Carrier for cell culture and cell culture method using same

Abstract

Provided is a carrier for cell culture, characterized in that the carrier comprises: two or more porous polymer membranes; a spacer member provided between the two or more porous polymer membranes and having one or more openings; and two protective members each having one or more openings and holding the two or more porous polymer membranes therebetween; and in that edges of the porous polymer membranes and an edge of the spacer member are melt-bonded and fixed, edges of the porous polymer membranes and edges of the protective members are melt-bonded and fixed by a seal member, and a space having an average thickness of 10 μm-1,500 μm is formed between two or more adjacent porous polymer membranes.

Description

Cell culture carrier and cell culture method using the same

[0001] The present invention relates to a carrier for cell culture and a method for cell culture using the same.

[0002] In recent years, proteins such as enzymes, hormones, antibodies, cytokines, and viruses (viral proteins) used in treatments and vaccines have been industrially produced using cultured cells. However, the technology for producing these proteins is expensive, which has been driving up healthcare costs. Therefore, there has been a need for innovative technologies to significantly reduce costs, such as techniques for culturing cells at high density and technologies to increase protein production.

[0003] As cells that produce proteins, scaffold-dependent adherent cells that adhere to culture substrates are sometimes used. Because these cells proliferate in a scaffold-dependent manner, they need to be cultured attached to the surface of a petri dish, plate, or chamber. Traditionally, culturing large quantities of these adherent cells required increasing the surface area for adhesion. However, increasing the culture area inevitably requires increasing the space needed, which has been a factor in increasing costs.

[0004] A cell culture method using a polyimide porous membrane has been developed as a way to culture adherent cells in large quantities while keeping the culture space small (for example, Patent Document 1). Prior to this application, polyimide porous membranes have been used for applications mainly related to batteries, such as filters, low dielectric constant films, and electrolyte membranes for fuel cells. Patent Documents 2 to 4 describe polyimide porous membranes that have excellent permeability to substances such as gases, high porosity, excellent smoothness of both surfaces, relatively high strength, and have many macrovoids that have excellent resistance to compressive stress in the thickness direction despite the high porosity. All of these are polyimide porous membranes made via amic acid.

[0005] To further increase the number of cells that can be cultured using a polyimide porous membrane, a cell culture module has also been developed in which two or more polyimide porous membranes are aggregated and housed in a casing having two or more culture medium inlets and outlets (Patent Document 5).

[0006] International Publication No. 2015 / 012415, International Publication No. 2010 / 038873, Japanese Patent Publication No. 2011-219585, Japanese Patent Publication No. 2011-219586, International Publication No. 2018 / 021368

[0007] Conventional cell culture modules have a structure in which multiple polymer porous membranes are stacked, resulting in poor cell adhesion between the stacked polymer porous membranes. This leads to insufficient oxygen supply, creating anaerobic conditions and hindering cell growth. Furthermore, when modules come into contact with each other or with the culture vessel during suspension culture, the casing comes into contact with the polymer porous membrane. As the culture time increases, the polymer porous membrane is damaged, and fragments are released into the culture medium.

[0008] As a result of diligent research, the inventors have succeeded in developing a new cell culture carrier that can solve the above problems. That is, although not limited thereto, the present invention preferably includes the following embodiments.

[0009] [1] A carrier for cell culture, comprising two or more polymer porous membranes, a spacer member having one or more openings provided between the two or more polymer porous membranes, and two protective members having one or more openings and sandwiching the two or more polymer porous membranes, wherein the polymer porous membrane is a polymer porous membrane having a surface layer A and a surface layer B having a plurality of pores, where the average pore diameter of the pores in surface layer A is smaller than the average pore diameter of the pores in surface layer B, the edges of the polymer porous membrane and the edges of the spacer member are welded and fixed, the edges of the polymer porous membrane and the edges of the protective member are welded and fixed by a sealing member, and a space of an average of 10 μm to 1500 μm is formed between adjacent two or more polymer porous membranes. [2] The carrier according to item 1, wherein the carrier is substantially circular, elliptical, or square. [3] The carrier according to item 1 or 2, wherein the diameter or one side of the carrier is 0.1 mm to 5.0 cm. [4] The carrier is 0.99 g / cm³ ３[5] A carrier according to any one of items 1 to 3, having the following densities: [5] A carrier according to any one of items 1 to 4, wherein the spacer member and / or protective member is selected from one or more of the group consisting of polyolefin, polyethylene, polypropylene, polyvinyl chloride, ethylene vinyl acetate resin, polystyrene, polyvinyl alcohol, polyethylene terephthalate, polyamide, polycarbonate, ionomer, polyurethane, polybutadiene, polyacrylonitrile, polybutylene terephthalate, and polyethylene naphthalate. [6] A carrier according to any one of items 1 to 5, wherein the spacer member and / or protective member is a mesh and / or nonwoven fabric and / or single-layer film and / or multilayer film. [7] A carrier according to any one of items 1 to 6, wherein the sealing member is a single-layer film, multilayer film, or nonwoven fabric selected from one or more of the group consisting of polypropylene, polyethylene, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, polyamide, and ethylene vinyl acetate copolymer. [8] The carrier according to any one of items 1 to 7, wherein the polymer porous membrane has a plurality of pores with an average pore diameter of 0.01 to 100 μm. [9] The carrier according to any one of items 1 to 8, wherein the average pore diameter of the surface layer A is 0.01 to 50 μm.

[10] The carrier according to any one of items 1 to 9, wherein the average pore diameter of the surface layer B is 20 to 100 μm.

[11] The carrier according to any one of items 1 to 10, wherein the total film thickness of the polymer porous membrane is 5 to 500 μm.

[12] The carrier according to any one of items 1 to 11, wherein the polymer porous membrane is a three-layer polymer porous membrane having a plurality of pores on a surface layer A and a surface layer B, and a macrovoid layer sandwiched between the surface layer A and the surface layer B, wherein the average pore diameter of the pores in the surface layer A is smaller than the average pore diameter of the pores in the surface layer B, and the macrovoid layer has partitions bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partitions and the surface layers A and B, and the pores in the surface layers A and B communicate with the macrovoids.

[13] The carrier according to any one of items 1 to 12, wherein the polymer porous membrane is a polyimide porous membrane.

[14] The carrier according to item 13, wherein the polyimide porous membrane is a polyimide porous membrane comprising a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

[15] The carrier according to item 13 or 14, wherein the polyimide porous membrane is a colored polyimide porous membrane obtained by molding a polyamic acid solution composition comprising a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine and a coloring precursor, and then heat-treating it at 250°C or higher.

[16] The carrier according to any one of items 1 to 12, wherein the polymer porous membrane is a polyethersulfone (PES) porous membrane.

[0010]

[17] A method for manufacturing a carrier according to items 1 to 16, comprising a first step of forming an opening in the sealing member, a second step of laminating the sealing member, the spacer member, the protective member and the polymer porous membrane, and a third step of welding the sealing member, the spacer member, the protective member and the polymer porous membrane.

[18] A method for manufacturing a carrier according to items 1 to 16, comprising a first step of forming an opening in the sealing member and the opening in the protective member, a second step of laminating the sealing member, the protective member and the polymer porous membrane, and a third step of welding the sealing member, the protective member and the polymer porous membrane.

[19] A manufacturing method according to item 17 or item 18, performed in the order of the first step, the second step and the third step.

[20] A cell culture method using a carrier according to items 1 to 16, comprising culturing the carrier while stirring it in a state where it is suspended and / or settled in a culture medium.

[21] The method according to item 20, characterized in that a part of the carrier is cultured while being irregularly exposed to the gas phase.

[0011] This invention allows suspended cells to be easily adsorbed, making it possible to culture cells simply and stably.

[0012] A schematic diagram (top view) showing a cell culture carrier (1, 1a, 1c) of one embodiment is shown. A schematic diagram showing cell culture carrier 1 of one embodiment. A schematic diagram of the A-A section of Figure 1 is shown. A schematic diagram showing cell culture carrier 1 of one embodiment. A perspective view showing each component spread out vertically. A schematic diagram showing cell culture carrier 1a of one embodiment. A schematic diagram of the A-A section of Figure 1 is shown. A schematic diagram showing cell culture carrier 1a of one embodiment. A perspective view showing each component spread out vertically. A schematic diagram (top view) showing cell culture carrier 1b of one embodiment is shown. A schematic diagram showing cell culture carrier 1b of one embodiment is shown. A schematic diagram of the B-B section of Figure 4A is shown. A schematic diagram showing cell culture carrier 1b of one embodiment. A perspective view showing each component spread out vertically. A schematic diagram showing cell culture carrier 1c of one embodiment. A schematic diagram of the A-A section of Figure 1 is shown. This is a schematic diagram (top view) showing a cell culture carrier 1d of one embodiment. This is a schematic diagram showing a cell culture carrier 1d of one embodiment. This shows a schematic diagram of the D-D section in Figure 6A. This is a schematic diagram (top view) showing a cell culture carrier 1e of one embodiment. This is a schematic diagram showing a cell culture carrier 1e of one embodiment. This shows a schematic diagram of the E-E section in Figure 7A. This is a schematic diagram (top view) showing a cell culture carrier 1f of one embodiment. This is a schematic diagram showing a cell culture carrier 1f of one embodiment. This shows a schematic diagram of the F-F section in Figure 8A. This is a schematic diagram showing a cell culture carrier 1f of one embodiment. This is a perspective view showing each component spread out vertically. This is a schematic diagram (top view) showing a cell culture carrier 1g of one embodiment. This is a schematic diagram showing a cell culture carrier 1g of one embodiment. This shows a schematic diagram of the E-E section in Figure 9A. This is a schematic diagram (top view) showing a cell culture carrier 1h of one embodiment. This is a schematic diagram showing a cell culture carrier 1h of one embodiment. This shows a schematic diagram of the H-H cross-section of Figure 10A. This is a schematic diagram (top view) showing a cell culture carrier 1i of one embodiment. This is a schematic diagram showing a cell culture carrier 1i of one embodiment. This is a perspective view showing each component spread out vertically. This is a schematic diagram showing the manufacturing process of the cell culture carrier 1 of one embodiment.

