 manufactured by Toyo Seiki Seisaku-sho, Ltd. can be used. The measurement sample is prepared by cutting out a 90 × 90 mm test piece from the resin sheet 230, and the measurement section diameter is 70 mm (permeation area is 38.46 cm ２ If the oxygen permeability is expected to be large, an aluminum mask is placed on the measurement sample in advance to reduce the actual permeation area to 5.0 cm. ２ It is more preferable to set the following.

[0027] From the viewpoint of improving the efficiency of cell observation, the resin sheet 230 is preferably transparent. Specifically, the total light transmittance measured in accordance with JIS K 7361-1 is preferably 50% or more and 100% or less, and more preferably 70% or more and 100% or less.

[0028] The resin sheet 230 preferably has an air bubble contact angle in water before sterilization of 90° or more and 120° or less, preferably 95° or more and 115° or less, and more preferably 100° or more and 110° or less. Furthermore, the resin sheet 230 preferably has an air bubble contact angle in water after sterilization of 90° or more, preferably 95° or more and 115° or less, and more preferably 100° or more and 110° or less.

[0029] The thickness of the resin sheet 230 is not particularly limited, but is preferably 10 μm or more and 500 μm or less, more preferably 20 μm or more and 300 μm or less, and even more preferably 20 μm or more and 200 μm or less. The greater the thickness, the greater the strength of the resin sheet 230. The smaller the thickness, the greater the oxygen permeability of the resin sheet 230.

[0030] The adhesive layer 240 adheres the resin sheet 230 to the lower surface of the bottom plate portion of the storage section 210. Known adhesives such as acrylic, silicone, urethane, and rubber adhesives can be used for the adhesive layer 240. Of these, acrylic adhesives and silicone adhesives are preferred.

[0031] 1-2. Coating Layer 300 The coating layer 300 is a coating made of a hydrophilic material and disposed inside the storage section 210. The coating layer 300 may be disposed so as to be in contact with the oxygen permeable section, or may be disposed so as to be in contact with a section that is not an oxygen permeable section, or may be disposed so as to be in contact with both of these. For example, in this embodiment, the coating layer 300 may be disposed so as to be in contact with the resin sheet 230 (oxygen permeable section) and also with the frame 220 (section that is not an oxygen permeable section).

[0032] The coating layer 300 can be a coating for imparting hydrophilicity to the inner surface of the storage section 210 to enhance cell adhesion. From this perspective, the coating layer 300 preferably contains a water-absorbent resin or gelatin. Examples of the water-absorbent resin include poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), PEG-acrylate, poly(vinylpyrrolidone) (PVP), polyethyleneimine (PEI), poly(L-lactide) (PLLA), poly(D-lactide) (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), polyalkoxyalkyl(meth)acrylate, polyhydroxyalkyl(meth)acrylate, and poly(2-methacryloyloxyphosphorylcholine) (PMPC). Of these, from the viewpoint of further enhancing cell adhesiveness, polyalkoxyalkyl(meth)acrylate, polyhydroxyalkyl(meth)acrylate, and gelatin are preferred, and polyalkoxyalkyl(meth)acrylate and polyhydroxyalkyl(meth)acrylate are more preferred.

[0033] Examples of polyalkoxyalkyl(meth)acrylates include polymethoxymethyl acrylate, poly(2-methoxyethyl acrylate) (PMEA), polymethoxypropyl acrylate, polymethoxybutyl acrylate, polyethoxymethyl acrylate, polyethoxyethyl acrylate, polyethoxypropyl acrylate, polyethoxybutyl acrylate, polypropoxymethyl acrylate, polypropoxyethyl acrylate, polypropoxypropyl acrylate, polypropoxybutyl acrylate, polybutoxymethyl acrylate, polybutoxyethyl acrylate, polybutoxypropyl acrylate, and polybutoxybutyl acrylate. acrylate, polymethoxymethyl methacrylate, polymethoxyethyl methacrylate, polymethoxypropyl methacrylate, polymethoxybutyl methacrylate, polyethoxymethyl methacrylate, polyethoxyethyl methacrylate, ethoxypropyl methacrylate, polyethoxybutyl methacrylate, polypropoxymethyl methacrylate, polypropoxyethyl methacrylate, polypropoxypropyl methacrylate, polypropoxybutyl methacrylate, polybutoxymethyl methacrylate, polybutoxyethyl methacrylate, polybutoxypropyl methacrylate, and polybutoxybutyl methacrylate. Of these, poly(2-methoxyethyl acrylate) (PMEA) is preferred.

[0034] Examples of polyhydroxyalkyl(meth)acrylates include polyhydroxyethyl methacrylate (PHEMA), polyhydroxypropyl methacrylate, and polyhydroxybutyl methacrylate, of which polyhydroxyethyl methacrylate (PHEMA) is preferred.

[0035] Of these, from the viewpoint of further enhancing cell adhesiveness, polymethoxyethyl acrylate, poly(hydroxyethyl methacrylate) and gelatin are preferred, and polymethoxyethyl acrylate is more preferred.

[0036] These may also be combined, for example, polyalkoxyalkyl(meth)acrylate and polyhydroxyalkyl(meth)acrylate may be used in combination. When combined, polymethoxyethyl acrylate and poly(hydroxyethyl methacrylate) are preferably combined. When these are combined, the ratio (mass ratio) of (polymethoxyethyl acrylate:poly(hydroxyethyl methacrylate)) is preferably 1:99 or more and 99:1 or less, more preferably 3:97 or more and 97:3 or less, and even more preferably 5:95 or more and 95:5 or less.

