Culture material and its uses

Abstract

To provide a culture member and a culture instrument having excellent morphological stability and oxygen supply capability, and allowing high-density culturing while holding the functions of cells.SOLUTION: A culture member cultures cells, tissues, or organs on a culture face thereof, the culture member containing resin, the culture face having at least one pore.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a culture device and its use. [Background technology]

[0002] Cells, tissues, and organs can only be cultured under conditions suitable for their growth. Therefore, they must be placed in culture vessels such as dishes, plates, and flasks along with culture medium containing the appropriate nutrients, and the culture vessels must be placed in incubators that can maintain the temperature, humidity, and gas concentrations at specified levels.

[0003] Furthermore, in order to efficiently carry out the above-mentioned culture, a sufficient and appropriate supply of oxygen is required. In order to supply oxygen to cells and carbon dioxide for adjusting the pH of the medium, gases such as oxygen and carbon dioxide must be supplied into the culture vessel, so that culture vessels made of materials with low gas permeability such as glass and polystyrene have openings at the top of the culture vessel, such as caps and lids, to ensure gas supply from inside the incubator to the inside of the vessel. However, cultured cells are usually attached to the bottom of the culture vessel or often float near the bottom, and the top surface is covered with culture medium, so the oxygen diffusion rate in the culture medium becomes the rate limiting factor, and oxygen supply to the cultured cells at the bottom in particular is insufficient, which has long been known to hinder cell proliferation and growth. In addition, when cell culture is performed by increasing the cell density in an attempt to create a state closer to in vivo, the supply of oxygen to the cultured cells is insufficient, which has been known to hinder cell proliferation and growth.

[0004] In order to promote the supply of oxygen to cells, there are measures such as increasing the oxygen partial pressure in the culture device, but a dedicated culture device for controlling the oxygen partial pressure is required, and it is generally more expensive than a culture device for culturing in the atmosphere. In addition, since the oxygen cylinder used to control the oxygen partial pressure is a combustion-supporting gas, there is a risk of oxidation heat generation, combustion, and explosion, and it is necessary to handle the oxygen cylinder with great care compared to nitrogen cylinders and carbon dioxide cylinders, which are non-flammable gases. As a simple and practical method for improving oxygen supply, a method is known in which a material with high oxygen permeability, such as polydimethylsiloxane (PDMS) (Non-Patent Document 1) or polybutadiene (Patent Document 1), is used on the culture surface. However, such a material with high oxygen permeability may be weak and easily broken, and may be easily bent when a culture medium is poured into the culture device, resulting in an unstable shape. When the culture device is bent, cells attached to the inner wall of the culture device may be peeled off due to the deformation of the container or the impact caused by the deformation, and the cells being cultured may gather at the bent area, making it impossible to culture cells efficiently. In addition, some materials used for the culture device are prone to drug sorption, which limits their use in drug discovery screening and diagnostic applications. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Utility Model Application Publication No. 1-112697 [Non-patent literature]

[0006] [Non-Patent Document 1] Yasuyuki Sakai, "Improvement of oxygen supply in liver culture", [online], Hepatocyte Research Society, [Retrieved April 9, 2019], Internet<http: / / hepato.umin.jp / kouryu / kouryu28.html> Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the above circumstances, and has an objective of providing a culture component and a culture instrument that have excellent shape stability and oxygen supply properties, and enable high-density culture while maintaining cellular function. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that the above problems can be solved by a culture device having the following configuration, and have completed the present invention. The present invention relates to, for example, the following [1] to

[11] . [1] A culture member for culturing cells, tissues, or organs on its culture surface, the culture member comprising a resin, and the culture surface having at least one pore. [2] The culture member according to [1], wherein the resin is a thermoplastic resin. [3] The culture member according to [1] or [2], wherein the resin is at least one selected from a polystyrene-based resin, a polyethylene terephthalate, and a 4-methyl-1-pentene polymer. [4] The culture member according to any one of [1] to [3], wherein the pores have an average pore size of 0.1 to 100 μm. [5] The number of the pores per unit area is 0.01 to 250 pores / cm 2 The culture member according to any one of [1] to [4], [6] The culture member according to any one of [1] to [5], wherein the culture member is in the form of a film or sheet. [7] A culture instrument, at least a culture surface of which is formed from the culture member according to any one of [1] to [6]. [8] The culture device according to [7], having at least one well. [9] The culture device according to [7] or [8], wherein the culture surface of the culture member has a coating layer made of at least one material selected from the group consisting of natural polymer materials, synthetic polymer materials, and inorganic materials.

[10] A step of contacting a cell, tissue or organ with the culture surface of the culture member according to any one of [1] to [6]; and A method for culturing cells, tissues, or organs, comprising the step of: supplying oxygen to the cells, tissues, or organs in contact with the culture surface.

[11] The resin is a thermoplastic resin, the pores have an average pore size of 0.1 to 100 μm, and the number of the pores per unit area is 0.01 to 250 pores / cm 2 The culture method according to

[10] , Effect of the Invention

[0009] According to the present invention, it is possible to provide a culture member and a culture instrument that have excellent shape stability and oxygen supplying properties, and that enable high-density culture while maintaining the functions of cells. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1A is a diagram showing an example of the arrangement of wells in a 24-well plate. FIG. 1B is a diagram showing the arrangement of pores in each well of a 24-well plate in Examples 1 to 4 and 9 to 14. FIG. 1C is a diagram showing the arrangement of pores in each well of a 24-well plate in Example 5. FIG. 1D is a diagram showing the arrangement of pores in each well of a 24-well plate in Example 6. FIG. 1E is a diagram showing the arrangement of pores in each well of a 24-well plate in Example 7. FIG. 1F is a diagram showing the arrangement of pores in each well of a 24-well plate in Example 8. [Diagram 2] FIG. 2 shows images of cells observed under a microscope one day after cell seeding in Examples 1 to 3 and Comparative Examples 4 and 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Unless otherwise specified, the description "A to B" regarding a numerical range means that the range is greater than or equal to A and less than or equal to B. For example, the description "1 to 5%" means greater than or equal to 1% and less than or equal to 5%.

[0012] [Culture materials] The culture component of the present invention (obtainable therefrom) is a component for culturing cells, tissues or organs (hereinafter also referred to as cells, etc.) on its culture surface, and is characterized in that the culture component contains a resin and the culture surface has at least one pore. Here, the culture member means a member constituting at least a part of a culture instrument used for culturing cells, etc. When the culture member is a part of the culture instrument, at least the culture surface for culturing cells, etc. is constituted by the culture member of the present invention. Here, the culture surface means a surface on which a medium is formed when culturing cells, etc., a surface on which cells, etc. are seeded, or a surface on which a medium is formed and cells, etc. are seeded. In other words, the culture surface is a concept that includes a surface on which a medium is to be formed and a surface on which cells, etc. are to be seeded.

[0013] The form of the culture member of the present invention is not particularly limited, and may be, for example, a film or sheet. When the culture member is in a film or sheet form, at least one surface of the film or sheet culture member is used as a culture surface, and the culture member can be suitably used as a culture instrument.

[0014] The culture member of the present invention means a member whose culture surface is not coated with a natural polymer material, a synthetic polymer material, or an inorganic material to serve as a scaffold for cells or the like.

[0015] The thickness of the culture member of the present invention is not particularly limited, and is preferably 20 to 500 μm, more preferably 25 to 400 μm, and particularly preferably 50 to 200 μm.

[0016] When the thickness of the culture member is within the above range, the pores can be easily formed, the strength tends to be excellent, and the oxygen supply property tends to be excellent.

[0017] When the culture component of the present invention is placed on the bottom surface of a container to prepare a culture tool such as a dish (also called a petri dish), flask, insert or plate, the thickness of the culture component is not particularly limited, but is preferably 20 μm to 400 μm, more preferably 20 μm to 300 μm, and even more preferably 20 μm to 200 μm. Although the thickness is appropriately selected depending on the form of the culture tool, by adjusting the thickness within the above upper and lower limits, it is easy to obtain an appropriate oxygen concentration in the medium necessary for cell proliferation, and it is easy to prepare a suitable culture tool with sufficient strength.

[0018] The culture member of the present invention may have a surface subjected to microfabrication other than pores in order to create spheroids or improve the scaffolding function of cells. The method of microfabrication can be appropriately selected from cutting, photolithography, electron beam direct writing, particle beam processing, scanning probe processing, etc., self-organization of fine particles, or nanoimprinting from a master formed by these methods, casting, injection molding, and plating. The shape of the microfabrication is not particularly limited, but it is preferable that the height from the groove to the mountain is 20 nm to 500 μm. In addition, it is possible to reduce the thickness of the thinnest part of the culture member to 20 μm compared to when the surface is not microfabricated.

[0019] The microfabricated culture member may be used as a microchannel device (also called a microchannel chip). A microchannel device is a general term for devices in which a microchannel or a reaction vessel is created by microfabricating the surface of a culture member, and the device is applied to bioresearch or chemical engineering. For example, devices called microTAS (micro Total Analysis Systems) or Lab on a Chip are exemplified, and applications as next-generation culture devices are being promoted. One embodiment of the present invention is a microchannel device including the culture member of the present invention.

[0020] The culture member of the present invention may be subjected to a surface modification treatment. The method used for the surface modification treatment is not particularly limited, but examples thereof include hydrophilic treatment such as corona treatment, plasma treatment, ozone treatment, and ultraviolet treatment, chemical vapor deposition, etching, addition of specific functional groups such as hydroxyl groups, amino groups, sulfone groups, thiol groups, and carboxyl groups, treatment with specific functional groups such as silane coupling, and surface roughening with an oxidizing agent. These surface modification treatments may be performed alone or in combination of two or more types. When performing surface modification treatment, it is preferable to perform it at least on the culture surface.