[0013] An embodiment of the present invention will be described below with reference to the drawings, but the scope of the present invention is not limited to the embodiment described herein, and various modifications can be made without departing from the spirit of the invention. Furthermore, if multiple upper and lower limits are given for a particular parameter, any combination of these upper and lower limits can be used to create a suitable numerical range.

[0014] In this specification, terms such as "first," "second," "third," etc., are used to distinguish one element from other elements. For example, the first element may be referred to as the second element, and similarly, the second element may be referred to as the first element, and this will not depart from the scope of the present invention.

[0015] 1. Carriers for cell culture

[0016] Figures 1 and 2 (Figures 2A and 2B) are schematic diagrams representing each component constituting the cell culture carrier 1 of the present invention in one embodiment (Figure 1: top view, Figure 2A: schematic diagram showing the A-A cross-section in Figure 1, Figure 2B: schematic diagram showing each component spread out vertically (perspective view)). Figures 3 to 11 are schematic diagrams showing the cell culture carrier (1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i) of the present invention in other embodiments. Note that the size and positional relationships of each component shown in the schematic diagrams of the carrier (1, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i) shown in the figures of this specification are exaggerated for illustrative purposes, and their height, width, and depth, or their positional relationships, can be adjusted as appropriate for the purpose. In addition, the components shown in the drawings may be used in combination. Furthermore, unless otherwise specified, the descriptions of components with the same number can generally be applied to the descriptions of other drawings as well.

[0017] In this specification, "carrier for cell culture" (also simply referred to as "carrier"; however, in the examples and comparative examples, it will be referred to as "carrier-type polymer porous membrane" or simply "carrier") means a substrate for seeding and culturing cells, applicable to cell culture vessels, cell culture apparatuses and cell culture systems, and especially cell culture vessels, cell culture apparatuses and cell culture systems used for suspension culture.

[0018] In one embodiment, the cell culture carrier 1 comprises two or more polymer porous membranes 10, a spacer member 20 having one or more openings 200 provided between the two or more polymer porous membranes 10, and two protective members 30 having one or more openings 300 and sandwiching the two or more polymer porous membranes 10. The polymer porous membrane 10 that can be applied to the cell culture carrier 1 in one embodiment is a polymer porous membrane having a surface layer A and a surface layer B having a plurality of pores, wherein the average pore diameter of the pores in surface layer A is smaller than the average pore diameter of the pores in surface layer B. In one embodiment, the polymer porous membrane 10 is a three-layer polymer porous membrane having a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore diameter of the pores in the surface layer A is smaller than the average pore diameter of the pores in the surface layer B, and the macrovoid layer has a partition wall bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, and it is preferable that the pores in the surface layers A and B communicate with the macrovoids. In one embodiment, the peripheral portion of the polymer porous membrane 10 and the peripheral portion of the spacer member 20 are welded and fixed. In one embodiment, the peripheral portion of the polymer porous membrane 10 and the spacer member 20 may be welded and fixed by a sealing member 40. In another embodiment, the spacer member 20 may also serve as a sealing member. In this case, the spacer member 20 is directly welded and fixed to the peripheral portion of the polymer porous membrane 10. In another embodiment, the sealing member 40 may also function as a spacer member. In this case, the sealing member 40 is directly welded and fixed to the edge of the polymer porous membrane 10. In another embodiment, the sealing member 40 may also function as a protective member. In this case, the sealing member 40 is directly welded and fixed to the edge of the polymer porous membrane 10. In another embodiment, the protective member 30 may also function as a sealing member. In this case, the protective member 30 is directly welded and fixed to the edge of the polymer porous membrane 10.Any one of the protective member 30, spacer member 20, and sealing member 40 can perform the other two roles. Furthermore, in one embodiment of the cell culture carrier 1, the edges of the polymer porous membrane 10 and the edges of the protective member 30 are welded and fixed by the sealing member 40. This creates a space between two or more adjacent polymer porous membranes 10, with an average size of 10 μm to 1500 μm. This space may contain culture medium or suspended cells.

[0019] The distance between two or more polymer porous membranes 10 refers to the average distance between two points located perpendicular to the plane of the membrane when two or more adjacent polymer porous membranes 10 are stacked in substantially parallel positions. The distance between two or more polymer porous membranes 10 may basically be determined by the sum of the thickness of the spacer member 20 and the thickness of the sealing members 40 provided above and below the spacer member 20. The thicknesses of the spacer member 20 and the sealing members 40 can be adjusted as appropriate and are not limited.

[0020] In the cell culture carrier 1, the distance between two or more polymer porous membranes 10 may be an average of 10 μm to 1500 μm, for example, 15 μm to 1000 μm, 20 μm to 800 μm, or 50 to 650 μm. Preferably, the distance is in the range of 15 μm to 1000 μm, more preferably in the range of 20 μm to 800 μm, and more preferably in the range of 50 to 650 μm. By forming a space within the above range between two or more polymer porous membranes 10, culture medium and oxygen can be efficiently supplied to the cells supported by the polymer porous membranes 10, making it possible to efficiently culture a larger number of cells.

[0021] In another embodiment, as shown in Figure 6, in the cell culture carrier 1d, two or more polymer porous membranes 10d may be provided with polymer porous membrane openings 11d in the central portion. This allows the culture medium to flow easily into the cell culture carrier 1d, promoting the inflow / outflow of the culture medium to the cells supported inside the carrier 1d, and further promoting the supply of nutrients and oxygen. Furthermore, in yet another embodiment, as shown in Figure 7 or Figure 8, the cell culture carrier 1e or 1f may be provided with one or more polymer porous membrane openings 11e or 11f in the peripheral portion away from the center. This allows the culture medium to flow easily into the cell culture carrier 1e or 1f, promoting the inflow / outflow of the culture medium to the cells supported inside the carrier 1e or 1f. In addition, by having the center of gravity of the carrier 1e or 1f at the periphery, the carrier 1e or 1f will float irregularly when cultured in suspension, and it is expected that the culture medium will be supplied evenly to the supported cells.

[0022] In one embodiment, the spacer member 20 is provided with one or more openings 200. For example, as shown in Figures 2A and 2B, the spacer member 20 may have a mesh-like structure, in which case the openings 200 may be, for example, square (e.g., square or rectangle). In this specification, a mesh-like structure means, for example, one having a grid-like structure in vertical, horizontal, and / or diagonal directions. The mesh-like structure may be a structure in which rod-shaped structures are alternately woven in vertical, horizontal, and / or diagonal directions, or a structure in which the intersections of rod-shaped structures are joined together. Also, the openings 200 of the spacer member 20 may be substantially circular, triangular, pentagonal, hexagonal, heptagonal, octagonal, or more polygonal. For example, as shown in Figures 3A and 3B, the spacer member 20a may have one substantially circular opening 200a, or it may have one square opening. In another embodiment, as shown in Figure 10B, the spacer member 20h may be formed of a material that has the ability to allow the culture medium to pass through, for example, a nonwoven fabric.

[0023] In one embodiment, the protective member 30 is provided with one or more openings 300. For example, as shown in Figures 2A and 2B, the protective member 30 may be a mesh structure, in which case the openings 300 may be quadrilateral (for example, square or rectangular). The mesh structure may be a structure in which rod-shaped structures are alternately woven in vertical, horizontal, and / or diagonal directions, or a structure in which the intersections of rod-shaped structures are joined together. Furthermore, the openings 300 of the protective member 30 may be approximately circular, triangular, pentagonal, hexagonal, heptagonal, octagonal, or polygonal in shape. For example, as shown in Figure 4 (Figures 4A to C), the protective member 30b may have one approximately circular opening 300b, or it may have one quadrilateral opening. Furthermore, in one embodiment, the carrier 1f may be equipped with a protective member 30f having a plurality of fan-shaped openings (300f, 300f') of different shapes, as shown in Figure 8 (Figures 8A to C), or the carrier 1g may be equipped with a protective member 30g having four fan-shaped openings (300g) of the same shape, as shown in Figure 9 (Figures 9A to B). Furthermore, in one embodiment, the carrier 1h may be equipped with a protective member 30h having 20 or more (for example, 100 to 500, 150 to 300) openings 300h (for example, Figure 10). Furthermore, in one embodiment, the carrier 1f may be equipped with a spacer member 20f having the same shape as the protective member 30f. In this case, the protective member 30f and the spacer member 20f may be stacked so that the orientation of the openings is the same (for example, Figure 8C). In the case of a protective member 30f having multiple fan-shaped openings (300d, 301d) of different shapes, such as the carrier 1f, the center of gravity of the carrier 1f is located at the edge, so when the carrier 1f is cultured in suspension, it is expected that it will float irregularly and that the culture medium will be evenly supplied to the supported cells.