[0037] The polyalkoxyalkyl(meth)acrylate and / or polyhydroxyalkyl(meth)acrylate preferably has a number average molecular weight (Mn) of 10,000 to 500,000, more preferably 20,000 to 400,000, even more preferably 30,000 to 350,000, and particularly preferably 30,000 to 250,000. The larger the number average molecular weight (Mn), the better the uniformity of the thickness of the coating layer, and the smaller the number average molecular weight (Mn), the better the dispersibility of the polyalkoxyalkyl(meth)acrylate and / or polyhydroxyalkyl(meth)acrylate in a solvent.

[0038] Examples of gelatin include cattle bone-derived gelatin, cattle hide-derived gelatin, pigskin-derived gelatin, donkey skin-derived gelatin, chicken skin-derived gelatin, and fish-derived gelatin. Pigskin-derived gelatin is preferred from the viewpoint of cell adhesiveness. These may be used alone or in combination of two or more.

[0039] The coating layer 300 has an air bubble contact angle in water of 30° to 100°, preferably 30° to 70°, and more preferably 30° to 55°. The larger the air bubble contact angle in water of the coating layer 300, the more appropriate the water content of the coating layer 300, making it less likely that cell contact will be impaired. The smaller the air bubble contact angle in water of the coating layer 300, the more appropriately hydrophilic the inner surface of the storage section 210 will be, improving cell adhesion. The culture substrate is applied to the surface of the coating layer 300 using an aqueous coating solution. Therefore, the smaller the air bubble contact angle in water of the coating layer 300, the easier it is to adhere the culture substrate to the coating layer 300, thereby preventing a decrease in cell adhesion due to detachment of the culture substrate. The air bubble contact angle in water can be adjusted by the material of the coating layer 300, the conditions of the pretreatment performed on the cell culture vessel 100 before placing the coating layer 300, and other factors.

[0040] The water contact angle of the coating layer 300 is preferably 40° to 90°, more preferably 45° to 90°, and more preferably 45° to 60°. The culture substrate is applied to the surface of the coating layer 300 using an aqueous coating liquid. Therefore, the smaller the water contact angle of the coating layer 300, the easier it is to adhere the culture substrate to the coating layer 300, thereby suppressing a decrease in cell adhesion due to detachment of the culture substrate. The water contact angle can be adjusted by the material of the coating layer 300, the conditions of the pretreatment performed on the cell culture vessel 100 before disposing the coating layer 300, and the like. Furthermore, the difference between the air bubble contact angle of the coating layer 300 in water and the air bubble contact angle of the resin sheet 230 (oxygen permeable portion) in water is preferably 35° to 75°, more preferably 40° to 70°, even more preferably 45° to 65°, and particularly preferably 45° to 60°. If the difference between these bubble contact angles is small, the adhesion of the coating layer 300 to the resin sheet 230 is high, and the coating layer 300 is less likely to peel off from the housing portion 210.

[0041] The difference in bubble contact angle between the coating layer 300 and the resin sheet 230 in water is the difference in bubble contact angle measured after the cell culture vessel 100 on which the coating layer 300 is formed is sterilized (e.g., terminal sterilization). Sterilization of the cell culture vessel 100 is performed by irradiation with electron beams, gamma rays, or the like. If the coating layer 300 or the substrate 200 (e.g., the resin sheet 230) is altered by these sterilization methods, the adhesion between them may decrease, causing the coating layer 300 to peel off, which may hinder cell adhesion. Furthermore, if the coating layer 300 is formed from a material containing hydrophilic groups such as hydroxyl groups or carboxyl groups, crosslinking may occur during sterilization, increasing the density of the hydrophilic groups. This may result in excessive hydrophilicity of the coating layer 300, increasing its water content, and reducing cell adhesion.

[0042] In contrast, if the difference between the bubble contact angle of the coating layer 300 after sterilization and the bubble contact angle of the resin sheet 230 is small, the difference in surface energy between the coating layer 300 and the substrate 200 (resin sheet 230) is small even after sterilization, and high adhesion between them can be maintained. Furthermore, by making the bubble contact angle of the coating layer 300 appropriately close to the bubble contact angle of the hydrophobic substrate 200 (resin sheet 230), it is possible to prevent a decrease in cell contactability due to an increase in water content caused by excessively increasing hydrophilicity of the coating layer 300 after sterilization.

[0043] The method for controlling the difference in bubble contact angle is not limited. For example, the difference can be adjusted by irradiating the polyalkoxyalkyl(meth)acrylate coating layer 300 with gamma rays, or by plasma treatment or corona treatment after gamma ray irradiation. Furthermore, the difference in bubble contact angle can be adjusted by storing the gelatin coating layer 300 at room temperature for about 2 to 3 weeks after formation to remove any radicals remaining in the gelatin.

[0044] In addition, the coating layer 300 made of polyhydroxyalkyl (meth)acrylate can adjust the difference in bubble contact angle by increasing the coating amount of polyhydroxyalkyl (meth)acrylate or by irradiating with gamma rays.