[0021] Among the surface modification treatments, hydrophilization treatments such as corona treatment, plasma treatment, ozone treatment, and ultraviolet treatment are preferred in order to increase the wettability of the surface of the culture member and to enable efficient cell culture. By hydrophilizing the surface of the culture member, for example, the adhesion between the culture member and the cells is improved, and the cells can grow uniformly on the surface of the culture member. In addition, by hydrophilizing the surface of the culture member, it becomes easier to coat the culture surface of the culture instrument with a natural polymer material, a synthetic polymer material, or an inorganic material. In particular, collagen can be uniformly loaded and adhered to the surface of the culture member, and even after the collagen loading treatment, the collagen does not peel off when washed with physiological saline or in the cell culture environment, and can be used for cell culture while maintaining a stable initial state. When performing plasma treatment, nitrogen, hydrogen, helium, oxygen, argon, etc. are used as the gas to be accompanied, and preferably at least one gas selected from nitrogen, helium, and argon is selected.

[0022] The method for producing the culture member of the present invention is not particularly limited, and the equipment used for production is also not limited. For example, a film or sheet containing a component containing a resin is formed, and the film or sheet is molded into a molded product having a desired shape as necessary, and the film, sheet, or molded product is formed with pores as described above to produce the culture member. The film, sheet, or other molded product to be the culture member can also be obtained by direct molding by a method such as extrusion molding, solution casting molding, injection molding, or blow molding.

[0023] Specifically, for example, the film or sheet may be formed by a typical inflation method or T-die extrusion method. The film or sheet is usually produced under heating. When the T-die extrusion method is used, the extrusion temperature varies depending on the type of resin, but is preferably 100°C to 400°C, and more preferably 150°C to 300°C. The roll temperature is preferably 20°C to 100°C, and more preferably 30°C to 90°C.

[0024] The film or sheet may be produced by a solution casting method in which a resin is dissolved in a solvent, poured onto a resin or metal, and slowly dried while leveling to form a film (sheet). There are no particular limitations on the solvent used, but a hydrocarbon solvent such as cyclohexane, hexane, decane, or toluene may be used. Two or more types of solvents may be mixed in consideration of the solubility of the resin and the drying efficiency. A polymer solution is applied by a method such as table coating, spin coating, dip coating, die coating, spray coating, bar coating, roll coating, or curtain flow coating, and then dried and peeled off to form a film or sheet.

[0025] The culture device of the present invention is preferably a cell culture device, more preferably a hepatocyte culture device.

[0026] <resin> The material forming the culture component of the present invention may contain a resin, and may consist of only a resin, or may consist of a composition containing a resin. The resin used as the material for the culture member is desirably one that is low in toxicity to cells, etc., and either natural or synthetic resins can be used as such a material. Among these, synthetic resins are preferred from the viewpoints of moldability and chemical resistance. The synthetic resin may be either a thermoplastic resin or a thermosetting resin, but thermoplastic resins are preferred because they are easy to mold.

[0027] The resin used as the material for the culture member can be appropriately selected from the viewpoints of shape stability, drug sorption, autofluorescence, transparency, oxygen permeability, etc. By selecting a culture material from such a viewpoint, a culture member can be obtained that can efficiently culture cells, can be used for drug discovery screening and diagnostic purposes, and also allows easy observation of cells.

[0028] <Thermoplastic resin> Thermoplastic resins that can be used as materials for the culture member are not particularly limited, and examples thereof include polyolefin-based resins; polymethacrylic-based resins such as polymethyl methacrylate resin; polyacrylic-based resins such as polymethyl acrylate resin; polystyrene-based resins; polyvinyl acetal resins; polyvinyl butyral resins; polyvinyl formal resins; polymethylpentene resins; polycarbonate resins; polyether ether ketone resins; polyether-based resins such as polyether ketone resins; polyester-based resins; polyamide-based resins such as nylon-6, nylon-66, and polymetaxylene adipamide; polyamideimide resins; polyimide resins; polyetherimide resins; styrene-based Examples of suitable elastomers include elastomers such as elastomers for polyolefins, elastomers for polyurethanes, elastomers for polyesters, elastomers for polyamides, norbornene resins, polytetrafluoroethylene resins, ethylene tetrafluoroethylene copolymers, polyvinylidene fluoride resins, polyvinyl fluoride resins, thermoplastic polyimide resins, polyvinylidene chloride resins, polyvinyl chloride resins, polyvinyl acetate resins, polysulfone resins, polyphenylene resins such as polyphenylene oxide resins and polyphenylene sulfide resins, polysulfone resins, polylactic acid resins, polyethersulfone resins, polyacrylonitrile resins, and styrene-acrylonitrile copolymer resins.

[0029] Examples of polyolefin resins include homopolymers or copolymers of α-olefins such as ethylene, propylene, 1-butene, 1-hexene, 4-methyl-1-pentene, and 1-octene; high-pressure low-density polyethylene; linear low-density polyethylene (LLDPE); high-density polyethylene; polypropylene; random copolymers of propylene and α-olefins having 2 to 10 carbon atoms; ethylene-vinyl acetate copolymers (EVA); and ionomer resins. Among these, 4-methyl-1-pentene polymers (homopolymers and copolymers of 4-methyl-1-pentene) are preferred because of their excellent balance of moldability, transparency, mechanical properties, breathability, light weight, and low drug sorption. In the present invention, 4-methyl-1-pentene homopolymers and copolymers of 4-methyl-1-pentene and other monomers are also collectively referred to as "4-methyl-1-pentene polymers."

[0030] Examples of polystyrene-based resins include homopolymers of styrene-based monomers (e.g., styrene, methylstyrene, ethylstyrene, isopropylstyrene, dimethylstyrene, paramethylstyrene, chlorostyrene, bromostyrene, vinyltoluene, and vinylxylene); and copolymers of styrene-based monomers and monomers copolymerizable with styrene-based monomers (hereinafter also referred to as "modified polystyrene"). Examples of monomers copolymerizable with styrene-based monomers include vinyl monomers (eg, acrylonitrile, methacrylonitrile, acrylic acid, methacrylic acid, methyl methacrylate, maleic anhydride, and butadiene). Examples of modified polystyrene include acrylonitrile-styrene copolymer (AS), methyl methacrylate-styrene copolymer, acrylonitrile-methyl methacrylate-styrene copolymer, acrylonitrile-butadiene-styrene copolymer (ABS), acrylonitrile-acrylic rubber-styrene copolymer (AAS), and acrylonitrile-ethylene propylene diene rubber-styrene copolymer (AES). Among these, styrene homopolymers are preferred because they have an excellent balance of moldability, transparency, mechanical properties, breathability, light weight, and low drug sorption.

[0031] Examples of polyester resins include resins obtained by polycondensation of dicarboxylic acids such as isophthalic acid, phthalic acid, terephthalic acid, 2,6-naphthalenedicarboxylic acid, adipic acid, and sebacic acid, and oxycarboxylic acids (e.g., p-oxybenzoic acid) with aliphatic glycols such as ethylene glycol, propylene glycol, butanediol, diethylene glycol, 1,4-cyclohexanedimethanol, and neopentyl glycol. One or more of the dicarboxylic acids, oxycarboxylic acids, and aliphatic glycols can be used. Representative examples of the polyester resin include polyethylene terephthalate (PET), polyethylene-2,6-naphthalenedicarboxylate (PEN), and polybutylene terephthalate (PBT). The resin may be a homopolymer or a copolymer containing 30 mol% or less of a third component. Among these, polyethylene terephthalate is preferred because it has an excellent balance of moldability, transparency, mechanical properties, breathability, light weight, and low drug sorption.

[0032] Among these, polyolefin-based resins, polystyrene-based resins, and polyester-based resins are preferred because they have an excellent balance of moldability, transparency, mechanical properties, breathability, light weight, and low drug sorption, and 4-methyl-1-pentene polymers (homopolymers and copolymers of 4-methyl-1-pentene), polystyrene-based resins, and polyethylene terephthalate are more preferred.

[0033] The above resins may be used alone or in combination of two or more.

[0034] The total light transmittance of the thermoplastic resin is preferably 70% or more, more preferably 80% or more, and even more preferably 90%. Specifically, the total light transmittance can be measured by the following method. Using a 2mm thick injection test piece made of thermoplastic resin, in accordance with JIS K7361, the total amount of transmitted light is measured using a haze / transmittance meter HM-150 (D65 light source) manufactured by Murakami Color Research Laboratory Co., Ltd., and the total light transmittance is calculated using the following formula. Total light transmittance (%) = 100 × (total transmitted light amount) / (incident light amount) If the total light transmittance is within the above range, the thermoplastic resin tends to have excellent transparency, which is preferable.

[0035] <4-methyl-1-pentene polymer> Hereinafter, 4-methyl-1-pentene polymer, which is a suitable example of the material for the culture member, will be described in more detail. The copolymer of 4-methyl-1-pentene and other monomers, which is an example of the 4-methyl-1-pentene polymer, may be any of a random copolymer, an alternating copolymer, a block copolymer, and a graft copolymer. As the copolymer of 4-methyl-1-pentene and other monomers, a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) is preferable because it has high strength, is not easily broken or cracked, and has little deflection even when used as a member.