[0024] The spacer members (20, 20a, 20f, 20g, 20h) and / or protective members (30, 30b, 30b', 30f, 30g, 30h) preferably have sufficient strength to not deform under normal culture conditions, such as stirring culture or shaking culture conditions, due to the movement of the culture medium. Furthermore, the spacer members (20, 20a, 20f, 20g, 20h) and / or protective members (30, 30b, 30b', 30f, 30g, 30h) preferably have a material that does not affect cell growth in cell culture. Such materials may be one or more selected from the group consisting of polyolefins, polyethylene, polypropylene, polyvinyl chloride, ethylene vinyl acetate resin, polystyrene, polyvinyl alcohol, polyethylene terephthalate, polyamide, polycarbonate, ionomer, polyurethane, polybutadiene, polyacrylonitrile, polybutylene terephthalate, and polyethylene naphthalate. The spacer members (20, 20a, 20f, 20g, 20h) and / or protective members (30, 30b, 30b', 30f, 30g, 30h) may be planar structures having a mesh structure made of the above material, and may be nonwoven fabric, single-layer film, multi-layer film, or a combination thereof.

[0025] In one embodiment of a cell culture carrier 1, the edges of the polymer porous membrane 10 and the edges of the spacer member 20 are welded and fixed together by a sealing member 40. The sealing member 40 is provided with an opening 400 through which culture medium and cells can pass. In this specification, "edge" refers to the peripheral region of the outer circumference of each member, and may refer to a range of 50% or less of the distance from the outer circumference to the center of gravity, for example, a range of 1% to 50%, 5% to 40%, or 10% to 30%.

[0026] In one embodiment of the cell culture carrier 1, the edges of the polymer porous membrane 10 and the edges of the protective member are welded and fixed together by the sealing member 40.

[0027] The material of the sealing member 40 can be any material that can weld, preferably by heat welding, the polymer porous membrane 10 and the spacer member 20. Furthermore, the material of the sealing member 40 can be any material that can weld the polymer porous membrane 10 and the protective member 30, and is not limited to, but may include, for example, polypropylene, polyethylene, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene, polyamide, ethylene-vinyl acetate copolymer, etc. The structure of the sealing member 40 may be a single-layer film, a multilayer film formed by combining multiple materials selected from the above, or a nonwoven fabric formed by combining multiple materials selected from the above. By appropriately selecting the material of the sealing member 40, the buoyancy of the carrier 1 in the culture medium can be adjusted. Any two or three of the protective member 30, spacer member 20, and sealing member 40 may be made of the same material.

[0028] Because the edges of carrier 1 are welded with a sealing member, the polymer porous membrane itself can be kept in a state where it is suppressed from continuously undulating due to the fluid. The sealing member that welds around the polymer porous membrane acts as part of the casing, preventing stress from being applied to the cells growing within the polymer porous membrane, and enabling stable cell culture without cell death.

[0029] In the modularized polymer porous membrane disclosed in Patent Document 5, peeling of the polymer porous membrane may occur due to frictional force between the polymer porous membrane and the surfaces inside the casing. On the other hand, in the carrier provided by the present application, the polymer porous membrane is covered with a sealing member, making it difficult for frictional force to occur between the polymer porous membranes, thus making it possible to suppress peeling of the polymer porous membrane.

[0030] The shape of the carrier 1 for cell culture can take any form, such as a substantially circular shape, an elliptical shape, a quadrangular shape, a triangular shape, a polygonal shape, etc., but a substantially circular shape, an elliptical shape or a quadrangular shape is preferred. In the present invention, the size of the carrier 1 for cell culture can take any size, but when it is substantially circular, for example, the diameter may be 0.1 mm to 5.0 cm, preferably 0.1 cm to 4.0 cm, more preferably 0.5 cm to 2.0 cm.

[0031] In another aspect, as shown by the carrier 1c in FIG. 5, the polymer porous membrane 10 may be provided in a mode where three or more are provided. From the viewpoint of being able to evenly seed cells and evenly grow or cultivate them, the polymer porous membrane 10 of the carrier 1 may be two, three, four, five, six or a plurality of sheets. Preferably two, four or six sheets, more preferably two or four sheets, and even more preferably two sheets.

[0032] In another aspect, three elliptical carriers 1b' may be combined, and their central portions may be sandwiched with a circular film (for example, FIG. 11B) and provided as a carrier 1i having a propeller shape (for example, FIG. 11A) that is welded.

[0033] In one embodiment, when the carrier for cell culture of the present invention is applied to the culture of cells that are preferably cultured under aerobic conditions, it may be formed to have a density that floats in the culture medium, and when applied to the culture of cells that are preferably cultured under anaerobic conditions, it may be formed to have a density that sinks in the culture medium. For example, when floating in the culture medium, the density of the carrier for cell culture is preferably 0.99 g / cm ３ or less. In this case, the carrier may be constructed by combining the above-mentioned respective members so that the density of the carrier for cell culture becomes 0.99 g / cm ３ or less. For example, when sinking in the culture medium, the density of the carrier for cell culture is preferably 1.001 g / cm ３ or more. In this case, the carrier may be constructed by combining the above-mentioned respective members so that the density of the carrier for cell culture becomes 1.001 g / cm ３ or more.

[0034] 2. Polymer porous membrane The polymer porous membrane applicable to the present invention will be described below.

[0035] In one aspect, the average pore diameter of the pores present in the surface layer A (hereinafter also referred to as "surface A" or "mesh surface") in the polymer porous membrane used in the present invention is not particularly limited. For example, it is 0.01 μm or more and less than 200 μm, 0.01 to 150 μm, 0.01 to 100 μm, 0.01 to 50 μm, 0.01 μm to 40 μm, 0.01 μm to 30 μm, 0.01 μm to 20 μm, or 0.01 μm to 15 μm, and preferably 0.01 μm to 15 μm.

[0036] In one aspect, the average pore diameter of the pores present in the surface layer B (hereinafter also referred to as "surface B" or "large-hole surface") in the polymer porous membrane used in the present invention is not particularly limited as long as it is larger than the average pore diameter of the pores present in the surface layer A. For example, it is more than 5 μm and 200 μm or less, 20 μm to 100 μm, 30 μm to 100 μm, 40 μm to 100 μm, 50 μm to 100 μm, or 60 μm to 100 μm, and preferably 20 μm to 100 μm.

[0037] The average pore diameter of the polymer porous membrane surface can be determined by measuring the pore area for 200 or more apertures from a scanning electron micrograph of the porous membrane surface and calculating the average diameter when the pore shape is assumed to be a perfect circle according to the following formula (Equation 1) from the average value of the pore area. (In the formula, Sa means the average value of the pore area.)

[0038] The thicknesses of the surface layers A and B are not particularly limited. For example, they are 0.01 to 50 μm, and preferably 0.01 to 20 μm.

[0039] The average pore diameter in the film-plane direction of the macrovoids in the macrovoid layer of the polymer porous membrane is not particularly limited, but is, for example, 10 to 500 μm, preferably 10 to 100 μm, and more preferably 10 to 80 μm. The thickness of the partitions in the macrovoid layer is not particularly limited, but is, for example, 0.01 to 50 μm, and preferably 0.01 to 20 μm. In one embodiment, at least one partition in the macrovoid layer has one or more pores with an average pore diameter of 0.01 to 100 μm, preferably 0.01 to 50 μm, that connect adjacent macrovoids. In another embodiment, the partitions in the macrovoid layer do not have pores.

[0040] The total film thickness of the polymer porous membrane surface used in the present invention is not particularly limited, but may be 5 μm or more, 10 μm or more, 20 μm or more, or 25 μm or more, and may be 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less, or 50 μm or less. Preferably, it is 5 to 500 μm, and more preferably 25 to 75 μm.

[0041] The thickness of the polymer porous membrane used in this invention can be measured using a contact-type thickness gauge.

[0042] The porosity of the polymer porous membrane used in the present invention is not particularly limited, but for example, it is 40% or more and less than 95%.

[0043] The porosity of the polymer porous membrane used in the present invention can be determined by measuring the film thickness and mass of a porous film cut to a predetermined size, and then calculating the basis weight mass according to the following formula (Equation 2). (In the formula, S represents the area of ​​the porous film, d represents the total film thickness, w represents the measured mass, and D represents the density of the polymer. If the polymer is polyimide, the density is 1.34 g / cm³.) ３ (Let's assume that.)

[0044] The polymer porous membrane used in the present invention is preferably a polymer porous membrane having a three-layer structure comprising a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore diameter of the pores in the surface layer A is 0.01 μm to 15 μm, the average pore diameter of the pores in the surface layer B is 20 μm to 100 μm, the macrovoid layer has partitions bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partitions and the surface layers A and B, the thickness of the partitions in the macrovoid layer and the surface layers A and B is 0.01 to 20 μm, the pores in the surface layers A and B communicate with the macrovoids, the total film thickness is 5 to 500 μm, and the porosity is 40% or more and less than 95%. In one embodiment, at least one partition in the macrovoid layer has one or more pores with an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm, that connect adjacent macrovoids. In another embodiment, the partition does not have such pores.