[0045] It is well known that plasma treatments, etc., can be applied to cell culture vessels to enhance cell adhesion. However, according to the inventors' findings, conventional plasma treatments of cell culture vessels often do not enhance cell adhesion, and even if they do, the effect tends to be lost within a short period of time. While the reason for this is unclear, it is thought that this is because highly oxygen-permeable materials, such as the resin sheet 230, are often amorphous, making it easy to incorporate functional groups formed by plasma treatment, etc., into their interiors. Perhaps due to these characteristics, the cell culture vessels are less likely to adhere to the culture substrate, or even if they do adhere, they tend to peel over time, or the adhesion of the cell culture vessels significantly decreases after storage. Therefore, it is believed that cell adhesion was not sufficiently enhanced. In contrast, by providing a coating layer 300 with an air bubble contact angle in water within the above range, adhesion of the culture substrate can be sufficiently enhanced and adhesion is less likely to decrease over time.

[0046] The amount of coating layer 300 attached is calculated by multiplying the concentration (μg / mL) of the material of coating layer 300 in the coating material by the amount of coating (mL) by the bottom area of ​​the well (cm ２ The amount of coating layer 300 applied is not particularly limited, but is preferably 0.1 μg / cm ２ 100 μg / cm or more ２ It is preferable that the concentration is 0.15 μg / cm or less. ２ More than 80μg / cm ２ More preferably, it is 0.2 μg / cm or less. ２ More than 70μg / cm ２ It is more preferable that the following conditions are satisfied: The greater the amount of coating layer 300 attached, the higher the cell adhesiveness can be; The smaller the amount of coating layer 300 attached, the higher the oxygen permeability can be;

[0047] After the coating layer 300 is disposed, the cell culture vessel 100 has an oxygen permeability coefficient through the coating layer 300 of 200 cm ３ × mm / (m ２ ×24hr×atm) or more 2400cm ３ × mm / (m２ × 24 hr × atm) or less, and ３ × mm / (m ２ ×24hr×atm) or more 2400cm ３ × mm / (m ２ × 24 hr × atm) or less, and ３ × mm / (m ２ ×24hr×atm) or more 2400cm ３ × mm / (m ２ From the same viewpoint, it is more preferable that the oxygen permeability through the coating layer 300 of the cell culture vessel 100 is 3000 cm ３ / (m ２ ×24hr×atm) or more 40000cm ３ / (m ２ × 24 hr × atm) or less, and ３ / (m ２ ×24hr×atm) or more 36000cm ３ / (m ２ × 24 hr × atm) or less, and ３ / (m ２ ×24hr×atm) or more 32000cm ３ / (m ２ × 24 hr × atm) or less is more preferable.

[0048] The oxygen permeability coefficient is determined by a differential pressure gas permeability measurement method at a temperature of 23°C and a relative humidity of 0% [cm ３ / (m ２ × 24h × atm)] is measured, and the oxygen permeability is divided by the thickness (mm) of the resin sheet 230. The device used for the measurement is not particularly limited as long as it uses a differential pressure gas permeability measurement method, but for example, a BT-3MT-C3 manufactured by Toyo Seiki Seisakusho Co., Ltd. can be used. The measurement sample is prepared by cutting out a 90 × 90 mm test piece from a 50 μm thick resin sheet, for example, and the measurement section diameter is 70 mm (permeation area is 38.46 cm ２ Because the oxygen permeability is large, an aluminum mask is applied to the sample in advance to reduce the actual permeation area to 5.0 cm. ２The resin sheet may or may not have been subjected to microfabrication or surface modification treatment, but is preferably one that has not been subjected to any treatment.

[0049] 1-3. Culture Substrate Layer When culturing cells, it is preferable to form a culture substrate layer on the surface of the coating layer 300. The cell culture vessel has good adhesion of the culture substrate layer, and the cells have high adhesiveness.

[0050] The culture substrate layer is preferably a layer containing a natural polymer. Examples of the natural polymer include collagen, laminin, laminin fragments, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan. Among these, laminin is preferred, and laminin fragments are more preferred, because of its excellent adhesion to the coating layer having the above-mentioned properties and its high cell adhesiveness.

[0051] The amount of the culture substrate layer to be attached is not particularly limited, but is preferably 0.1 μg / cm ２ 100 μg / cm or more ２ It is preferable that the concentration is 0.2 μg / cm or less. ２ More than 70μg / cm ２ More preferably, it is 0.3 μg / cm or less. ２ 50μg / cm or more ２ It is more preferable that the following conditions are satisfied: The greater the amount of attached culture substrate layer, the higher the cell adhesiveness can be; The smaller the amount of attached culture substrate layer, the higher the oxygen permeability can be;

[0052] 2. Method for Manufacturing Cell Culture Vessel The cell culture vessel 100 can be manufactured by forming a coating layer 300 on a substrate 200 having an oxygen-permeable portion (resin sheet 230).

[0053] The substrate 200 may be prepared by a known method. For example, the substrate 200 may be prepared by attaching the resin sheet 230 to the bottom surface of the frame 220 via the adhesive layer 240.

[0054] The coating layer 300 can be formed by applying a coating material containing the material of the coating layer 300 described above to the surface of the substrate 200 on which cells are cultured (the inner surface of the storage section 210).