[0036] The 4-methyl-1-pentene polymer is preferably at least one polymer selected from a 4-methyl-1-pentene homopolymer and a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), and more preferably a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

[0037] Examples of the olefin include ethylene, propylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-octadecene, and 1-eicosene. The olefin can be appropriately selected according to the physical properties required for the culture member. For example, from the viewpoint of moderate oxygen permeability and excellent rigidity, the olefin is preferably an α-olefin having 8 to 18 carbon atoms, and more preferably at least one selected from 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, and 1-octadecene. When the carbon number of the olefin is within the above range, the processability of the polymer is improved, and the appearance of the culture member due to cracks or cracks at the ends tends to be less likely to occur. In addition, the rate of defective products of the culture member is reduced.

[0038] The olefin may be one or more. From the viewpoint of material strength, the carbon number is preferably 2 or more, and more preferably 10 or more. When combining two or more different α-olefins, it is particularly preferable to combine at least one selected from 1-tetradecene and 1-hexadecene with at least one selected from 1-heptadecene and 1-octadecene.

[0039] The content of structural units derived from 4-methyl-1-pentene in the 4-methyl-1-pentene polymer is preferably 60 to 100 mol %, more preferably 80 to 98 mol %. In addition, when the 4-methyl-1-pentene polymer is a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), the content of the structural units derived from at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) in the copolymer is preferably 0 to 40 mol%, more preferably 2 to 20 mol%. The content of these structural units is defined as 100 mol% of the total repeating structural units in the 4-methyl-1-pentene polymer. When the content of the structural units is within the above range, a homogeneous culture surface with excellent processability can be obtained, and the film has a good balance between toughness and strength, so that bending is reduced.

[0040] The 4-methyl-1-pentene polymer may have structural units (hereinafter also referred to as "other structural units") other than the structural units derived from 4-methyl-1-pentene and the structural units derived from ethylene and the α-olefin having 3 to 20 carbon atoms, within a range that does not impair the effects of the present invention. The content of the other structural units is, for example, 0 to 10.0 mol%. When the 4-methyl-1-pentene polymer has other structural units, the other structural units may be one type or two or more types.

[0041] Examples of monomers that derive other structural units include cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins. Examples of cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins include compounds described in paragraphs

[0035] to

[0041] of JP2013-169685A.

[0042] The 4-methyl-1-pentene polymers may be used alone or in combination of two or more kinds.

[0043] Commercially available 4-methyl-1-pentene polymers can also be used. Specific examples include TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31, and DX845 (all trademarks) manufactured by Mitsui Chemicals, Inc. In addition, 4-methyl-1-pentene polymers manufactured by other manufacturers that satisfy the above requirements can also be preferably used. These commercially available products can be used alone or in combination of two or more.

[0044] 4-methyl-1-pentene polymers usually have a melting point of 200°C to 240°C and are highly heat resistant. In addition, they do not undergo hydrolysis and have excellent water resistance, boiling water resistance, and steam resistance, so that culture components such as culture utensils containing 4-methyl-1-pentene polymers can be sterilized with high-pressure steam. 4-methyl-1-pentene polymers also have a high visible light transmittance (usually 90% or more) and do not emit autofluorescence, so that culture utensils containing 4-methyl-1-pentene polymers are easy to observe cultured cells. Furthermore, they show excellent chemical resistance to most chemicals and are not prone to sorbing drugs, so they are suitable for use in drug discovery screening and diagnostic applications. 4-methyl-1-pentene polymers can be heat sealed, and are easy to thermally bond not only to themselves but also to other materials. In addition, they can be thermoformed, so they can be easily molded into culture utensils of any shape, and for example, they can be easily molded using an imprint method or an insert method.

[0045] The weight average molecular weight (Mw) of the 4-methyl-1-pentene polymer measured by gel permeation chromatography (GPC) using standard polystyrene as a reference material is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,000,000, and even more preferably 30,000 to 500,000. Here, the sample concentration during GPC measurement can be, for example, 1.0 to 5.0 mg / ml. The molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer is preferably 1.0 to 30, more preferably 1.1 to 25, and even more preferably 1.1 to 20. The solvent used in GPC is preferably orthodichlorobenzene. An example of the measurement conditions is the condition shown in the examples described below, but the measurement conditions are not limited to these conditions.

[0046] By setting the weight average molecular weight (Mw) to the above upper limit or less, in the resin molding method described below, the occurrence of defects such as gels in the film produced by melt molding is easily suppressed, and a film with a uniform surface is easily produced. In addition, when produced by a solution casting method, the solubility in a solvent is improved, and defects such as gels in the film are easily suppressed, and a film with a uniform surface is easily produced.

[0047] In addition, by setting the weight-average molecular weight (Mw) to the above lower limit or more, the culture member tends to have sufficient strength. Furthermore, by setting the molecular weight distribution within the above range, the stickiness of the surface of the culture member produced tends to be suppressed, and the toughness of the culture member tends to be sufficient, making it easier to suppress the occurrence of cracks during bending during molding or cutting.

[0048] When two or more types of 4-methyl-1-pentene polymers are used, the weight average molecular weight (Mw) and molecular weight distribution (Mw / Mn) of each resin may be within the above range.

[0049] Since 4-methyl-1-pentene-1 polymer has the excellent properties described above, a culture instrument in which at least the culture surface is formed from the culture component of the present invention does not adversely affect culture, and has good stability, light transmittance, and moldability, and can be sterilized, making it an extremely excellent material for culture components.

[0050] <Method for producing 4-methyl-1-pentene polymer> The method for producing the 4-methyl-1-pentene polymer may be any method that can polymerize 4-methyl-1-pentene, olefin, and other monomers. In addition, a chain transfer agent, such as hydrogen, may be coexisted in order to control the molecular weight and molecular weight distribution. The equipment used for the production is not limited. The polymerization method may be a known method, and may be a gas phase method, a slurry method, a solution method, or a bulk method. The slurry method or the solution method is preferable. In addition, the polymerization method may be a single-stage polymerization method or a multi-stage polymerization method such as a two-stage polymerization method, in which a plurality of polymers having different molecular weights are blended into the polymerization system. In either the single-stage or multi-stage polymerization method, when hydrogen is used as a chain transfer agent, it may be added all at once or in portions, for example, at the initial, middle, or final stages of polymerization. The polymerization may be carried out at room temperature or may be heated as necessary, but from the viewpoint of polymerization efficiency, it is preferable to carry out the polymerization at 20°C to 80°C, and particularly preferable to carry out the polymerization at 40°C to 60°C. There are no limitations on the catalyst used in the production, but from the viewpoint of polymerization efficiency, it is preferable to use, for example, the solid titanium catalyst component (I) described in WO 2006 / 054613.

[0051] <Production method of polystyrene resin> The polystyrene resin can be produced, for example, by the polymerization method shown below. Examples of the polymerization method for polystyrene resins include known styrene polymerization methods such as bulk polymerization, solution polymerization, suspension polymerization, etc. These polymerization methods may be batch polymerization or continuous polymerization, and continuous polymerization is preferred from the viewpoint of productivity. A specific example of such a polymerization method is a method in which a raw material solution containing monomers is prepared by adding and mixing a styrene-based monomer and, if necessary, a polymerization solvent, a polymerization initiator, a chain transfer agent, etc. The raw material solution is continuously fed into a facility equipped with a single reactor or two or more reactors arranged in series and / or parallel, and a devolatilizer for a devolatilization step for removing volatile components such as unreacted monomers, and polymerization is allowed to proceed stepwise.

[0052] Examples of the reactor include a complete mixing type reactor, a laminar flow type reactor, a loop type reactor in which a part of the polymerization liquid is withdrawn while the polymerization is proceeding, etc. There is no particular limitation on the order of arrangement of these reactors.

[0053] When polymerizing the polystyrene-based resin, a polymerization solvent, a polymerization initiator such as an organic peroxide, and a chain transfer agent may be used as necessary from the viewpoint of controlling the polymerization reaction. A polymerization solvent is generally used to adjust the polymerization rate, molecular weight, etc. The polymerization solvent is not particularly limited, and examples thereof include alkylbenzenes such as benzene, toluene, ethylbenzene, and xylene, ketones such as acetone and methyl ethyl ketone, and aliphatic hydrocarbons such as hexane and cyclohexane. The amount of the polymerization solvent used is not particularly limited, but from the viewpoints of controlling gelation, improving productivity, increasing molecular weight, etc., it is usually preferably 1 to 50 mass % and more preferably 3 to 20 mass % relative to 100 mass % of the total polymerization solution in the polymerization reactor.

[0054] When the polymerization raw materials are polymerized to obtain the polystyrene-based resin, a polymerization initiator and a chain transfer agent can be contained in the polymerization raw material composition. The polymerization initiator is not particularly limited, and examples thereof include organic peroxides such as peroxyketals such as 2,2-bis(t-butylperoxy)butane, 1,1-bis(t-butylperoxy)cyclohexane, and n-butyl-4,4-bis(t-butylperoxy)valerate, dialkyl peroxides such as di-t-butyl peroxide, t-butylcumyl peroxide, and dicumyl peroxide, diacyl peroxides such as acetyl peroxide and isobutyryl peroxide, peroxydicarbonates such as diisopropyl peroxydicarbonate, peroxyesters such as t-butyl peroxyacetate, ketone peroxides such as acetylacetone peroxide, and hydroperoxides such as t-butyl hydroperoxide. The polymerization initiator is preferably used in an amount of 0.005 to 0.08% by mass based on the styrene-based monomer. The chain transfer agent is not particularly limited, but examples thereof include α-methylstyrene dimer, n-dodecyl mercaptan, t-dodecyl mercaptan, and n-octyl mercaptan. The chain transfer agent is preferably used in an amount of 0.01 to 0.50% by mass based on the styrene monomer.