[0045] The polymer porous membrane used in the present invention is preferably sterilized. The sterilization treatment is not particularly limited, but examples include dry heat sterilization, steam sterilization, sterilization with disinfectants such as ethanol, and any other sterilization treatment such as electromagnetic wave sterilization such as ultraviolet light or gamma rays.

[0046] The polymer porous membrane used in the present invention is not particularly limited as long as it has the structural characteristics described above, but is preferably a polyimide porous membrane or a polyethersulfone (PES) porous membrane.

[0047] 2-1. Polyimide Porous Membrane Polyimides are a general term for polymers that contain imide bonds in their repeating units, and usually refer to aromatic polyimides in which aromatic compounds are directly linked by imide bonds. Aromatic polyimides have a rigid and strong molecular structure because aromatic compounds have a conjugated structure via imide bonds, and because the imide bonds have strong intermolecular forces, they have very high levels of thermal, mechanical, and chemical properties.

[0048] The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane containing (as the main component) a polyimide obtained from tetracarboxylic dianhydride and a diamine, and more preferably a polyimide porous membrane consisting of a polyimide obtained from tetracarboxylic dianhydride and a diamine. "Contained as the main component" means that the polyimide porous membrane does not essentially contain any components other than the polyimide obtained from tetracarboxylic dianhydride and a diamine, or it may contain other components, but these are additional components that do not affect the properties of the polyimide obtained from tetracarboxylic dianhydride and a diamine.

[0049] In one embodiment, the polyimide porous membrane that can be used in the present invention also includes a colored polyimide porous membrane obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component and a coloring precursor, and then heat-treating it at 250°C or higher.

[0050] Polyamic acids are obtained by polymerizing a tetracarboxylic acid component and a diamine component. Polyamic acids are polyimide precursors that can be cyclized to polyimides by thermal or chemical imidation.

[0051] Polyamic acids can be used even if a portion of the amic acid is imidized, as long as this does not affect the present invention. In other words, polyamic acids may be partially thermally imidized or chemically imidized.

[0052] When thermally imidizing polyamic acid, fine particles such as imidation catalysts, organophosphorus-containing compounds, inorganic fine particles, and organic fine particles may be added to the polyamic acid solution as needed. Similarly, when chemically imidizing polyamic acid, fine particles such as chemical imidizing agents, dehydrating agents, inorganic fine particles, and organic fine particles may be added to the polyamic acid solution as needed. It is preferable to carry out the process under conditions that prevent the precipitation of coloring precursors when these components are added to the polyamic acid solution.

[0053] In this specification, "coloring precursor" means a precursor that is partially or completely carbonized by heat treatment at 250°C or higher to produce a colored product.

[0054] The coloring precursors that can be used in the production of the above-mentioned polyimide porous membrane are preferably those that can be uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution and then thermally decomposed and carbonized to produce a colored product by heat treatment at 250°C or higher, preferably 260°C or higher, more preferably 280°C or higher, more preferably 300°C or higher, preferably in the presence of oxygen such as air, and more preferably those that produce a black colored product, and more preferably carbon-based coloring precursors.

[0055] When heated, the colored precursors appear to be carbonides, but structurally they contain heteroatoms other than carbon, and include layered structures, aromatic cross-linked structures, and disordered structures containing tetrahedral carbon.

[0056] The carbon-based coloring precursor is not particularly limited and includes, for example, tar or pitch such as petroleum tar, petroleum pitch, coal tar, and coal pitch, coke, polymers obtained from monomers containing acrylonitrile, and ferrocene compounds (ferrocene and ferrocene derivatives). Among these, polymers and / or ferrocene compounds obtained from monomers containing acrylonitrile are preferred, and polyacrylonitrile is preferred as the polymer obtained from monomers containing acrylonitrile.

[0057] Furthermore, in another embodiment, the polyimide porous membrane that can be used in the present invention also includes a polyimide porous membrane obtained by molding a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component, and then heat-treating it, without using the above-mentioned coloring precursor.

[0058] A porous polyimide film produced without using a coloring precursor may be manufactured, for example, by casting a polyamic acid solution consisting of 3 to 60% by mass of polyamic acid having an intrinsic viscosity number of 1.0 to 3.0 and 40 to 97% by mass of an organic polar solvent into a film, immersing or contacting it with a solidification solvent in which water is an essential component to produce a porous polyamic acid film, and then heat-treating the porous polyamic acid film to imide it. In this method, the solidification solvent in which water is an essential component may be water, or a mixture of 5% by mass or more and less than 100% by mass of water and more than 0% by mass and 95% by mass or less of an organic polar solvent. Furthermore, after the imide iteration, at least one side of the obtained porous polyimide film may be subjected to plasma treatment.

[0059] In the production of the above-mentioned porous polyimide membrane, any tetracarboxylic dianhydride can be used and can be appropriately selected according to the desired properties. Specific examples of tetracarboxylic dianhydrides include pyromellitic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA), 2,3,3',4'-biphenyltetracarboxylic dianhydride (a-BPDA), oxydiphthalic dianhydride, diphenylsulfone-3,4,3',4'-tetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl) sulfide dianhydride, 2,2-bis(3,4-dicarboxyphenyl)-1,1,1,3,3,3-hexafluoropropane dianhydride, 2,3,3',4'-benzophenonetetracarboxylic dianhydride, 3,3',4,4'-benzophenonetetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)methane dianhydride, and 2,2-bis(3,4-dicarboxyphenyl)propane Examples include dianhydrides, p-phenylenebis(trimellitic acid monoester anhydride), p-biphenylenebis(trimellitic acid monoester anhydride), m-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, 1,3-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)biphenyl dianhydride, 2,2-bis[(3,4-dicarboxyphenoxy)phenyl]propane dianhydride, 2,3,6,7-naphthalenetetracarboxylic acid dianhydride, 1,4,5,8-naphthalenetetracarboxylic acid dianhydride, and 4,4'-(2,2-hexafluoroisopropylidene)diphthalic acid dianhydride. Furthermore, it is also preferable to use aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfonetetracarboxylic acid. These can be used individually or in combination of two or more.

[0060] Among these, at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydrides and pyromellitic dianhydrides is particularly preferred. As the biphenyltetracarboxylic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride can be suitably used.

[0061] Any diamine can be used in the production of the above-mentioned polyimide porous membrane. Specific examples of diamines include: 1) Benzene diamines with one benzene ring, such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, and 2,6-diaminotoluene;2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenylmethane, 3,3'-dimethyl-4,4'-diaminobiphenyl, 2,2'-dimethyl-4,4'-diaminobiphenyl, 2,2'-bis(trifluoromethyl)-4,4'-diaminobiphenyl, 3,3'-dimethyl-4,4'-diaminodiphenylmethane, 3,3'-dicarboxy-4,4'-diaminodiphenylmethane, 3 ,3',5,5'-tetramethyl-4,4'-diaminodiphenylmethane, bis(4-aminophenyl) sulfide, 4,4'-diaminobenzanilide, 3,3'-dichlorobenzidine, 3,3'-dimethylbenzidine, 2,2'-dimethylbenzidine, 3,3'-dimethoxybenzidine, 2,2'-dimethoxybenzidine, 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, 3,3'-diaminodiphenyl Sulfide, 3,4'-diaminodiphenyl sulfide, 4,4'-diaminodiphenyl sulfide, 3,3'-diaminodiphenyl sulfone, 3,4'-diaminodiphenyl sulfone, 4,4'-diaminodiphenyl sulfone, 3,3'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-di Diamines with two benzene rings, such as aminodiphenylmethane, 2,2-bis(3-aminophenyl)propane, 2,2-bis(4-aminophenyl)propane, 2,2-bis(3-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 2,2-bis(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 3,3'-diaminodiphenyl sulfoxide, 3,4'-diaminodiphenyl sulfoxide, and 4,4'-diaminodiphenyl sulfoxide;3) 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, 1,4-bis(3-aminophenoxy)benzene, 1,4-bis(4-aminophenoxy)benzene, 1,3-bis(3-aminophenoxy)-4-trifluoromethylbenzene, 3,3'-diamino-4-(4-phenyl)phenoxybenzophenone, 3,3'-diamino-4,4'-di(4-phenylphenoxy)benzophenone, 1,3 Benzene-nuclear diamines such as bis(3-aminophenylsulfide)benzene, 1,3-bis(4-aminophenylsulfide)benzene, 1,4-bis(4-aminophenylsulfide)benzene, 1,3-bis(3-aminophenylsulfone)benzene, 1,3-bis(4-aminophenylsulfone)benzene, 1,4-bis(4-aminophenylsulfone)benzene, 1,3-bis[2-(4-aminophenyl)isopropyl]benzene, 1,4-bis[2-(3-aminophenyl)isopropyl]benzene, and 1,4-bis[2-(4-aminophenyl)isopropyl]benzene;4) 3,3'-bis(3-aminophenoxy)biphenyl, 3,3'-bis(4-aminophenoxy)biphenyl, 4,4'-bis(3-aminophenoxy)biphenyl, 4,4'-bis(4-aminophenoxy)biphenyl, bis[3-(3-aminophenoxy)phenyl] ether, bis[3-(4-aminophenoxy)phenyl] ether, bis[4-(3-aminophenoxy)phenyl] ether, bis[4-(4-aminophenoxy)phenyl] ether, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(4-A Minophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(4-aminophenoxy)phenyl]ketone, bis[3-(3-aminophenoxy)phenyl]sulfide, bis[3-(4-aminophenoxy)phenyl]sulfide, bis[4-(3-aminophenoxy)phenyl]sulfide, bis[4-(4-aminophenoxy)phenyl]sulfide, bis[3-(3-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone [nophenoxy)phenyl]sulfone, bis[4-(4-aminophenoxy)phenyl]sulfone, bis[3-(3-aminophenoxy)phenyl]methane, bis[3-(4-aminophenoxy)phenyl]methane, bis[4-(3-aminophenoxy)phenyl]methane, bis[4-(4-aminophenoxy)phenyl]methane, 2,2-bis[3-(3-aminophenoxy)phenyl]propane, 2,2-bis[3-(4-aminophenoxy)phenyl]propane, 2,2- Diamines with four benzene rings, such as bis[4-(4-aminophenoxy)phenyl]propane, 2,2-bis[3-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[3-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[4-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, and 2,2-bis[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane.