[0055] The coating material may be any treatment liquid containing the above-mentioned materials and an aqueous solvent. While the type of solvent is not particularly limited, when the coating material contains polyalkoxyalkyl(meth)acrylate and / or polyhydroxyalkyl(meth)acrylate, the solvent preferably contains water and a primary alcohol. Using a solvent containing a primary alcohol can improve the uniformity of the coating material. Examples of primary alcohols include methanol, ethanol, 1-propanol, and 1-butanol. The volume ratio of water to primary alcohol in the solvent (water:primary alcohol) is preferably 20:80 or more and 80:20 or less, and more preferably 40:60 or more and 60:40 or less. When the coating layer 300 is made of gelatin, the solvent may be water alone.

[0056] The concentration of the material for the coating layer 300 in the coating material is preferably 0.0005 mg / mL to 0.5 mg / mL, more preferably 0.001 mg / mL to 0.3 mg / mL, and even more preferably 0.02 mg / mL to 0.3 mg / mL. The higher the concentration of the coating layer 300, the more effective it is at improving cell adhesion. Setting the concentration of the coating layer 300 within an appropriate range makes it easier to form the coating layer 300 uniformly.

[0057] The amount of coating material to be applied may be adjusted in accordance with the amount of coating layer 300 to be applied.

[0058] Before applying the coating material, the surface of the substrate 200 on which the cells are cultured may be pretreated. Examples of pretreatments that can be used include plasma treatment, corona discharge, and UV ozone treatment. These pretreatments can adjust the bubble contact angle of the coating layer 300 in water and its adhesion to the substrate. For example, in the case of plasma treatment, the treatment conditions can be an irradiation time of 1 second to 15 seconds, an output of 10 V to 150 V, and a distance between the bottom of the container and the torch of 1 mm to 15 mm.

[0059] It is preferable to sterilize the substrate 200 after forming the coating layer 300 by applying and drying the coating material. The sterilization is more preferably performed after packaging the cell culture vessel (terminal sterilization). The sterilization can be performed by a known method such as irradiation with electron beams or gamma rays, for example, by irradiation with gamma rays at a dose of 12 kGy.

[0060] The bubble contact angle of the resin sheet 230 and the coating layer 300 in water after sterilization can also be adjusted by adjusting the conditions of the sterilization treatment and post-sterilization treatment. For example, when the coating layer 300 contains a polyalkoxyalkyl(meth)acrylate and / or a polyhydroxyalkyl(meth)acrylate, the bubble contact angle of the coating layer 300 in water after sterilization can be adjusted by sterilizing the coating layer 300 with electron beam irradiation or by pre-treating the coating layer 300 before applying a coating material. Furthermore, when the coating layer 300 contains gelatin, storing the coating layer 300 at room temperature for about 2 to 3 weeks after sterilization can remove radicals remaining in the gelatin and adjust the bubble contact angle of the coating layer 300 in water.

[0061] 3. Method for Culturing Cells The cell culture vessel described above can be used for culturing cells and the like.

[0062] When culturing cells, it is preferable to form a culture substrate layer on the surface of the coating layer 300. The above-mentioned cell culture vessel has good adhesion of the culture substrate layer and high cell adhesiveness.

[0063] The culture substrate layer is preferably a layer containing natural polymers, examples of which include collagen, laminin, laminin fragments, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan.

[0064] Laminin is a cruciform molecule consisting of α, β, and γ chains associated at a coiled-coil region, with several species of each chain. Therefore, it is referred to as a combination of five α chains (LAMA1-5), four β chains (LAMB1-4), and three γ chains (LAMC1-3), with laminin 511 representing laminin α5β1γ1. Each laminin exerts its own unique function. It is preferable to use laminin 511 or laminin 521. The origin of laminin is not particularly limited, and laminins derived from various organisms can be used. Either full-length laminin or laminin fragments can be used, with laminin fragments being preferred from the viewpoint of enhancing cell adhesiveness.

[0065] The culture substrate layer can be formed by applying a treatment solution containing the above-mentioned natural polymer and water and drying it.

[0066] Cell culture can be carried out by placing a drug (culture medium) and target cells in each well 224 and placing them in the culture space of a culture device (incubator) set to predetermined conditions such as temperature.

[0067] 4. Other Embodiments It should be noted that the above-described embodiments each represent an example of the present invention, and the present invention is not limited to the above-described embodiments. Needless to say, various other embodiments are possible within the scope of the concept of the present invention.

[0068] Fig. 3A is a bottom perspective view showing an example of another cell culture vessel 500. Fig. 3B is a top perspective view of the cell culture vessel 500. Fig. 4 is a cross-sectional view of a portion of the cell culture vessel 500.

[0069] The cell culture vessel 500 is used, for example, for culturing neurons. The cell culture vessel 500 has chambers 524a and 524b in which cell bodies 540 of undeveloped neurons induced to differentiate from stem cells are placed, and a channel 524c connecting the chambers 524a and 524b. The cell culture vessel 500 is placed in the culture space of a culture device (e.g., an incubator) with a drug (culture medium) contained in the chambers 524a, 524b, and the channel 524c, and a mass of cell bodies 540 of neurons, which are the target cells to be cultured, contained in one of the chambers 524a and 524b.

[0070] The cell bodies 540 housed in the chamber grow axons 542 along the flow channels 524c. At this time, the axons 542 self-organize and bundle together to form nerve tissue (a process for culturing cells).

[0071] The cell culture vessel 500 has a frame 520 and a resin sheet 530. The resin sheet 530 is a sheet made of a transparent resin that is oxygen permeable.