[0055] As the volatilizing apparatus, for example, a typical volatilizing apparatus such as a flash drum, a twin-screw volatilizing apparatus, a thin-film evaporator, an extruder, etc. can be used, and generally, a vacuum volatilizing tank with a heater or a volatilizing extruder, etc., is used. As the arrangement of the volatilizing apparatus, for example, one using only one vacuum volatilizing tank with a heater, one connecting two vacuum volatilizing tanks with a heater in series, and one connecting a vacuum volatilizing tank with a heater and a volatilizing extruder in series can be mentioned. In order to reduce the volatile components as much as possible, one connecting two vacuum volatilizing tanks with a heater in series or one connecting a vacuum volatilizing tank with a heater and a volatilizing extruder in series is preferable.

[0056] The conditions for the devolatilization step are not particularly limited, and for example, when the polymerization of the polystyrene resin is carried out by bulk polymerization, the polymerization can be allowed to proceed until the final amount of unreacted styrene monomer in the polystyrene resin is preferably 50 mass% or less, more preferably 40 mass% or less. The devolatilization treatment can remove volatile matters such as unreacted materials (styrene monomers) and / or solvents.

[0057] The temperature for the devolatilization treatment is usually about 190 to 280° C. The pressure for the devolatilization treatment is preferably 0.1 to 50 kPa, more preferably 0.13 to 13 kPa, still more preferably 0.13 to 7 kPa, and particularly preferably 0.13 to 1.3 kPa. Desirable devolatilization methods include, for example, a method of devolatilization under reduced pressure while heating, or devolatilization through an extruder designed to remove volatile components.

[0058] <Polyethylene terephthalate> Polyethylene terephthalate (PET) is a polymer obtained by polycondensation of at least one selected from terephthalic acid and its ester-forming derivatives with at least one selected from ethylene glycol and its ester-forming derivatives. Examples of ester-forming derivatives of terephthalic acid include alkyl esters of terephthalic acid such as dimethyl terephthalate. Examples of ester-forming derivatives of ethylene glycol include fatty acid esters such as ethylene glycol fatty acid esters.

[0059] As long as the properties are not impaired, polyethylene terephthalate may be a copolymer of at least one selected from terephthalic acid and its ester-forming derivatives with at least one other dicarboxylic acid and its ester-forming derivative, or may be a copolymer of at least one selected from ethylene glycol and its ester-forming derivatives with at least one other diol and its ester-forming derivative. Examples of dicarboxylic acids and their ester-forming derivatives used as copolymerization components include isophthalic acid, adipic acid, oxalic acid, sebacic acid, decanedicarboxylic acid, naphthalenedicarboxylic acid, and alkyl esters thereof. Examples of diols and their ester-forming derivatives used as copolymerization components include ethylene glycol, propylene glycol, neopentyl glycol, 1,5-pentanediol, 1,6-hexanediol, decamethylene glycol, cyclohexanedimethanol, cyclohexanediol, polyethylene glycol having a molecular weight of 400 to 6000, long-chain glycols such as poly-1,3-propylene glycol, and polytetramethylene glycol, and fatty acid esters thereof. These copolymerization components may be used alone or in combination of two or more. These copolymerization components are preferably 20% by mass or less of the raw materials forming the polyethylene terephthalate.

[0060] <Method for producing polyethylene terephthalate> Polyethylene terephthalate can be produced by known polycondensation methods, ring-opening polymerization methods, and the like. Either batch polymerization method or continuous polymerization method may be applied, but continuous polymerization method is preferred in that the amount of carboxyl end groups can be reduced and the effect of improving fluidity is greater. Either transesterification reaction or direct polymerization reaction may be applied, but direct polymerization reaction is preferred in terms of cost. In order to efficiently proceed with the esterification reaction, transesterification reaction, and / or polycondensation reaction, it is preferable to add a catalyst during these reactions. Specific examples of catalysts include organotitanium compounds such as methyl ester, tetra-n-propyl ester, tetra-n-butyl ester, tetraisopropyl ester, tetraisobutyl ester, tetra-tert-butyl ester, cyclohexyl ester, phenyl ester, benzyl ester, tolyl ester, or mixed esters of these of titanic acid, dibutyltin oxide, methylphenyltin oxide, tetraethyltin, hexaethylditin oxide, cyclohexahexylditin oxide, didodecyltin oxide, triethyltin hydrochloride, and the like. Examples of the catalyst include tin compounds such as tin oxide, triphenyltin hydroxide, triisobutyltin acetate, dibutyltin diacetate, diphenyltin dilaurate, monobutyltin trichloride, dibutyltin dichloride, tributyltin chloride, dibutyltin sulfide, and butylhydroxytin oxide; alkylstannoic acids such as methylstannoic acid, ethylstannoic acid, and butylstannoic acid; zirconium compounds such as zirconium tetra-n-butoxide; and antimony compounds such as antimony trioxide and antimony acetate. The catalyst may be used alone or in combination of two or more. Among these, organic titanium compounds and tin compounds are preferred, and tetra-n-butyl ester of titanic acid is more preferred. The amount of catalyst added is generally 0.01 to 0.2 parts by mass relative to 100 parts by mass of polyethylene terephthalate.

[0061] The type of polyethylene terephthalate is not particularly limited as long as it does not impair the effects obtained by the present invention, but from the viewpoints of mechanical strength and moldability, crystalline polyethylene terephthalate (C-PET) is preferred.

[0062] When a film-shaped polyethylene terephthalate is used as a culture member, the film-shaped polyethylene terephthalate may or may not be stretched. When a film-shaped polyethylene terephthalate is stretched, the crystal orientation is caused, which is preferable from the viewpoint of mechanical properties. The stretching method is preferably biaxial stretching.

[0063] When the culture member is made of a composition containing a resin, the resin is preferably 90% by mass or more and less than 100% by mass, more preferably 95% by mass or more and less than 100% by mass, and particularly preferably 99% by mass or more and less than 100% by mass, based on 100% by mass of the culture member. If a large amount of components other than the resin is contained, not only the oxygen permeability decreases, but also the transparency and strength decrease.

[0064] The material forming the culture member of the present invention may contain components other than resin, such as additives such as heat resistance stabilizers, light resistance stabilizers, processing aids, plasticizers, antioxidants, lubricants, defoamers, antiblocking agents, colorants, modifiers, antibacterial agents, antifungal agents, and antifogging agents.

[0065] <pore> The culture member of the present invention has at least one pore on its culture surface. Here, the pore in the culture member means a minute hole contained in the culture member that penetrates the culture member, and is different from the unevenness generated by the surface processing of the culture member.

[0066] The opening shape of the pores is not particularly limited, and may be any shape such as a circle, an ellipse, a square, a rectangle, a hexagon, a cloud, a cross, etc. From the viewpoint of processability, among these, a circle or an ellipse is preferable. When the culture member of the present invention has a plurality of pores, the opening shapes of the plurality of pores may be substantially the same or different, but are preferably substantially the same.

[0067] The number of pores is not particularly limited, but the pore density (number per unit area) is 0.01 to 250 pores / cm 2 is preferably 0.1 to 230 particles / cm 2 More preferably, the density is 0.3 to 200 particles / cm. 2 When the pore density is within the above range, the oxygen supply is excellent and the metabolic activity of cells tends to be high.

[0068] The average pore size of the pores is not particularly limited, but is preferably 0.1 to 100 μm, more preferably 1 to 90 μm, and even more preferably 3 to 85 μm. When the average pore size of the pores is within the above range, the medium is less likely to leak, the oxygen supply is excellent, and the metabolic activity of the cells tends to be high.

[0069] The method for measuring the average pore size is not particularly limited, and known methods can be used. Examples of methods for measuring the average pore size include a method for determining the average pore size by gas adsorption, mercury intrusion, bubble point method, etc., a method for observing pores with a microscope, electron microscope, etc., measuring the pore size, and taking the average value as the average pore size, and a method for estimating the diameter of the instrument used for perforation, such as a needle or laser beam, as the average pore size. When measuring the pore size by observation and when estimating the diameter of the instrument used for perforation as the average pore size, the pore size refers to the diameter of the maximum inscribed circle relative to the opening shape of the pore, for example, when the opening shape of the pore is substantially circular, it refers to the diameter of the circle, when it is substantially elliptical, it refers to the minor axis of the ellipse, when it is substantially square, it refers to the length of the side of the square, and when it is substantially rectangular, it refers to the length of the short side of the rectangle. Because it is simple, a method for estimating the diameter of the instrument used for perforation, such as a needle or laser beam, as the average pore size is preferred.

[0070] The culture member of the present invention preferably has pores with an average pore size of 0.1 to 100 μm at 0.01 to 250 pores / cm 2 and more preferably has an average pore size of 1 to 90 μm and a pore density of 0.1 to 230 pores / cm. 2 and more preferably has an average pore size of 3 to 85 μm and a pore density of 0.3 to 200 pores / cm. 2 When the average pore size and pore density of the pores are within the above ranges, the oxygen supply is excellent and the metabolic activity of the cells is high.

[0071] The opening area of ​​the pores is preferably 0.005 to 8000 μm 2 , more preferably 0.1 to 7000 μm 2 , more preferably 5 to 6000 μm 2 When the opening area of ​​the pores is within the above range, the medium is less likely to leak, the oxygen supply is excellent, and the metabolic activity of the cells tends to be high.

[0072] The total opening area ratio of the pores (total opening area per well / total surface area per well of the culture surface of the culture member × 100 (%)) is preferably 0.000001 to 0.3%, more preferably 0.000005 to 0.1%, and even more preferably 0.00001 to 0.08%. When the total opening area ratio of the pores is within the above range, the medium is less likely to leak, oxygen supply is excellent, and the metabolic activity of the cells tends to be high.