[0062] These can be used individually or in mixtures of two or more. The diamines used can be appropriately selected according to the desired properties.

[0063] Among these, aromatic diamine compounds are preferred, and 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether and paraphenylenediamine, 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, and 1,4-bis(3-aminophenoxy)benzene can be suitably used. In particular, at least one diamine selected from the group consisting of benzenediamine, diaminodiphenyl ether and bis(aminophenoxy)phenyl is preferred.

[0064] The porous polyimide membrane that can be used in the present invention is preferably formed from a polyimide obtained by combining a tetracarboxylic dianhydride and a diamine, which has a glass transition temperature of 240°C or higher, or 300°C or higher and no clear transition point, from the viewpoint of heat resistance and dimensional stability at high temperatures.

[0065] The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane made of the following aromatic polyimides from the viewpoint of heat resistance and dimensional stability at high temperatures: (i) an aromatic polyimide made of at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units and an aromatic diamine unit; (ii) an aromatic polyimide made of a tetracarboxylic acid unit and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units and bis(aminophenoxy)phenyl units; and / or (iii) an aromatic polyimide made of at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units and bis(aminophenoxy)phenyl units.

[0066] The polyimide porous membrane used in the present invention is preferably a three-layer polyimide porous membrane having a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore diameter of the pores in the surface layer A is 0.01 μm to 15 μm, the average pore diameter of the pores in the surface layer B is 20 μm to 100 μm, the macrovoid layer has partitions bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partitions and the surface layers A and B, the thickness of the partitions in the macrovoid layer and the surface layers A and B is 0.01 to 20 μm, the pores in the surface layers A and B communicate with the macrovoids, the total film thickness is 5 to 500 μm, and the porosity is 40% or more and less than 95%. Here, at least one partition in the macrovoid layer has one or more pores with an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm, that connect adjacent macrovoids.

[0067] For example, the polyimide porous membranes described in International Publication No. 2010 / 038873, Japanese Patent Publication No. 2011-219585, or Japanese Patent Publication No. 2011-219586 can also be used in the present invention.

[0068] 2-2. Polyethersulfone (PES) Porous Membranes The PES porous membranes that can be used in the present invention contain polyethersulfone and are typically substantially composed of polyethersulfone. Polyethersulfone may be synthesized by methods known to those skilled in the art, for example, by polycondensation reaction of a divalent phenol, an alkali metal compound and a dihalogenodiphenyl compound in an organic polar solvent, or by pre-synthesizing an alkali metal disal of a divalent phenol and polycondensation reaction with a dihalogenodiphenyl compound in an organic polar solvent.

[0069] Examples of alkali metal compounds include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides, and alkali metal alkoxides. Sodium carbonate and potassium carbonate are particularly preferred.

[0070] Examples of divalent phenol compounds include hydroquinone, catechol, resorcinol, 4,4'-biphenol, bis(hydroxyphenyl)alkanes (e.g., 2,2-bis(hydroxyphenyl)propane and 2,2-bis(hydroxyphenyl)methane), dihydroxydiphenyl sulfones, dihydroxydiphenyl ethers, or compounds in which at least one hydrogen atom of the benzene ring is substituted with a lower alkyl group such as a methyl group, ethyl group, or propyl group, or a lower alkoxy group such as a methoxy group or ethoxy group. Two or more of the above compounds can be used as divalent phenol compounds.

[0071] Polyethersulfone may be a commercially available product. Examples of commercially available products include Sumika Excel 7600P (manufactured by Sumitomo Chemical Co., Ltd.) and Sumika Excel 5900P (manufactured by Sumitomo Chemical Co., Ltd.).

[0072] The logarithmic viscosity of the polyethersulfone is preferably 0.5 or higher, more preferably 0.55 or higher, from the viewpoint of good formation of macrovoids in the porous polyethersulfone film, and preferably 1.0 or lower, more preferably 0.9 or lower, even more preferably 0.8 or lower, and particularly preferably 0.75 or lower, from the viewpoint of ease of manufacturing the porous polyethersulfone film.

[0073] Furthermore, from the viewpoint of heat resistance and dimensional stability at high temperatures, it is preferable that the PES porous membrane, or polyethersulfone as a raw material thereof, has a glass transition temperature of 200°C or higher, or that no clear glass transition temperature is observed.

[0074] The method for producing a PES porous membrane that can be used in the present invention is not particularly limited, but for example, it may include the steps of: casting a polyethersulfone solution containing 0.3% to 60% by mass of polyethersulfone having a logarithmic viscosity of 0.5 to 1.0 and 40% to 99.7% by mass of an organic polar solvent into a film shape, immersing or contacting it with a solidification solvent having polyethersulfone as a poor solvent or non-solvent as an essential component to produce a solidified film having pores; and heat-treating the solidified film having pores obtained in the above step to coarseen the pores to obtain a PES porous membrane, wherein the heat treatment includes raising the temperature of the solidified film having pores to above the glass transition temperature of the polyethersulfone, or to 240°C or higher.

[0075] The PES porous membrane that can be used in the present invention is preferably a PES porous membrane having a surface layer A, a surface layer B, and a macrovoid layer sandwiched between surface layer A and surface layer B, wherein the macrovoid layer has partitions bonded to surface layers A and B, and a plurality of macrovoids surrounded by the partitions and surface layers A and B, the average pore diameter in the direction of the membrane plane being 10 μm to 500 μm, the partitions of the macrovoid layer having a thickness of 0.1 μm to 50 μm, and surface layers A and B each having a thickness of 0.1 μm to The PES porous membrane is 50 μm thick, and of the surface layers A and B, one has multiple pores with an average pore diameter of more than 5 μm and 200 μm or less, and the other has multiple pores with an average pore diameter of 0.01 μm or more and less than 200 μm, the surface opening ratio of one of the surface layers A and B is 15% or more, and the surface opening ratio of the other surface layer is 10% or more, the pores of the surface layers A and B communicate with the macrovoids, and the PES porous membrane has a total thickness of 5 μm to 500 μm and a porosity of 50% to 95%.

[0076] <Method for Manufacturing a Cell Culture Carrier> Figure 6 is a schematic diagram showing a method for manufacturing a cell culture carrier 1 in one embodiment. The following description is an example of the manufacturing process for the carrier 1 and is not limited thereto. For example, in one embodiment, the present invention provides a method for manufacturing the carrier described above, comprising: a first step of forming an opening in the sealing member; a second step of laminating the sealing member, the spacer member, the protective member and the polymer porous membrane; and a third step of welding the sealing member, the spacer member, the protective member and the polymer porous membrane. In another embodiment, the present invention provides a method for manufacturing the carrier described above, comprising: a first step of forming an opening in the sealing member and an opening in the protective member; a second step of laminating the sealing member, the protective member and the polymer porous membrane; and a third step of welding the sealing member, the protective member and the polymer porous membrane. Furthermore, the method may also include a step of cutting the carrier into an arbitrary shape. Each step is described below.

[0077] (Process for forming the opening of the sealing member (first step)) An opening of any size is formed in the sealing member forming layer 401 (Figure 6(A)). The opening formed here becomes the opening 400 of the sealing member 40. For example, in the embodiment to be described later, multiple openings with a diameter of 6 mm are formed in the sealing member forming layer 401.

[0078] (Lamination process (second process)) (1) protective member forming layer 301, (2) sealing member forming layer 401, (3) polymer porous film forming layer 100, (4) sealing member forming layer 401, (5) spacer member forming layer 201, (6) sealing member forming layer 401, (7) polymer porous film forming layer 100, (8) sealing member forming layer 401, and (9) protective member forming layer 301 are laminated in the order of (1) → (9) from the bottom layer to the top layer (Figure 6 (B)).