[0072] Frame 520 has a frame portion 522 and a bottom plate portion 528. At positions of bottom plate 528 corresponding to chamber 524 a, chamber 524 b, and flow path 524 c, through-holes are formed in the shapes of chamber 524 a, chamber 524 b, and flow path 524 c. Frame 520 is a one-piece molded product made by injection molding from the same synthetic resin as frame 220, for example.

[0073] 3B , resin sheet 530 is placed on the bottom surface of frame 520 (the bottom surfaces of chambers 524a, 524b, and flow path 524c) and pressed to be in close contact with the bottom surface of frame 520. This forms cell culture vessel 500 having resin sheet 530 as the bottom material of chambers 524a, 524b, and flow path 524c.

[0074] 4, a coating layer 300 is disposed on the surface of the resin sheet 530 on the side where the cells are to be placed (the surface on the chamber 524a, chamber 524b, and flow channel 524c side). The coating layer 300 has the above-described properties.

[0075] Such a cell culture vessel 500 can also be used to form a culture substrate layer and culture cells. The coating layer 300 having the above-described properties enhances cell adhesion.

[0076] For example, the above-mentioned cell culture vessel is a cell culture vessel for growing or propagating cells, but a container for storing or transporting cells or for observing cells may also be used as a cell culture vessel. Furthermore, the cells to be cultured by the cell culture vessel are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. The cells may be living cells or dead cells.

[0077] The present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[0078] The glass transition point (Tg) of the material used below is a value determined from the peak top of the loss modulus measured using a dynamic viscoelasticity measuring device (DVA-225 manufactured by IT Measurement & Control Co., Ltd.) in a tensile mode at a temperature of −30 to 200° C. (heating rate of 3° C. / min), a frequency of 1 Hz, and in an air atmosphere.

[0079] The number average molecular weight (Mn) of the materials used below is a value measured by gel permeation chromatography (GPC). Specifically, the number average molecular weight (Mn) of a polymer dissolved in dimethylformamide (DMF) was calibrated with a polystyrene standard under the following conditions to obtain the molecular weight. Column: Two TSKgel SuperAWM-H columns (manufactured by Tosoh Corporation) Column temperature: 40°C Mobile phase: GPC DMF (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 10 mM LiBr added Detector: Differential refractometer Flow rate: 0.6 mL / min. Sample concentration: 1.0 mg / mL Injection volume: 20 μL Column calibration: Monodisperse polystyrene (EasiVial PS-H polystyrene (manufactured by Agilent Technologies, Inc.)) Molecular weight calibration: Relative calibration method (polystyrene equivalent)

[0080] 1. Preparation of cell culture vessels 4-methyl-1-pentene polymer (TPX ("TPX" is a registered trademark of Mitsui Chemicals, Inc.), molecular weight (Mw) = 428,000, molecular weight distribution (Mw / Mn) = 4.1, Tg = 40°C) was fed into an extruder equipped with a full-flight screw and a T-die, and extrusion molding was performed by setting the extrusion temperature to 270°C, the roll temperature to 60°C, and adjusting the roll rotation speed to obtain a resin sheet with a thickness of 50 μm.

[0081] The thickness of the resin sheet was determined as the average thickness obtained by measuring five points on the resin sheet using a Digimatic Micrometer (MDC-25MXT, manufactured by Mitutoyo Corporation).

[0082] A 24-well culture plate (cell culture vessel) was prepared by adhering the resin sheet obtained above to the bottom of a 24-well vessel frame (frame) made of polystyrene (hereinafter simply referred to as "PS") with a glass transition temperature of 94°C via an acrylic adhesive. The culture plate was then placed in the chamber of an atmospheric pressure plasma surface treatment device (manufactured by Sekisui Chemical Co., Ltd.), and the chamber was filled with a nitrogen gas stream to plasma-treat the culture plate (irradiation time: 2 seconds, output: 10 V, distance between the vessel bottom and the torch: 2 mm).

[0083] The oxygen permeability and oxygen permeability coefficient of the resin sheet after sterilization were measured using a differential pressure gas permeability measuring device (BT-3MT-C3, manufactured by Toyo Seiki Seisaku-sho, Ltd.) under an environment of 23°C temperature and 0% RH. The measurement diameter was 70 mm (permeation area was 38.47 cm ２ Since the oxygen permeability coefficient was expected to be large, an aluminum mask was placed on the sample in advance to reduce the actual permeation area to 5.0 cm. ２ It was decided.

[0084] 2. Preparation of Coating Material 2-1. Types of Coating Material The following materials were prepared. (1) Polyalkoxyalkyl(meth)acrylate PMEA1: Poly(2-methoxyethyl acrylate) number average molecular weight Mn 74,000 (manufactured by POLYMER SOURCE) PMEA2: Poly(2-methoxyethyl acrylate) number average molecular weight Mn 305,000 (manufactured by POLYMER SOURCE) (2) Polyhydroxyalkyl(meth)acrylate Poly(2-hydroxyethyl methacrylate) (PHEMA) (manufactured by Sigma-Aldrich Japan) (3) Gelatin Powdered gelatin (B-Matrix Gelatin LS-250, manufactured by Nitta Gelatin Co., Ltd.)