[0073] When the culture member of the present invention has a plurality of pores, the pore sizes of the plurality of pores may be substantially the same or may vary, but it is preferable that they are highly uniform. The coefficient of variation can be used as an index of the uniformity of the pore size. The coefficient of variation of the pore size (standard deviation ÷ average value × 100 (%)) is preferably 30% or less, and more preferably 20% or less. When the pore size is highly uniform, the medium is less likely to leak, the oxygen supply is excellent, and the metabolic activity of the cells tends to be high.

[0074] When the culture member of the present invention has a plurality of pores, the arrangement of the pores is not particularly limited, and the pores may be arranged in a fixed pattern or randomly. Examples of the arrangement in a fixed pattern include 60° staggered, 45° staggered, parallel, herringbone, etc., and the pores may be arranged at equal intervals on the sides or circumference of a plurality of polygons or circles of different sizes that share a common center. Arranging in a fixed pattern is preferable because it tends to prevent the medium from leaking, has excellent oxygen supply, and has a high metabolic activity of cells.

[0075] The interval between the multiple pores is not particularly limited, but is preferably larger than the average pore size of the pores, and more preferably is a constant interval. The interval between the multiple pores in the same well is preferably 200 μm to 8 mm, more preferably 300 μm to 7 mm, and even more preferably 400 μm to 6 mm. When the interval between the multiple pores is within the above range, the medium is less likely to leak, oxygen supply is excellent, and metabolic activity of the cells tends to be high.

[0076] The multiple pores may be in communication with each other inside the culture member.

[0077] The method for forming the pores is not particularly limited, and any known method can be used. Examples of the method for forming the pores include a method of heating a very thin needle such as a microsyringe to pierce the film and melt it to open holes, and a method using a laser.

[0078] There are various types of lasers such as carbon dioxide laser, YAG laser, semiconductor laser, argon laser, etc., and there is no particular limitation as long as it can perform perforation. Among them, it is preferable to use a YAG laser in the wavelength region of 100 to 1100 nm, since it is possible to perforate small holes with a small diameter, and it is more preferable to use an ultraviolet YAG laser in the wavelength region of 100 to 400 nm, since it is absorbed and perforated even in transparent materials through which visible light passes, and a deep ultraviolet YAG laser in the wavelength region of 260 to 270 nm is even more preferable. When a deep ultraviolet YAG laser in the wavelength region of 260 to 270 nm is used, the output is preferably 10 to 200 mW. When the output range is within the above range, the workability is good, and perforation of a desired diameter can be achieved while avoiding excessive irradiation.

[0079] The scanning speed of the laser beam is preferably 1 to 100 mm / s. When the moving speed of the laser beam is within the above range, workability is good and perforation of a desired diameter can be achieved while avoiding excessive irradiation.

[0080] The perforation of the pores may be performed after obtaining a molded body from the resin-containing component, or simultaneously with the preparation of the molded body from the resin-containing component. The molded body with the perforated pores may be used as a culture member as is, or may be stretched before use. Preferably, a film or sheet is molded from the resin-containing component, the film or sheet is perforated with pores, and the perforated film or sheet is molded into a culture member having a desired shape. Alternatively, preferably, the pores are perforated after the molded body having a desired shape is prepared by molding the resin-containing component.

[0081] Examples of methods for drilling pores simultaneously with the production of a molded body from a component containing a resin include a method in which an organic solvent solution of a resin is cast onto a substrate, the organic solvent is evaporated while condensation occurs on the surface of the casting liquid, and the microdroplets produced by the condensation are evaporated to obtain a culture member having pores; and a method in which a molten thermoplastic resin is discharged from a spinneret together with a heated gas by a melt blowing method, the thermoplastic resin is stretched by the heated gas to form a fibrous resin, and a culture member having pores is obtained.

[0082] <Cells, tissues, or organs> The cells in the present invention are not particularly limited, and in the case of animal cells, they may be floating cells or adhesive cells, and examples thereof include fibroblasts, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, nerve cells, corneal epithelial cells, oral mucosa cells, retinal pigment cells, periodontal ligament stem cells, myofibroblasts, cardiac muscle cells, hepatocytes, splenic endocrine cells, skin keratinocytes, skin fibroblasts, subcutaneous fat-derived precursor cells, kidney cells, bottom root sheath cells, nasal mucosa epithelial cells, vascular endothelial precursor cells, vascular endothelial cells, vascular smooth muscle cells, osteoblasts, chondrocytes, skeletal muscle cells, immortalized cells, cancer cells, keratinocytes, embryonic stem cells (ES cells), EBV-transformed B cells, and induced pluripotent stem cells (iPS cells). They may be either primary cultured cells or established, passaged cells. Since the skin, kidney, liver, brain, nerve tissue, cardiac tissue, skeletal muscle tissue, cancer stem cells, etc. have a high oxygen requirement and the cells that compose them also have a high oxygen requirement, the cells in the present invention are preferably cells that compose the skin, kidney, liver, brain, nerve tissue, cardiac tissue, or skeletal muscle tissue, or cancer stem cells. As the cells, tissues, or organs, hepatocytes, renal cells, cardiac muscle cells, nerve cells, or cancer stem cells are preferred, and hepatocytes are more preferred.

[0083] In the present invention, the term "tissue" refers to a group of similar cells that perform a similar function. The tissue is not particularly limited, and examples thereof include epithelial tissue, connective tissue, muscle tissue, and nerve tissue. Due to their high oxygen demand, the tissue is preferably hepatic lobule, cardiac muscle tissue, nerve tissue, or skeletal muscle tissue, and more preferably hepatic lobule.

[0084] In the present invention, an organ means a tissue that gathers together to perform a common task. The organ is not particularly limited, and may be, for example, the lung, heart, liver, kidney, spleen, pancreas, gallbladder, esophagus, stomach, skin, brain, etc. Since the organ has a high oxygen requirement, the organ is preferably the skin, kidney, liver, or brain, and more preferably the liver.

[0085] The cells, etc. are preferably cells, since they are suitable for culturing on the culture surface of the culture tool. In the present invention, the cells, etc. are preferably aerobic, and more preferably do not include anaerobic ones. The origin of the cells, etc. is not particularly limited, and may be any organism such as an animal, a plant, a fungus, a protozoan, or a bacterium, but animals or plants are preferred, animals are more preferred, and mammals are particularly preferred. The culture tool in the present invention has suitable oxygen supply properties and also retains cell adhesive properties, so the cells, etc. are preferably adherent, and more preferably adherent cells.

[0086] <Liver cells> The hepatocytes in the present invention may be any cells present in the liver, including hepatocytes, and specifically include vascular endothelial cells, vascular smooth muscle cells, adipocytes, blood cells, hepatic mononuclear cells, hepatic macrophages (including Kupffer cells), hepatic stellate cells, intrahepatic bile duct epithelial cells, embryonic sac fibroblasts, etc. The hepatocytes are a cell population containing, for example, 20% or more, 30% or more, 40% or more, or 50% or more of hepatic parenchymal cells.

[0087] The hepatocytes may be primary cultured cells or established subcultured cells, but it is preferable to use primary cultured cells as the hepatocytes. The type of established subcultured cells is not particularly limited, and examples thereof include SSP-25, RBE, HepG2, TGBC50TKB, HuH-6, HuH-7, ETK-1, Het-1A, PLC / PRF / 5, Hep3B, SK-HEP-1, C3A, THLE-2, THLE-3, HepG2 / 2.2.1, SNU-398, SNU-449, SNU-182, SNU-475, SNU-387, SNU-423, FL62891, DMS153, etc.

[0088] The hepatocytes may be derived from any mammal, but are particularly preferably human, bovine, canine, feline, porcine, miniature pig, rabbit, hamster, rat, or mouse cells, and more preferably human, rat, mouse, or bovine cells.

[0089] The hepatocytes may be a cell population containing cell types other than hepatocytes, for example, a cell population containing 20% ​​or more, 30% or more, 40% or more, or 50% or more of hepatocytes.

[0090] <culture> In the present invention, the term "culture" is used in a broad sense to include not only proliferation and maintenance of cells, etc., but also processes such as seeding, subculturing, differentiation induction, and self-organization induction of cells, etc. There are no limitations on the medium, etc. used for culture, and a medium may be selected according to the characteristics of the cells, etc.

[0091] <Cell Culture> The cell culture may be two-dimensional (including the case where the cells spontaneously form layers) or three-dimensional. The culture member of the present invention has suitable oxygen supplying properties for culture, so that the cells can be sufficiently supplied with oxygen not only in two-dimensional culture but also in three-dimensional culture where the cells are stacked three-dimensionally, and the cells can proliferate, differentiate, and furthermore, the cell self-organization phenomenon can easily occur.

[0092] Three-dimensional culture is the intentional cultivation of cells in a three-dimensional space. It can be either a scaffold type, in which cells are cultivated in a scaffold, or a scaffold-free type, in which cells are cultivated in a suspended state as a mass (spheroid), but the scaffold type is preferred. In the case of the scaffold type, Matrigel is used as a scaffold material because it allows efficient cultivation of cells. TM , collagen gel, laminin, alginate hydrogel, and vitrigel are preferred.

[0093] There are no limitations on the medium used for culture, but in order to culture the cells efficiently, it is preferable to culture the cells in the presence of serum (eg, fetal bovine serum).