[0079] (Welding process (third step)) The laminated members are welded to a hot press device, such as a press molding device or a hot press device, at a temperature of 100°C to 260°C and a load of 0 to 100 kN / m. ２ The components and sealing members are then heat-welded together (Figure 6(C)). Here, heat welding is used as the means of welding, but depending on the type of component, other welding processes such as ultrasonic welding, vibration welding, induction welding, high-frequency welding, semiconductor laser / diode laser welding, and spin welding may also be used.

[0080] (Cutting process (fourth step)) The heat-welded members are cut into any shape to form the carrier 1. For example, the carrier 1 is obtained by cutting to a size larger than the opening 400 formed in the seal member forming layer. For example, in the embodiment described later, the carrier 1 was cut to a diameter of 8 mm. Cutting may be done using any cutting machine, for example, by cutting with a pinnacle die.

[0081] <Method for cell culture using a carrier> In one embodiment, the present invention provides a method for cell culture using the above-mentioned carrier, comprising culturing the carrier on which cells have been seeded while stirring it in a state in which it is suspended and / or settled in a culture medium.

[0082] In another embodiment, the cell culture method described above is also provided, in which a portion of the carrier is cultured while being intermittently exposed to the gas phase.

[0083] The types of cells that can be used in the present invention are selected from the group consisting of, for example, animal cells, insect cells, plant cells, yeasts, and bacteria. Animal cells are broadly classified into cells derived from animals belonging to the phylum Chordata and cells derived from invertebrates (animals other than those belonging to the phylum Chordata). The origin of animal cells in this specification is not particularly limited. Preferably, it means cells derived from animals belonging to the phylum Chordata. The phylum Chordata includes the superclass Agnatha and the superclass Gnathostomata, and the superclass Gnathostomata includes the classes Mammalia, Aves, Amphibia, Reptilia, etc. Preferably, it is cells derived from animals belonging to the class Mammalia, generally referred to as mammals. Mammals are not particularly limited, but preferably include mice, rats, humans, monkeys, pigs, dogs, sheep, goats, etc.

[0084] The types of animal cells that can be used in the present invention are not limited, but are preferably selected from the group consisting of pluripotent stem cells, tissue stem cells, somatic cells, and germ cells.

[0085] In this specification, "pluripotent stem cells" is intended to be a general term for stem cells that have the ability to differentiate into cells of any tissue (pluripotency). While not limited to these, pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (EG cells), germ cells (GS cells), etc. Preferably, ES cells or iPS cells are used. iPS cells are particularly preferred for reasons such as the absence of ethical issues. Any known pluripotent stem cells can be used, but for example, the pluripotent stem cells described in International Publication No. 2009 / 123349 (PCT / JP2009 / 057041) can be used.

[0086] "Tissue stem cells" refer to stem cells whose differentiation is limited to a specific tissue lineage, but which possess the ability to differentiate into a variety of cell types (pluripotency). For example, hematopoietic stem cells in the bone marrow become the basis of blood cells, and neural stem cells differentiate into nerve cells. There are also various other types, such as hepatic stem cells that form the liver and skin stem cells that become skin tissue. Preferably, tissue stem cells are selected from mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, skin stem cells, or hematopoietic stem cells.

[0087] "Somatic cells" refer to cells other than germ cells that make up a multicellular organism. In sexual reproduction, they are not passed on to the next generation. Preferably, somatic cells are selected from hepatocytes, pancreatic cells, muscle cells, osteocytes, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, kidney cells, lung cells, or blood cells such as lymphocytes, erythrocytes, leukocytes, monocytes, macrophages, or megakaryocytes.

[0088] "Germ cells" refer to cells that play a role in reproduction by transmitting genetic information to the next generation. This includes, for example, gametes for sexual reproduction, namely eggs, oocytes, sperm, spermatophores, and spores for asexual reproduction.

[0089] The cells may be selected from a group consisting of sarcoma cells, cell lines, and transformed cells. "Sarcoma" refers to cancer that arises in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, and includes soft tissue sarcomas and malignant bone tumors. Sarcoma cells are cells derived from sarcomas. "Cell lines" refer to cultured cells that have been maintained outside the body for a long period of time, have acquired certain stable properties, and are capable of semi-permanent subculturing. There are various cell lines derived from various tissues of various organisms, including humans, such as PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human fetal kidney), HL-60 cells (derived from human leukocytes), HeLa cells (derived from human cervical cancer), Vero cells (derived from African green monkey kidney epithelial cells), MDCK cells (derived from canine kidney tubular epithelial cells), HepG2 cells (human liver cancer cell line), BHK cells (neonatal hamster kidney cells), and NIH3T3 cells (derived from mouse fetal fibroblasts). "Transformed cells" refer to cells whose genetic properties have been altered by introducing nucleic acids (DNA, etc.) from outside the cell.

[0090] In this specification, “adherent cells” generally refers to cells that need to adhere themselves to a suitable surface for proliferation, and are also called adherent cells or scaffold-dependent cells. In some embodiments of the present invention, the cells used are adherent cells. The cells used in the present invention are adherent cells, and more preferably, cells that can be cultured in a suspended state in a culture medium. Adherent cells that can be cultured in suspension can be obtained by acclimatizing adherent cells to a state suitable for suspension culture by known methods, and examples include CHO cells, HEK293 cells, Vero cells, NIH3T3 cells, and cell lines derived from these cells. Even if not listed herein, the cells used in the present invention are not particularly limited as long as they are adherent cells that can be cultured in suspension by acclimatization.

[0091] <Use of Carrier in Cell Culture Apparatus> The cell culture carrier of the present invention can be used with commercially available cell culture apparatus and systems. For example, it can be used with cell culture apparatuses where the culture container consists of a flexible bag, and can be used while suspended in the culture container. Furthermore, the cell culture carrier of the present invention can be used for culture in agitated culture vessels such as spinner flasks. In addition, it can be used with both open and closed culture vessels. For example, it can be used with a range of vessels from petri dishes, flasks, plastic bags, and test tubes to large tanks. Examples include cell culture dishes from BD Falcon and Nunc® Cell Factory from Thermo Scientific.

[0092] The present invention will be described more specifically below based on examples. However, the present invention is not limited to these examples. Those skilled in the art can easily modify and change the present invention based on the description herein, and such modifications fall within the technical scope of the present invention.

[0093] The polyimide porous membrane used as a polymer porous membrane in the following examples was prepared by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component, 3,3',4,4'-biphenyltetracarboxylic acid dianhydride (s-BPDA), and a diamine component, 4,4'-diaminodiphenyl ether (ODA), and a coloring precursor, polyacrylamide, and then heat-treating it at 250°C or higher. The obtained polyimide porous membrane was a three-layer polyimide porous membrane having a surface layer A and a surface layer B having multiple pores, and a macrovoid layer sandwiched between surface layers A and B. The average pore diameter of the pores in surface layer A was 6 μm, the average pore diameter of the pores in surface layer B was 46 μm, the film thickness was 25 μm, and the porosity was 73%.

[0094] <Configuration of "Carrier-type polymer porous membrane" and "Modular polymer porous membrane" prepared in the Examples and Comparative Examples> The carriers for cell culture were prepared as follows. In the Examples and Comparative Examples, the spacer member and the protective member were both made of the same material mesh.

[0095] The following polyimide porous membranes and nonwoven fabrics were used as polymer porous membranes: • Polyimide porous membrane (X): A polyimide porous membrane with a diameter of 0.8 cm. • Polyimide porous membrane (X2): A polyimide porous membrane with a diameter of 3.0 cm. • Laminated polyimide porous membrane (W): Four 1.0 × 1.0 cm polyimide porous membranes were laminated in the order of A side / B side / / A side / B side / / A side / B side / / B side / A side / / B side / A side / / B side / A side. • Nonwoven fabric (M): A core-sheath type composite fiber nonwoven fabric (polyethylene / polypropylene) with a diameter of 0.8 cm (made using raw cotton manufactured by Ube Eximo Co., Ltd.).

[0096] The following meshes were used as spacer members or protective members: ・Mesh (Y1): Spacer member (polypropylene) processed to an outer diameter of 0.8 cm and an inner diameter of 0.6 cm (manufactured by NBC Mesh Tech, part number: ESP10TC) ・Mesh (Y2): Spacer member (polypropylene) with an outer diameter of 0.8 cm (manufactured by NBC Mesh Tech, part number: ESP10TC) ・Mesh (Y3): Spacer member (polyamide) with an outer diameter of 0.8 cm (manufactured by Tokyo Screen, part number: NB20) was used. ・Mesh (Y4): Spacer member (polypropylene) of 1.0 × 1.0 cm (manufactured by NBC Mesh Tech, part number: ESP10TC) was used. ・Mesh (Y5): Spacer member (polypropylene) with an outer diameter of 0.8 cm (manufactured by Kureba, part number: #10) The following washers were used as spacer members or protective members. - Washer (Y6): A spacer member (polypropylene) machined to have an outer diameter of 3.0 cm and an inner diameter of 2.6 cm. The spacer member may also function as a sealing member (Z1). Alternatively, the sealing member (Z1) may also function as a spacer member. Alternatively, the sealing member (Z1) may also function as a protective member. Alternatively, the protective member may also function as a sealing member (Z1).