[0085] 2-2. Preparation of coating material 1 g of PHEMA was dissolved in a mixed solution of 10 mL of ethanol and 10 mL of water for injection, and stirred with a stirrer for 1 hour. The solution was then diluted with ethanol and water (volume ratio 1:1) and sterilized by filtration to prepare coating materials. At this time, the concentration of PHEMA after dilution was changed to obtain coating materials 1-1 to 1-4.

[0086] 50 mg of powdered gelatin (B-Matrix Gelatin LS-250, manufactured by Nitta Gelatin Co., Ltd.) was added to 50 mL of water for injection (Japanese Pharmacopoeia, manufactured by Otsuka Pharmaceutical Co., Ltd.) and allowed to stand at room temperature for 30 minutes to allow swelling. The bottle containing the swollen gelatin was then heated in a 50°C water bath for 1 hour to dissolve the gelatin, which was then diluted with water for injection to prepare a coating material. The concentration of the diluted gelatin was varied to obtain coating materials 1-5 to 1-7 and 2-5.

[0087] To 3 mg of either the polyalkoxyalkyl(meth)acrylate or polyhydroxyalkyl(meth)acrylate, 0.5 mL of ethanol and 0.5 mL of water for injection were added to prepare an ethanol / aqueous solution of polyalkoxyalkyl(meth)acrylate or polyhydroxyalkyl(meth)acrylate at a concentration of 3 mg / mL. This was then diluted with ethanol and water (volume ratio 1:1) to obtain coating materials 2-1 to 2-4.

[0088] Coating material 2-6 was prepared by mixing an 80 vol% ethanol / aqueous solution of poly(2-methoxyethyl acrylate) (PMEA1) with a concentration of 30 μg / mL and a 20 vol% ethanol / aqueous solution of poly(2-hydroxyethyl methacrylate) (PHEMA) with a concentration of 30 μg / mL. The ratio (mass ratio) of PMEA1 to PHEMA in coating material 2-6 was 80:20.

[0089] A 20 vol% ethanol / aqueous solution of poly(2-methoxyethyl acrylate) (PMEA1) with a concentration of 30 μg / mL and an 80 vol% ethanol / aqueous solution of poly(2-hydroxyethyl methacrylate) (PHEMA) with a concentration of 30 μg / mL were mixed to prepare coating material 2-7. The ratio (mass ratio) of PMEA1 to PHEMA in coating material 2-7 was 20:80.

[0090] A 0.1 M hydrochloric acid solution (for volumetric analysis, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted 100-fold with water for injection (Japanese Pharmacopoeia, manufactured by Otsuka Pharmaceutical Co., Ltd.) to prepare a 0.001 M hydrochloric acid solution. The prepared hydrochloric acid solution was sterilized by filtration. A 3 mg / mL collagen solution (Cell Matrix Type I-P, derived from porcine tendon, manufactured by Nitta Gelatin Co., Ltd.) was diluted with the above 0.001 M hydrochloric acid solution to prepare coating materials 2-8 to 2-9.

[0091] 3. Coating Layer Arrangement 3-1. Experiment 1 In Experiment 1, when preparing the cell culture vessels, the culture plates with the resin sheets attached were packed in gamma-ray-resistant bags and sterilized by irradiating with 10 kGy of gamma rays. Then, each of the coating materials 1-1 to 1-9 was applied to each hole of the sterilized cell culture vessel using a pipette in an amount of 200 μl. The vessels were then dried overnight at room temperature to obtain cell culture vessels 1-1 to 1-9 with coating layers arranged thereon.

[0092] 3-2. Experiment 2 In Experiment 2, the culture plates were not sterilized before the coating material was applied. Instead, the cell culture vessels after the coating material was applied and dried were packaged in gamma-ray-resistant bags and sterilized by irradiating with 12 kGy of gamma rays (terminal sterilization), yielding cell culture vessels 2-1 to 2-9 each having a coating layer. Cell culture vessel 2-5 was stored in an environment of 23°C and 50% RH for 3 weeks before the subsequent evaluation.

[0093] Furthermore, a cell culture vessel without a coating layer was packed in a gamma-ray resistant bag and sterilized by irradiating with gamma rays of 12 kGy to obtain a cell culture vessel 2-10.

[0094] 4. Measurement and Evaluation 4-1. Evaluation of Experiment 1 4-1-1. Bubble Contact Angle in Water The bubble contact angle in water on the obtained coating layer surface (cell culture vessel 1-10 is the surface of the resin sheet on which the cells are placed) was evaluated using a fully automated contact angle meter (manufactured by KRUSS, device name: DSA30). The value measured 1 second after the bubbles made contact was taken as the bubble contact angle.

[0095] 4-1-2. Water Contact Angle The water contact angle of the resulting coating layer surface (cell culture vessel 1-10: the surface of the resin sheet on which the cells were placed) was measured in accordance with JIS R 3257:1999. Under constant temperature and humidity conditions of 25±5°C and 50±10% RH, a 1.8 μL droplet of distilled water was dropped onto the surface, and the angle of the contact interface between the surface and the water droplet was measured within 1 minute using a contact angle meter (A-XE model, manufactured by Kyowa Interface Science Co., Ltd.). The measurement position was the center of the well, and the average value of the values ​​obtained from measurements at three wells was used as the water contact angle of the cell culture vessel. The water contact angle was measured immediately after the coating layer was formed.