[0094] When the culture member of the present invention is used for culture, in other words, when culture is performed using the culture device of the present invention, the cell culture density is preferably 0.1×10 5 cells / cm 2 ~10.0×10 5 cells / cm 2 and more preferably 0.5×10 5 cells / cm 2 ~5.0×10 5 cells / cm 2 and more preferably 1.0×10 5 cells / cm 2 ~4.0×10 5 cells / cm 2 It is. The above range is preferable because the drug metabolic activity is enhanced more than when the cell culture density is outside the above range. The culture member of the present invention has excellent oxygen supplying properties, and therefore can be used for suitable cell culture even when the cell culture density is high. Generally, the cell density of a living organism is 2.5×10 5 cells / cm 2 However, the culture member of the present invention is preferable because it is possible to culture cells at a cell culture density similar to that in a living body, thereby enabling in vitro culture under conditions closer to those in vivo.

[0095] <Culture equipment> In the present invention, a culture instrument means any instrument used for culturing cells, etc. At least a part of the culture instrument is composed of the culture member. The culture instrument may be entirely composed of the culture member, or only a part of the culture instrument may be composed of the culture member. When only a part of the culture instrument is composed of the culture member, at least the culture surface for culturing cells, etc. is composed of the culture member of the present invention.

[0096] The culture device is typically used in a device such as an incubator, a mass culture device, or a perfusion culture device.

[0097] As the culture tool, various known culture tools can be used, and the shape and size are not particularly limited. Examples of the culture tool include culture vessels such as dishes, flasks, plates, bottles, bags, and tubes, as well as inserts, cups, insoles, and slides, and the culture vessels are preferred.

[0098] The culture tool is preferably a culture tool having at least one well, more preferably a culture vessel having at least one well, even more preferably a plate having at least one well, and even more preferably a plate having wells such as 6 wells, 12 wells, 24 wells, 48 ​​wells, 96 wells, 384 wells, 1536 wells, etc. In general, a culture tool having a well-like recessed shape on the bottom surface needs to have a thick bottom surface to stabilize the complex shape of the bottom surface, and it is difficult to sufficiently supply oxygen to cells, etc. When the culture member of the present invention is used, even a plate having wells such as 1 well, 6 wells, 12 wells, 24 wells, 48 ​​wells, 96 wells, 384 wells, 1536 wells, etc. has a stable shape and sufficient oxygen supply to cells, etc.

[0099] The culture tool is preferably a tool with a bottom surface as a culture surface in order to hold or store the culture medium. When the culture tool is a dish, flask, insert, or plate, the bottom surface is the culture surface, so the culture member of the present invention preferably constitutes at least a part or all of the bottom surface, side surface, and top surface. When at least the bottom surface (culture surface) is constituted by the culture member of the present invention, oxygen can be more efficiently supplied to the culture medium through the culture member, and cells, etc. in the culture medium can be more efficiently grown. In addition, cells can be cultured at a higher density while maintaining their functions.

[0100] The shape of the bottom of the culture instrument is not particularly limited, and examples thereof include flat bottom, round bottom (U bottom), flat bottom (F bottom), conical bottom (V bottom), flat bottom + curved edge, etc. When processing into a round bottom (U bottom), flat bottom (F bottom), conical bottom (V bottom), flat bottom + curved edge, etc., it may be processed at once by general injection molding or press molding, or it is also possible to prepare a film or sheet and perform secondary processing such as vacuum molding or pressure molding. The shape of the bottom is selected according to the purpose of the culture, but when cells are cultured two-dimensionally, a flat bottom is usually desirable, and when cells are cultured three-dimensionally, a round bottom (U bottom) or conical bottom (V bottom) is usually desirable.

[0101] The portions of the culture instrument other than the culture member may be made of a material other than the culture member. The material other than the culture member is not particularly limited, and known materials can be used. Examples of such materials include polydimethylsiloxane (PDMS), thermosetting resin, cyclic olefin polymer, cyclic olefin copolymer, glass, etc.

[0102] The culture device may be a culture device having a culture surface coated with a natural polymer material, a synthetic polymer material, or an inorganic material.

[0103] The coated culture instrument may be obtained, for example, by coating a culture instrument with a natural polymeric material, a synthetic polymeric material, or an inorganic material by known methods, or by using an already coated culture component on at least the culture surface of the culture instrument.

[0104] The coated culture instrument has better adhesiveness and proliferation of cells, etc. This is believed to be because the natural polymeric material, synthetic polymeric material, or inorganic material coated on the culture surface serves as a scaffold for cells, etc. Therefore, when culturing adherent cells, etc., it is a preferred embodiment to coat a culture member or culture instrument with a natural polymeric material, synthetic polymeric material, or inorganic material and use it as a culture instrument.

[0105] The natural polymer materials, synthetic polymer materials, and inorganic materials are not particularly limited, and examples of natural polymer materials include collagen, gelatin, alginic acid, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, fibronectin, laminin, fibrinogen, osteopontin, tenascin, vitronectin, thrombospondin, agarose, elastin, keratin, chitosan, fibrin, fibroin, and sugars; synthetic polymer materials include polyglucose acid, polylactic acid, polyethylene glycol, polycaprolactone, synthetic peptides, and synthetic proteins; synthetic polymer materials include polyethylene glycol, polyhydroxyethyl methacrylate, and polyethyleneimine; and inorganic materials include β-tricalcium phosphate and calcium carbonate.

[0106] Furthermore, examples of the natural polymeric material, synthetic polymeric material, or inorganic material include vitrigel obtained by vitrifying a hydrogel such as a conventional extracellular matrix component and then rehydrating it, etc. For example, collagen vitrigel composed of a high-density collagen fiber network made from collagen, which is one of the extracellular matrix components, can be mentioned.

[0107] From the viewpoints of improving cell adhesion and cell proliferation, maintaining cell function for a longer period, etc., coating with proteins or peptides such as collagen, gelatin, laminin, polylysine, etc. is preferred, and coating treatment with collagen or polylysine is more preferred. These coatings may be used alone or in combination of two or more.

[0108] The culture tool of the present invention may be disinfected or sterilized to prevent contamination. The method of disinfection or sterilization is not particularly limited, and includes physical disinfection methods such as steam circulation, boiling, intermittent irradiation, and ultraviolet light, chemical disinfection using gases such as ozone and disinfectants such as ethanol, heat sterilization methods such as high-pressure steam and dry heat, irradiation sterilization methods such as gamma ray and high-frequency, and gas sterilization methods such as ethylene oxide gas and hydrogen peroxide gas plasma. Among them, ethanol disinfection, high-pressure steam sterilization, gamma ray sterilization, and ethylene oxide gas sterilization are preferred because they are easy to operate and can be sufficiently sterilized. These disinfection and sterilization methods may be performed alone or in combination of two or more.

[0109] The method for producing the culture instrument of the present invention is not particularly limited, and when the entire culture instrument is composed of the culture member, it can be produced by the same method as the method for producing the culture member. When a part of the culture instrument is formed of the culture member, the culture instrument can be obtained by appropriately joining the culture member and other members. There is no particular limit to the method for joining, and the culture member and other members may be formed integrally, or may be adhered to each other via an adhesive or pressure sensitive adhesive.

[0110] The culture instrument of the present invention is preferably a cell culture instrument, and more preferably a liver cell culture instrument.

[0111] <Culture method> The method for culturing cells or the like of the present invention is a culture member for culturing cells, tissues, or organs on its culture surface, the culture member containing a resin, and the culture surface having at least one pore, comprising the steps of: contacting the cells, tissues, or organs with the culture surface of the culture member; and supplying oxygen to the cells, tissues, or organs in contact with the culture surface. The method of contacting cells, etc. with the culture surface of the culture component is not particularly limited as long as it is possible to contact the cells, etc. with the culture surface of the culture component, and examples thereof include seeding the cells, etc. on the culture surface of the culture component or culture instrument. The method of supplying oxygen to cells, etc. in contact with the culture surface is not particularly limited as long as it is possible to supply oxygen to cells, etc. in contact with the culture surface. For example, oxygen may be supplied to a culture incubator containing a culture component or culture equipment, and oxygen may be supplied to the cells, etc. via the culture component. The resin is a thermoplastic resin, the pores have an average pore size of 0.1 to 100 μm, and the number of the pores per unit area is 0.01 to 250 pores / cm 2 It is preferable that: The method for culturing cells or the like is preferably a method for culturing cells, and more preferably a method for culturing hepatocytes. EXAMPLES

[0112] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these. The methods for adjusting the collagen coating solution, the cell type and culture medium preparation, the method for measuring oxygen permeability, the method for evaluating the presence or absence of bending, the method for evaluating cell adhesion, the method for measuring the oxygen concentration in the culture medium, and the method for measuring metabolic activity are described below.

[0113] [Preparation of collagen coating solution] A 0.1 M hydrochloric acid solution (for volumetric analysis, Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted 100-fold with water for injection (Japanese Pharmacopoeia, Otsuka Pharmaceutical Co., Ltd.) to prepare a 0.001 M hydrochloric acid solution, which was then sterilized by filtration. A 3 mg / mL collagen solution (Cell Matrix Type I-P, derived from porcine tendon, Nitta Gelatin Co., Ltd.) was diluted 6-fold with a 0.001 M hydrochloric acid solution to prepare a 0.5 mg / mL collagen solution.