[0097] The following sealing components were used: • Sealing component (Z1): A sealing component (polypropylene / polyethylene) processed to have an outer diameter of 0.8 cm and an inner diameter of 0.6 cm (manufactured by Shimojima Co., Ltd., product number: HEIKO® Sweet Pack Kamasu MB) • Sealing component (Z2): A sealing component (nylon / polyethylene) processed to have an outer diameter of 0.8 cmΦ and an inner diameter of 0.6 cmΦ (manufactured by Fukusuke Kogyo Co., Ltd., product number: Nylon Poly New L Type NO. 3B4) • Washer (Y6): A component (polypropylene) processed to have an outer diameter of 3.0 cm and an inner diameter of 2.6 cm

[0098] (Example 1) A carrier-type polymer porous membrane (hereinafter also referred to as "carrier 1") was obtained by alternately stacking the following polyimide porous membrane (X), mesh (Y1), and sealing member (Z1) in the order Y1, Z1, X, Z1, Y1, Z1, X, Z1, Y1, then heat welding them together and sterilizing them with gamma rays.

[0099] (Example 2) The carrier-type polymer porous membrane (hereinafter also referred to as "carrier 2") used a polyimide porous membrane (X), a mesh (Y2), and a sealing member (Z1). The members were alternately stacked in the order Y2, Z1, X, Z1, Y2, Z1, X, Z1, Y2, heat-sealed, and then sterilized with gamma rays to obtain the membrane.

[0100] (Example 3) A carrier-type polymer porous membrane (hereinafter also referred to as "carrier 3") was prepared by laminating a polyimide porous membrane (X) and a sealing member (Z2) in the order Z2XZ2 and heat welding them to create a heat-welded body (Z2XZ2). Then, the mesh (Y3) described below and the heat-welded body (Z2XZ2) were alternately laminated in the order Y3, Z2, X, Z2, Y3, Z2, X, Z2, Y3 and heat-welded, and then sterilized with gamma rays to obtain carrier 3.

[0101] (Example 4) A carrier-type polymer porous membrane (hereinafter also referred to as "carrier 4") was obtained by alternately stacking a polyimide porous membrane (X) and a sealing member (Z1) in the order Z1, X, Z1, X, Z1, heat welding, and then sterilizing with gamma rays. In this example, the sealing member (Z1) also functions as a spacer member and a protective member.

[0102] (Example 5) A carrier-type polymer porous membrane (hereinafter also referred to as "carrier 5") was obtained by alternately stacking polyimide porous membranes (X), mesh (Y1), and sealing members (Z1) in the order Y1, Z1, X, Z1, X, Z1, Y1, heat welding, and then sterilizing with gamma rays. In this example, the sealing members (Z1) provided between the polymer porous membranes (X) also function as spacer members.

[0103] (Example 6) A carrier-type polymer porous membrane (hereinafter also referred to as "carrier 6") was obtained by alternately stacking a polyimide porous membrane (X), a mesh (Y1), and a sealing member (Z1) in the order Y1, Z1, X, Z1, X, Z1, Y1, Z1, X, Z1, Y1, Z1, X, Z1, Y1, then heat-sealing them together and sterilizing them with gamma rays.

[0104] (Example 7) A carrier-type polymer porous membrane (hereinafter also referred to as "carrier 7") was obtained by alternately stacking polyimide porous membranes (X), mesh (Y1), and sealing members (Z1) in the order Y1, Z1, X, Z1, X, Z1, X, Z1, X, Z1, Y1, heat welding, and then sterilizing with gamma rays. In this example, the sealing members (Z1) provided between the polymer porous membranes (X) also function as spacer members.

[0105] (Example 8) A carrier-type polymer porous membrane (hereinafter also referred to as "carrier 8") was obtained by alternately laminating a polyimide porous membrane (X) and a nonwoven fabric (M) in the order of M, X, M, X, M, then heat-sealing them together and sterilizing them with gamma rays.

[0106] (Example 9) The carrier-type polymer porous membrane (hereinafter also referred to as "carrier 9") used a polyimide porous membrane (X), a mesh (Y5), and a sealing member (Z1). The members were alternately stacked in the order Y5, Z1, X, Z1, Y5, Z1, X, Z1, Y5, heat-sealed, and then sterilized with gamma rays to obtain the membrane.

[0107] (Example 10) For the carrier-type polymer porous membrane (hereinafter also referred to as "carrier 9"), a polyimide porous membrane (X2) and a washer (Y6) were used. Each member was alternately laminated in the order of Y6, X2, Y6, X2, Y6, and after heat welding, it was obtained by sterilization with gamma rays. In this example, the washer (Y6) also functions as a sealing member.

[0108] (Comparative Example 1) For the carrier-type polymer porous membrane (hereinafter also referred to as "carrier C1"), a polyimide porous membrane (X) and the mesh (Y2) described below were alternately laminated in the order of Y2, X, Y2, X, Y2, and after heat welding, it was obtained by sterilization with gamma rays.

[0109] (Comparative Example 2) For the carrier-type polymer porous membrane (hereinafter also referred to as "carrier C2"), a polyimide porous membrane (X) and a mesh (Y1) were alternately laminated in the order of Y1, X, Y1, X, Y1, heat welded, and then obtained by sterilization with gamma rays.

[0110] (Comparative Example 3) The modularized polymer porous membrane (hereinafter also referred to as "module C1") was obtained by alternately laminating a polyimide porous membrane (W) and a mesh (Y4) in the order of W, Y4, W, Y4, W, Y4, W, Y4, W, and then accommodating the laminate in a polyethylene casing having 16 2 mm × 2 mm medium outflow inlets on one side, and then sterilizing with gamma rays.

[0111] (Comparative Example 4) For the carrier-type polymer porous membrane (hereinafter also referred to as "carrier C3"), a polyimide porous membrane (X), a mesh (Y1), and a sealing member (Z1) were alternately laminated in the order of Z1, X, Z1, Y1, Z1, X, Z1, heat welded, and then obtained by sterilization with gamma rays.

[0112] The densities of the carriers in Examples 1, 2, 4, 5, 6, 7, 8, 9, 10, and Comparative Examples 1, 3, and 4 were adjusted to be 0.99 g / cm ３ as follows and prepared to float during culture. The densities of the carriers in Example 3 and Comparative Example 2 were adjusted to be 1.001 g / cm ３ or more and prepared to sediment during culture.

[0113] Table 1 shows the composition of the examples and comparative examples. The following abbreviations are used: PP for polypropylene, PE for polyethylene, PPI for polyimide porous membrane, and PA for polyamide.

[0114] <Test 1: Confirmation of peeling of polymer porous film>

[0115] <Experimental Materials and Methods> (1) Shaking Carrier-type and Modular Polymer Porous Membranes 20 mL of Ham's F-12 (containing L-glutamine and phenol red) (product number: 087-08335, Fujifilm Wako Pure Chemical Industries) was added to an Erlenmeyer flask (Nalgene Single-Use PETG Erlenmeyer Flasks with Baffled Bottom, product number: 4116-0125, Thermo Scientific).

[0116] Add 20 carrier-type polymer porous membranes each from Example 2, Example 3, Example 4, Comparative Example 1, and Comparative Example 2, and 1 modular polymer porous membrane from Comparative Example 3 to an Erlenmeyer flask, and heat at 37°C and 5% CO2. ２ The device was shaken at 110 rpm for 7 days in the presence of the substance.

[0117] (2) Confirmation of polymer porous membrane peeling: The entire amount of the culture medium was collected, and after centrifugation at 300 g for 3 minutes, the medium was collected to obtain peeled polymer porous membrane material. The results of visual confirmation of the presence or absence of peeled material are shown in Table 2.

[0118] In Examples 2, 3, 4, and 8, the edges of the polymer porous membrane and the edges of the spacer member, as well as the edges of the polymer porous membrane and the edges of the protective member, are welded and fixed together by the sealing member. As a result, it was found that even when polymer porous materials come into contact with each other, or with the polymer porous membrane and the culture vessel during suspension culture while shaking, the polymer porous membrane is not destroyed, and no fragments are released into the culture medium.

[0119] On the other hand, in Comparative Examples 1, 2, and 3, it was found that the polymer porous membrane was destroyed during shaking, and numerous fragments were released into the culture medium.

[0120] <Experiment 2: Culture of Human Fibroblast Cell Lines> <Experimental Materials and Methods> (1) Expanded Culture of Human Fibroblasts 10 mL of FGM-2 Fibroblast Growth Medium-2 Bullet Kit (product number: CC-3132, LONZA) was added to a Falcon® Dish (untreated, Corning), and the seeding density was 3,500 cells / cm². ２ Human fibroblasts were seeded and the temperature was set to 37°C and 5% CO2. ２ Static culture was performed for 7 days in the presence of [the substance].

[0121] (2) Evaluation of cell adhesion and proliferation rate to carrier-type polymer porous membrane Add 3 mL of culture medium and 20 carriers to a flat-bottom tube (Tube 30 ml, product number: 62.555.001, SARSTEDT) and add 4.0 × 10⁶ human fibroblasts. ５ Sow the seeds individually and maintain a temperature of 37°C and 5% CO2. ２ Static culture was performed for 1 day in the presence of [the substance].