[0096] 4-1-3. Uniform application of coating material A collagen layer was formed by applying a 0.3 mg / ml collagen solution to the surface of the coating layer. The formed collagen layer was colored red using a picrosirius red staining kit. The coloring was visually confirmed, and the uniform application was evaluated according to the following criteria: ◎: The coloring was uniform, with the periphery and center of every hole in the cell culture vessel being about the same. ◯: Some holes in the cell culture vessel were colored darker at the periphery than at the center. ×: Some holes were not colored.

[0097] 4-1-4. Oxygen Permeability and Oxygen Permeability Coefficient The oxygen permeability and oxygen permeability coefficient of the resin sheet after the coating layer was formed were measured using a differential pressure gas permeability measuring device (BT-3MT-C3, manufactured by Toyo Seiki Seisaku-sho, Ltd.) under an environment of 23°C and 0% RH. The measurement diameter was 70 mm (permeation area was 38.47 cm). ２ Since the oxygen permeability coefficient was expected to be large, an aluminum mask was placed on the sample in advance to reduce the actual permeation area to 5.0 cm. ２ It was decided.

[0098] 4-1-5. Cell Adhesion The following materials were prepared: Laminin 1: Mouse-derived laminin (full-length laminin, manufactured by Corning) Laminin 2: iMatrix-511 (laminin α5β1γ1 fragment, manufactured by Matrixome)

[0099] 0.2 mL of a 1.5 mg / mL laminin 1 aqueous solution was added to the surface of the coating layer, and the vessel was left to stand at 37°C for 60 minutes, after which it was washed with Dulbecco's PBS (-). Next, 0.5 mL of culture medium containing human iPS cells was seeded onto each culture surface of the cell culture vessel using a micropipette. The vessel was then covered with a polystyrene lid and placed in an incubator at 37°C and 5% CO ２ After 4 days, the cell culture vessels were removed from the incubator, and each culture surface was observed under a microscope to observe how the cells adhered and spread. The cell adhesiveness was evaluated according to the following criteria. Micrographs of cells observed under a phase-contrast microscope after 4 days of culture in the cell culture vessels are shown in Figures 5A to 5C. Note that Figure 5A is a micrograph of a cell culture vessel that was evaluated as ⊚, Figure 5B is a micrograph of a cell culture vessel that was evaluated as O, and Figure 5C is a micrograph of a cell culture vessel that was evaluated as ×. ◎: Cells adhered and spread sufficiently, and the appearance was uniform within and between wells. ○: Cells adhered and spread, although there were some uneven areas within and between wells. △: There were many uneven areas within and between wells. ×: Cells did not adhere in most areas of the entire well, forming cell clumps.

[0100] 4-2. Evaluation of Experiment 2 4-2-1. Air bubble contact angle in water The air bubble contact angle in water on the coating layer surface (in the case of cell culture vessel 2-10, the surface of the resin sheet on which the cells were placed) and the substrate surface before and after terminal sterilization was measured in the same manner as in Experiment 1.

[0101] 4-2-2. Water contact angle The water contact angles of the substrate and coating layer surfaces (in the case of cell culture vessel 2-10, the surface of the resin sheet on which the cells were placed) were measured in the same manner as in Experiment 1. The water contact angles of the substrate and coating layer were measured immediately after the coating layer was formed.

[0102] 4-2-3. Oxygen permeability and oxygen permeability coefficient The oxygen permeability and oxygen permeability coefficient of the resin sheet after terminal sterilization were measured in the same manner as in Experiment 1.

[0103] 4-2-4. Cell Adhesion Measurements were performed in the same manner as in Experiment 1, using either laminin 1 or laminin 2. Figures 6A to 6C show micrographs of cells cultured in cell culture vessels for four days, taken using a phase-contrast microscope. Note that Figure 6A is a micrograph of a cell culture vessel rated as ◎, Figure 6B is a micrograph of a cell culture vessel rated as ◯, and Figure 6C is a micrograph of a cell culture vessel rated as ×.

[0104] The materials and evaluation results of the cell culture vessels in Experiment 1 are shown in Tables 1 and 2, and the materials and evaluation results of the cell culture vessels in Experiment 2 are shown in Tables 3 to 5. In Tables 1 to 5, the unit of oxygen permeability is cm ３ × mm / (m ２ × 24hr × atm), and the unit of oxygen permeability is cm ３ / (m ２ × 24 hr × atm).

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] As is clear from Tables 1 to 5, the oxygen permeability coefficient is 200 cm ３ × mm / (m ２ ×24h×atm) or more 2400cm ３ × mm / (m ２ A cell culture vessel having an oxygen permeable portion with a contact angle of air bubbles in water of 30° or more and 100° or less (×24h×atm) or less had high cell adhesion.

[0111] This application claims priority from Japanese Patent Application No. 2024-002531, filed January 11, 2024, and Japanese Patent Application No. 2024-171160, filed September 30, 2024. The matters described in the specification, claims, and drawings of those applications as originally filed are incorporated herein by reference.

[0112] The cell culture vessel according to the present invention can be applied to the culture of various cells.