[0114] [Cell types and medium preparation] Culture medium was added to a centrifuge tube (50 ml) containing a cell suspension containing primary frozen rat hepatocytes. The culture medium was prepared by adding 1.5 ml of fetal bovine serum (FBS, Fujifilm Wako Pure Chemical Industries), 0.15 ml of L-proline (for culture, Fujifilm Wako Pure Chemical Industries) diluted to 3.0 g / ml with water for injection (Fuso Pharmaceutical Industries), and 1 × 10 ethanol (for molecular biology, Fujifilm Wako Pure Chemical Industries). -3 The following was added to the flask: 1.5 μL of dexamethasone (for biochemistry, Fujifilm Wako Pure Chemical Industries) solution diluted to 36 mM; 21 μL of hydrocortisone (for culture, Fujifilm Wako Pure Chemical Industries) solution diluted to 36 mM with ethanol; 15 μL of epidermal growth factor (EGF, for cell biology, Fujifilm Wako Pure Chemical Industries) solution diluted to 20 μg / mL with BSA solution diluted to 1.0 mg / mL with water for injection; 8.7 μL of insulin solution (10 mg / mL in HEPESS, SIGMA); 0.3 mL of penicillin-streptomycin solution (containing 5000 units / mL penicillin and 5000 μg / mL streptomycin, for culture, Fujifilm Wako Pure Chemical Industries); The cells were adjusted by adding 13 mL of a culture-grade solution (containing L-glutamine, 15 mg / mL phenol red, 110 mg / mL sodium pyruvate, and 3700 mg / mL sodium bicarbonate; Fujifilm Wako Pure Chemical Industries, Ltd.). The cell density was adjusted by adjusting the cell number of a cell suspension containing frozen primary rat hepatocytes, and was set to 4.0 × 10 unless otherwise specified. 5 cells / cm 2 In Example 9 and Comparative Examples 1, 3, and 5, the cell density was 1.0 × 10 5 cells / cm2 The cells were cultured at a cell density of 1000×.

[0115] [Oxygen permeability measurement] The oxygen permeability of the film before perforation was measured using a Toyo Seiki Seisakusho differential pressure gas permeability measuring device MT-C3 under an environment of 23°C temperature and 0% RH. The measurement area was 70 mm (permeation area was 38.46 cm). 2 An aluminum mask was applied to the sample in advance, and the actual transmission area was set to 5.0 cm 2 It was decided.

[0116] [Whether or not there is deflection] One day after cell seeding, the culture vessels were removed from the incubator and the bottom of each of the four culture vessels was examined from the side to see whether the film sagged in the culture environment. If there was no change in any of the four culture vessels compared to when the vessel was made and no sagging of the film was observed, it was rated as "no bending," and if there was a change in at least one of the four culture vessels compared to when the vessel was made and sagging of the film was observed, it was rated as "bending."

[0117] [Evaluation of cell adhesion] One day after cell seeding, three representative wells from each of the four culture vessels were observed under a microscope to observe the state of cells adhering and proliferating. In all four culture vessels, the cases where the cells were uniformly adhering and proliferating over the entire culture surface and maintaining their morphology were evaluated as ◯, and the cases where the cells were not uniformly adhering over the entire culture surface, not proliferating, or not maintaining their morphology in at least one of the four culture vessels were evaluated as ×.

[0118] [Measurement of oxygen concentration in culture medium] One day after cell seeding, the medium in the culture vessel was removed, 0.5 mL of new medium was added, and the oxygen partial pressure (hPa) in the medium was measured using a fluorescent oxygen sensor (FireSting oxygen monitor, manufactured by BAS Co., Ltd.). The measurement was performed in a humidified incubator, and the sensor was installed at a height of 80 μm from the bottom of the culture vessel using a jack, and measurements were taken for one hour. The oxygen partial pressure (hPa) in the medium one hour after the start of measurement was divided by 1013 hPa (1 atmosphere of air) and multiplied by 100 to calculate the value (%), which was the oxygen concentration in the medium. Measurements were performed in three representative wells, and the average value was calculated.

[0119] [Metabolic activity measurement] After removing the medium from the culture vessel 24 hours after cell seeding, Luciferin-CEE diluted in the medium was added and cultured for an additional 3 hours. After culture, the cells were transferred to a 96-well plate together with the medium containing Luciferin-CEE, and a mixture of Luciferin Detection Regent and Reconstitution Buffer was added. The cells were reacted at room temperature in the dark for 1 hour. After 1 hour, the luminescence amount (Relative Light Unit, RLU) was measured using a luminometer. Measurements were performed on 3 representative wells, and the average value was calculated.

[0120] The protein amount was measured by removing the Luciferin-CEE solution diluted with the medium, adding 200 μL of PBS(-) to the medium, and then recovering the cells in an Eppendorf tube using a cell scraper and centrifuging (4°C, 22,000×g, 10 min). The supernatant was then removed, 100 μL of 0.1 M sodium hydroxide solution was added, and the amount of protein was measured using a Pierce TM The protein amount was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). The absorbance at a wavelength of 450 nm was measured using a plate reader (SPECTRA max PLUS384, Molecular Devices).

[0121] The metabolic activity (pmol / L) of the Luciferin-CEE solution obtained by the luminometer was calculated using P450-Glo TMMeasurement was performed using a CYP1A1 Assay kit (Promega), and metabolic activity (pmol / min / mg protein) was calculated by dividing the absorbance by the protein amount obtained and the reaction time of the Luciferin-CEE solution.

[0122] [Example 1] A 50 μm thick unstretched film of a polystyrene (hereinafter referred to as PS) homopolymer (manufactured by Okura Kogyo Co., Ltd., product name: Ceromer S-2, oxygen permeability 3310 cm) was cut into a rectangle of 12 cm × 8 cm. 3 / (m 2 Using a UV laser processing machine (manufactured by Takano Corporation), laser processing was performed under the conditions of wavelength 266 nm, output 50 mW, scanning speed 10 mm / sec., and beam diameter 5 μm, forming 24 pores with a diameter of 5 μm in the PS film. The arrangement of the pores will be described later. Using a digital microscope (Keyence Corporation, VHX-1000), the perforated film was photographed from above, and the pore size was measured based on the photographed image. Although the individual measurements of the pore size of the 24 pores are not shown, the variation in pore size was within 10%. The perforated film was plasma-treated using an atmospheric pressure plasma surface treatment device (manufactured by Sekisui Chemical Co., Ltd.) by filling the chamber with a nitrogen gas flow (treatment speed 2 m / min, output 4.5 kW, 2 round trips). The plasma-treated perforated film was then attached to the bottom of a PS 24-well culture plate frame, with the bottom of each well open, via a medical adhesive (manufactured by 3M). One well (circular shape with a diameter of 16 mm, area approximately 2 cm) was placed in the plate. 2 ) with a pore density of 0.5 pores / cm 2 We created a 24-well cell culture vessel. The holes were designed and drilled in advance so that they would be arranged in each well as shown in Figure 1B when the perforated film was attached to the frame of the 24-well culture plate. Specifically, one hole was placed at the center of a circle along the inside of the bottom of the circular well. The cell culture vessel was packed in a gamma-ray resistant bag and sterilized by irradiating with 10 kGy of gamma rays. 0.5 mL of 0.5 mg / mL collagen solution was then added to the culture surface of the culture vessel, and the excess collagen solution was then removed. After leaving it to stand at room temperature for 30 to 60 minutes, it was washed with Dulbecco's PBS (-) and dried overnight at room temperature. The collagen-coated cell culture vessel produced by the same method was designated as culture vessel 1, and five identical vessels were produced.

[0123] Next, a medium (0.5 mL) containing rat primary frozen hepatocytes was seeded on each of the culture surfaces of five culture vessels 1 using a micropipette, covered with a polystyrene lid, and brought into an incubator to start culturing at 37°C and 5% CO2. The presence or absence of bending and cell adhesion were evaluated for four culture vessels. Then, the oxygen concentration in the medium was measured using one of the four culture vessels 1. The metabolic activity was measured for the remaining three vessels. The results are shown in Table 1. The metabolic activity values ​​are shown in Table 1 as the average values ​​of the results for the three vessels. Furthermore, the cell adhesion of the remaining vessel was evaluated after seven days of culture. The results are shown in Table 1. Figure 2 shows the results of observing the cells one day after cell seeding using a phase contrast microscope.

[0124] [Example 2] A cell culture vessel was produced in the same manner as in Example 1, except that the laser processing conditions were changed to an output of 100 mW and a beam diameter of 20 μm. The hole diameter was 20 μm and the hole density was 0.5 / cm. 2 A 24-well cell culture vessel was created and evaluated. The results are shown in Table 1.

[0125] [Example 3] A cell culture vessel was produced in the same manner as in Example 2, except that the laser processing conditions were changed to a beam diameter of 40 μm. The hole diameter was 40 μm and the hole density was 0.5 / cm. 2 A 24-well cell culture vessel was created and evaluated. The results are shown in Table 1.

[0126] [Example 4] A cell culture vessel was produced in the same manner as in Example 2, except that the laser processing conditions were changed to a beam diameter of 80 μm. The hole diameter was 80 μm and the hole density was 0.5 / cm. 2A 24-well cell culture vessel was created and evaluated. The results are shown in Table 1.

[0127] [Example 5] A cell culture vessel was prepared in the same manner as in Example 2, except that 120 20 μm holes were formed in the PS film, and an evaluation was carried out. 2 ) with 5 pores (pore diameter 20 μm) per pore, resulting in a pore density of 2.5 pores / cm 2 A 24-well cell culture vessel was created. The holes were designed and perforated in advance so that they would be arranged as shown in Figure 1C in each well when the perforated film was attached to the frame of the 24-well culture plate. Specifically, one hole was placed at the center of a circle along the inside of the bottom of the circular well, and four holes were placed at the positions where a concentric circle (radius 5 mm) of the circle intersected with two orthogonal diagonals of the circle. The results are shown in Table 1.