[0122] After one day of incubation, the carrier was placed in an Erlenmeyer flask (Nalgene Single-Use PETG Erlenmeyer Flasks with Baffled Bottom, product number: 4116-0125, Thermo Scientific) and 25 mL of culture medium was added. 37°C, 5% CO2 ２ The culture was performed for 6 days at 80 rpm in a shaker-equipped incubator under normal conditions, with the culture medium being completely replaced twice a week.

[0123] The number of cells in the carrier was measured using CCK8 on days 1 and 7 of culture.

[0124] <Evaluation of Cell Adhesion and Proliferation Rate> Cell adhesion and proliferation rates were calculated using the following formulas.

[0125] Cell adhesion rate = (CCK8 equivalent cell count / seeded cell count (4.0 × 10) ５ )) × 100

[0126] The cell adhesion rate in Example 2 is expressed as a relative value based on that value.

[0127] Proliferation rate = Equivalent number of CCK8 cells after 7 days of culture / Equivalent number of CCK8 cells after 1 day of culture

[0128] The results of the cell culture showed that in Examples 2, 5, 6, and 7, the cell adhesion rate was high, and cells proliferated until the 7th day of culture. On the other hand, in Comparative Example 4, because the surface of the carrier-type polymer porous membrane lacked a mesh structure, the cell adhesion rate was low, and it was found that cells hardly proliferated at all.

[0129] (3) Culture of human fibroblasts using an Erlenmeyer flask. 25 mL of culture medium was added to an Erlenmeyer flask (Nalgene Single-Use PETG Erlenmeyer Flasks with Baffled Bottom, product number: 4116-0125, Thermo Scientific). A carrier was placed inside the Erlenmeyer flask and cultured at 37°C in 5% CO2. ２ The cells were moistened in a shaker-equipped incubator in the presence of [unspecified substance]. After removing the inserts from the incubator, 2 mL of culture medium was added to the inside of each well. Human fibroblasts cultured in the above method (1) were detached from a Falcon® Dish (untreated, Corning) to obtain a cell suspension with a cell density of 20,000 cells / cm³. ２ Seeds were sown in an Erlenmeyer flask and stored at 37°C in 5% CO2. ２ The culture was performed with shaking for 51 days in the presence of the gas. By culturing with shaking, a portion of the carrier can be intermittently exposed to the gas phase during the culture process.

[0130] The culture medium was shaken at 30 rpm for the first two days, and then at 80 rpm from the third day onward. The culture medium was changed twice a week, and the entire volume of the medium was collected at that time to obtain the culture supernatant. On day 31 of culture, the number of cells in the carrier was measured using CCK8.

[0131] <Evaluation of Culture Supernatant> The glucose and lactate concentrations in the collected culture medium were measured using Cedex® Bio (Roche). The consumption and production amounts of each component were calculated using the following formulas.

[0132] Glucose consumption = Glucose concentration in unused culture medium - Glucose concentration in recovered culture medium

[0133] Lactic acid production = Lactic acid concentration in recovered culture medium - Lactic acid concentration in unused culture medium

[0134] Table 4 shows the metabolic ratio of lactate and glucose, and the number of cells on day 31 of cell culture.

[0135] Cell culture results confirmed cell proliferation in Examples 1, 2, and 3. In particular, in Example 1, the presence of an air layer between the polyimide porous membranes facilitated the exchange of the culture medium, resulting in a low lactic acid / glucose metabolic ratio and a rapid cell proliferation rate.

[0136] <Experiment 3: Culture of Human Adipose-Derived Mesenchymal Stem Cell Lines> <Experimental Materials and Methods> (1) Expanded Culture of Human Adipose Tissue-Derived Mesenchymal Stem Cells 10 mL of Cellartis (Trademark Registered) MSC Xeno-Free Culture Medium (Product No.: Y50200, Takara Bio) was added to a Corning (Registered Trademark) Dish (Culture Surface Treated, Corning Corporation), and the seeding density was 1,700 cells / cm². ２ Human adipose-derived mesenchymal stem cells were seeded and prepared at 37°C and 5% CO2. ２ Static culture was performed for 7 days in the presence of [the substance].

[0137] (2) Evaluation of cell adhesion rate and proliferation rate to carrier-type polymer porous membrane. 5 mL of culture medium and a culture area of ​​20 cm² were placed in a deep 6-well plate (Greiner Bio-One GmbH, ThinCert® cell culture insert, ThinCert plate, product number 657110). ２ Adding carriers, human adipose tissue-derived mesenchymal stem cells 4.0 × 10 ５ Sow the seeds individually and maintain a temperature of 37°C and 5% CO2. ２ Static culture was performed for 1 day in the presence of [the substance].

[0138] After one day of incubation, the carrier was placed in a storage bottle (Corning Incorporated, Costar® 125 mL polystyrene storage bottle, product number 8388) and 25 mL of culture medium was added. 37°C, 5% CO2 ２ In the presence of [substance name], the culture medium was completely replaced twice a week, and static incubation was performed for 6 days.

[0139] Cell adhesion and proliferation rates were calculated by measuring the number of carrier cells using CCK8 on days 1 and 7 of culture.

[0140] <Evaluation of Cell Adhesion and Proliferation Rate> Cell adhesion and proliferation rates were calculated using the following formulas.

[0141] Cell adhesion rate = (CCK8 equivalent cell count / seeded cell count (4.0 × 10) ５ )) × 100

[0142] The cell adhesion rate in Example 9 is expressed as a relative value based on that value.

[0143] Proliferation rate = Equivalent number of CCK8 cells after 7 days of culture / Equivalent number of CCK8 cells after 1 day of culture

[0144] Table 5 shows the relative values ​​of cell adhesion and proliferation rates. The carrier in Example 9 floated, the carrier in Example 10 floated, and the carrier in Comparative Example 3 settled.

[0145] The cell culture results showed that in Examples 9 and 10, the cell adhesion rate was high, and cells proliferated until day 7 of culture. On the other hand, in Comparative Example 3, the cell adhesion rate was low, and it was found that efficient culture was not possible.

[0146] 1, 1a, 1b, 1b', 1c, 1d, 1e, 1f, 1g, 1h, 1i Carrier 10, 10b', 10d, 10e, 10f Polymer porous membrane 11d, 11e, 11f Polymer porous membrane opening 100 Polymer porous membrane forming layer 20, 20a, 20f, 20g, 20h Spacer member 200, 200a Spacer member opening 201 Spacer member forming layer 30, 30b, 30b', 30f, 30g, 30h Protective member 300, 300b, 300f, 300f', 300g Protective member opening 301 Protective member forming layer 40 Seal member 400 Seal member opening 401 Seal member forming layer 50 Circular film

Claims

1. A carrier for cell culture, comprising: two or more polymer porous membranes; a spacer member having one or more openings provided between the two or more polymer porous membranes; and two protective members having one or more openings and sandwiching the two or more polymer porous membranes, wherein the polymer porous membrane is a polymer porous membrane having a surface layer A and a surface layer B having a plurality of pores, where the average pore diameter of the pores in surface layer A is smaller than the average pore diameter of the pores in surface layer B; the edges of the polymer porous membrane and the edges of the spacer member are welded and fixed; the edges of the polymer porous membrane and the edges of the protective member are welded and fixed by a sealing member; and a space of an average of 10 μm to 1500 μm is formed between adjacent two or more polymer porous membranes.

2. The carrier according to claim 1, wherein the carrier is substantially circular, elliptical, or square in shape.

3. The carrier according to claim 1, wherein the diameter or side length of the carrier is 0.1 mm to 5.0 cm.

4. The carrier is 0.99 g / cm³ ３ The carrier according to claim 1, having the following density.

5. The carrier according to claim 1, wherein the spacer member and / or protective member is selected from one or more of the group consisting of polyolefin, polyethylene, polypropylene, polyvinyl chloride, ethylene vinyl acetate resin, polystyrene, polyvinyl alcohol, polyethylene terephthalate, polyamide, polycarbonate, ionomer, polyurethane, polybutadiene, polyacrylonitrile, polybutylene terephthalate, and polyethylene naphthalate.

6. The carrier according to claim 1, wherein the spacer member and / or protective member is a mesh and / or nonwoven fabric and / or single-layer film and / or multi-layer film.

7. The carrier according to claim 1, wherein the sealing member is a single-layer film, multilayer film, or nonwoven fabric selected from one or more of the group consisting of polypropylene, polyethylene, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, polyamide, and ethylene vinyl acetate copolymer.

8. A method for manufacturing a carrier according to claim 1, comprising: a first step of forming an opening in the sealing member; a second step of laminating the sealing member, the spacer member, the protective member and the polymer porous film; and a third step of welding the sealing member, the spacer member, the protective member and the polymer porous film.

9. A method for manufacturing a carrier according to claim 1, comprising: a first step of forming an opening in the sealing member and an opening in the protective member; a second step of laminating the sealing member, the protective member and the polymer porous film; and a third step of welding the sealing member, the protective member and the polymer porous film.

10. The manufacturing method according to claim 8 or claim 9, wherein the first step, the second step, and the third step are carried out in that order.

11. A method for culturing cells using the carrier described in claim 1, comprising culturing the carrier while stirring it in a culture medium suspended and / or settled.

12. The method according to claim 11, characterized in that a portion of the carrier is cultured while being intermittently exposed to the gas phase.

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