[0113] REFERENCE SIGNS LIST 100 Cell culture vessel 200 Substrate 210 Storage section 220 Frame 222 Frame section 224 Well 230 Resin sheet 240 Adhesive layer 300 Coating layer 500 Cell culture vessel 520 Frame 522 Frame section 528 Bottom plate section 524a, 524b Chamber 524c Flow path 530 Resin sheet 540 Cell body 542 Axon 544 Nerve tissue

Claims

1. A cell culture container having a substrate with an oxygen permeation part and a coating layer disposed on the surface of the substrate on the side where cells are arranged, wherein the oxygen permeation part is made of resin and has an oxygen permeability coefficient of 200 cm ３ ×mm / (m ２ ×24 h×atm) or more and 2400 cm ３ ×mm / (m ２ ×24 h×atm) or less, and the bubble contact angle of the coating layer in water is 30° or more and 100° or less.

2. The difference between the bubble contact angle of the coating layer in water and the bubble contact angle of the oxygen permeable part in water is 35° or more and 75° or less. The cell culture container according to claim 1.

3. The water contact angle of the coating layer is 40° or more and 90° or less. The cell culture container according to claim 1.

4. The coating layer contains polyalkoxyalkyl (meth)acrylate and / or polyhydroxyalkyl (meth)acrylate. The cell culture container according to claim 1.

5. The polyalkoxyalkyl (meth)acrylate includes at least one compound selected from the group consisting of polymethoxymethyl acrylate, poly(2-methoxyethyl acrylate), polymethoxypropyl acrylate, polymethoxybutyl acrylate, polyethoxymethyl acrylate, polyethoxyethyl acrylate, polyethoxypropyl acrylate, polyethoxybutyl acrylate, polypropoxymethyl acrylate, polypropoxyethyl acrylate, polypropoxypropyl acrylate, polypropoxybutyl acrylate, polybutoxymethyl acrylate, polybutoxyethyl acrylate, polybutoxypropyl acrylate, polybutoxybutyl acrylate, polymethoxymethyl methacrylate, polymethoxyethyl methacrylate, polymethoxypropyl methacrylate, polymethoxybutyl methacrylate, polyethoxymethyl methacrylate, polyethoxyethyl methacrylate, ethoxypropyl methacrylate, polyethoxybutyl methacrylate, polypropoxymethyl methacrylate, polypropoxyethyl methacrylate, polypropoxypropyl methacrylate, polypropoxybutyl methacrylate, polybutoxymethyl methacrylate, polybutoxyethyl methacrylate, polybutoxypropyl methacrylate, and polybutoxybutyl methacrylate. The cell culture container according to claim 4.

6. The number average molecular weight Mn of the polyalkoxyalkyl (meth)acrylate and / or polyhydroxyalkyl (meth)acrylate is 10,000 or more and 500,000 or less. The cell culture container according to claim 4.

7. The coating layer contains gelatin. The cell culture container according to claim 1.

8. The coating amount of the coating layer is 0.1 μg / cm ２ or more and 100 μg / cm ２ or less. The cell culture container according to claim 1.

9. The cell culture container according to claim 1, wherein the base material contains at least one resin selected from the group consisting of polystyrene, polyethylene, polypropylene, 4-methyl-1-pentene-based (co)polymers, and cyclic olefin-based (co)polymers.

10. The cell culture container according to claim 1, wherein the bubble contact angle of the oxygen permeable part in water is 100° or more.

11. The cell culture container according to claim 1, wherein the oxygen permeable part contains at least one resin selected from the group consisting of polyethylene, polypropylene, polyethylene terephthalate, and 4-methyl-1-pentene-based (co)polymers.

12. The cell culture container according to claim 1, wherein the base material has a frame body having through holes and a resin sheet disposed on the upper surface or the bottom surface of the through holes.

13. The cell culture container according to claim 12, wherein the oxygen permeable part is formed on the resin sheet.

14. The cell culture container according to claim 12, wherein the resin sheet has a thickness of 10 μm or more and 500 μm or less.

15. The cell culture container according to claim 12, wherein the resin sheet is formed of a thermoplastic resin of a type different from that of the base material.

16. The sterility assurance level (SAL) of the medical device measured in accordance with BS EN 556-1:2001 is 10 －２ or less, The cell culture container according to claim 1.

17. A step of preparing a substrate having an oxygen permeable portion, and a step of applying a coating material to the surface of the substrate on the side where cells are cultured and drying the applied coating material to dispose a coating layer, the method for manufacturing a cell culture vessel comprising: the oxygen permeable portion is made of resin and has an oxygen permeability coefficient of 200 cm ３ ×mm / (m ２ ×24 h×atm) or more and 2400 cm ３ ×mm / (m ２ ×24 h×atm) or less; and the bubble contact angle of the disposed coating layer in water is 30° or more and 100° or less. A method for manufacturing a cell culture vessel.

18. The method for manufacturing a cell culture container according to claim 17, comprising a step of packaging the cell culture container after disposing the coating layer and a step of sterilizing the packaged cell culture container.

19. The method for manufacturing a cell culture container according to claim 17, wherein the coating layer contains polyalkoxyalkyl (meth)acrylate and / or polyhydroxyalkyl (meth)acrylate.

20. The method for manufacturing a cell culture container according to claim 17, wherein the coating material contains gelatin and a solvent.

21. A method for culturing cells, comprising a step of preparing the cell culture container according to any one of claims 1 to 16, a step of forming a culture substrate layer containing a natural polymer selected from the group consisting of collagen, laminin, laminin fragment, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan on the surface of the coating layer, and a step of culturing cells in the cell culture container.

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