[0128] [Example 6] A cell culture vessel was prepared in the same manner as in Example 2, except that 480 20 μm holes were formed in the PS film, and an evaluation was carried out. 2 ) with 20 pores (pore diameter 20 μm) and a pore density of 10 pores / cm 2 A 24-well cell culture vessel was created. The pores were designed and perforated in advance so that they would be arranged in each well as shown in Figure 1D when the perforated film was attached to the frame of the 24-well culture plate. Specifically, 4 pores were evenly arranged on concentric circles with radii of 1.6 mm, 3.2 mm, and 4.8 mm along the inside of the bottom of the circular well, and 8 pores were evenly arranged on a concentric circle with a radius of 6.4 mm. The results are shown in Table 1.

[0129] [Example 7] A cell culture vessel was prepared in the same manner as in Example 2, except that 2,400 20 μm holes were formed in the PS film, and an evaluation was carried out. 2 ) with 100 pores (pore diameter 20 μm) and a pore density of 50 pores / cm 2A 24-well cell culture vessel was created. The pores were designed and perforated in advance so that they would be arranged as shown in Figure 1E in each well when the perforated film was attached to the frame of the 24-well culture plate. Specifically, 4 pores were evenly arranged on a concentric circle with a radius of 0.8 mm along the inside of the bottom of the circular well, 16 pores on concentric circles with radii of 1.6 mm and 3.2 mm, and 32 pores on concentric circles with radii of 4.8 mm and 6.4 mm. The results are shown in Table 1.

[0130] [Example 8] A cell culture vessel was prepared in the same manner as in Example 2, except that 9,600 20 μm holes were formed in the PS film, and an evaluation was carried out. 2 ) with 400 pores (pore diameter 20 μm) and a pore density of 200 pores / cm 2 A 24-well cell culture vessel was created. The pores were designed and perforated in advance so that they would be arranged as shown in Figure 1F in each well when the perforated film was attached to the frame of the 24-well culture plate. Specifically, 8 pores were evenly arranged on concentric circles with radii of 0.8 mm, 1.6 mm, 2.1 mm, and 2.7 mm, 16 pores on concentric circles with radii of 3.2 mm, 3.7 mm, and 4.3 mm, 32 pores on concentric circles with radii of 4.8 mm and 5.3 mm, and 64 pores on concentric circles with radii of 5.9 mm, 6.4 mm, 6.9 mm, and 7.5 mm along the inside of the circular well bottom. The results are shown in Table 1.

[0131] [Example 9] Cell seeding concentration was 1.0 x 10 5 cells / cm 2 Except for changing the above, the evaluation was carried out in the same manner as in Example 2. The results are shown in Table 1.

[0132] [Example 10] A cell culture vessel was prepared and evaluated in the same manner as in Example 3, except that collagen treatment was not performed. The results are shown in Table 1.

[0133] [Example 11] Instead of the PS film, a 50 μm-thick TPX film (hereinafter referred to as PMP, manufactured by Mitsui Chemicals Tocello Co., Ltd., product name: Opulent X-88B, oxygen permeability 38240 cm 3 / (m 2 A cell culture vessel was prepared in the same manner as in Example 2, except that a 200-millimeter x 24 h x atm pore size and a pore density of 0.5 cells / cm were used. 2 A 24-well cell culture vessel was created and evaluated. The results are shown in Table 1.

[0134] [Example 12] A cell culture vessel was produced in the same manner as in Example 11, except that the laser processing conditions were changed to a beam diameter of 40 μm. The hole diameter was 40 μm and the hole density was 0.5 / cm. 2 A 24-well cell culture vessel was created and evaluated. The results are shown in Table 1.

[0135] [Example 13] Instead of the PS film, a biaxially oriented polyethylene terephthalate film (hereinafter referred to as PET, manufactured by Toray Industries, Inc., product name: Lumirror #50-T60, oxygen permeability 30 cm 3 / (m 2 A cell culture vessel was prepared in the same manner as in Example 2, except that a 200-millimeter x 24 h x atm pore size and a pore density of 0.5 cells / cm were used. 2 A 24-well cell culture vessel was created and evaluated. The results are shown in Table 1.

[0136] [Example 14] A cell culture vessel was produced in the same manner as in Example 13, except that the laser processing conditions were changed to a beam diameter of 40 μm. The hole diameter was 40 μm and the hole density was 0.5 / cm. 2 A 24-well cell culture vessel was created and evaluated. The results are shown in Table 1.

[0137] [Comparative Example 1] A cell culture vessel was produced in the same manner as in Example 1, except that the laser processing conditions were changed to an output of 150 mW and a beam diameter of 120 μm, and collagen coating was not performed. The pore diameter was 120 μm and the pore density was 0.5 / cm.2 A 24-well cell culture vessel was prepared and culture evaluation was performed. The results are shown in Table 2.

[0138] [Comparative Example 2] A cell culture vessel was produced and evaluated in the same manner as in Example 1, except that a PS film without pores formed by laser processing was used. The results are shown in Table 2.

[0139] [Comparative Example 3] Cell seeding concentration was 1.0 x 10 5 cells / cm 2 Except for changing the above, the evaluation was carried out in the same manner as in Comparative Example 2. The results are shown in Table 2.

[0140] [Comparative Example 4] A commercially available 24-well PS culture vessel (manufactured by Corning, oxygen permeability 200 cm) with a PS bottom thickness of 1000 μm was used. 3 / (m 2 A cell culture vessel was prepared and evaluated in the same manner as in Comparative Example 2, except that a 100% aqueous solution of 100% sodium hydroxide was used. The results are shown in Table 2.

[0141] [Comparative Example 5] Cell seeding concentration was 1.0 x 10 5 cells / cm 2 Except for changing the above, the evaluation was carried out in the same manner as in Comparative Example 4. The results are shown in Table 2.

[0142] [Comparative Example 6] A cell culture vessel was produced and evaluated in the same manner as in Example 11, except that a TPX film without holes formed by laser processing was used. The results are shown in Table 2.

[0143] [Comparative Example 7] A cell culture vessel was produced and evaluated in the same manner as in Example 13, except that a PET film without holes formed by laser processing was used. The results are shown in Table 2.

[0144] [Table 1]

[0145] [Table 2]

[0146] Table 1 shows that long-term culture of rat primary frozen hepatocytes is possible for up to 7 days when using a culture vessel in which the culture member of the present invention is placed at the bottom of the culture vessel. In addition, this culture vessel had no bending of the bottom, excellent shape stability, and no leakage of medium components. Furthermore, the cells adhered and proliferated uniformly over the entire culture surface, maintaining their morphology, demonstrating that this culture vessel has excellent cell adhesiveness.

[0147] Looking more closely at the effect of culturing primary frozen rat hepatocytes using the culture material of the present invention, the oxygen concentration in the medium was high after one day of culture, and oxygen was effectively supplied via the culture material. In addition, the metabolic activity value, which evaluated the metabolic activity of drugs as a function of the hepatocytes, was high, and it was revealed that the cell function was maintained normally. Furthermore, 5 cells / cm 2 It was shown that cell function can be maintained normally even in high-density culture.

[0148] Furthermore, Examples 1 to 9 showed that increasing the pore size and pore density improved the oxygen supply capacity from the bottom of the culture vessel, improved the saturated oxygen concentration, and also improved the metabolic activity value.

[0149] On the other hand, in Comparative Example 1, the pore size was 120 μm, so liquid leakage occurred from the pores, and culture evaluation was not possible. In Comparative Examples 2 to 5, polystyrene containers without pores were used, so the saturated oxygen concentration and metabolic activity value decreased, indicating that the cells could not express normal functions. In particular, 5 cells / cm 2In the high-density culture (Comparative Example 2 and Comparative Example 4), the metabolic activity value further decreased, indicating that the cells could not express normal functions. In Comparative Example 6, in which a polyethylene terephthalate container without pores was used, the saturated oxygen concentration decreased, and the metabolic activity value also decreased, indicating that the cells could not express normal functions.

[0150] Examples 1 to 9 and Comparative Example 2 as well as Examples 13, 14 and Comparative Example 6 showed that the use of a culture component having pores on the bottom surface of the culture vessel improved the oxygen supply capacity, improved the saturated oxygen concentration, and further improved the metabolic activity value.

[0151] From Example 10, the culture vessel of the present invention can culture 4×10 5 cells / cm 2 It was revealed that high-density culture of this strain showed high saturated oxygen concentration and metabolic activity.

[0152] From the above results, it became clear that the culture material of the present invention has excellent shape stability, oxygen supply property, and also good cell adhesion property, and is capable of culturing cells, etc., while retaining their functions even at high density.

Claims

1. A culture vessel having at least one well, at least a part or all of the bottom surface of which is formed from the following culture member: A culture member for culturing a cell, tissue, or organ on its culture surface, the culture member comprising a resin, the culture surface having at least one pore; The average pore size of the pores is 0.1 to 100 μm, The total opening area ratio of the pores (total opening area per well / total surface area per well of the culture surface of the culture member × 100 (%)) is 0.000001 to 0.3%; Culture material.

2. A culture vessel as described in claim 1, wherein the thickness of the culture member is 20 μm to 400 μm.

3. The culture vessel according to claim 1 or 2, wherein the resin is a thermoplastic resin.

4. The culture vessel according to any one of claims 1 to 3, wherein the resin is at least one selected from a polystyrene-based resin, a polyethylene terephthalate, and a 4-methyl-1-pentene polymer.

5. The number of pores per unit area is 0.01 to 250 pores / cm 2 The culture vessel according to any one of claims 1 to 4,

6. The culture vessel according to any one of claims 1 to 5, wherein the culture surface of the culture member has a coating layer of at least one material selected from the group consisting of natural polymer materials, synthetic polymer materials, and inorganic materials.

7. A step of contacting a cell, tissue or organ with the culture surface of the culture vessel according to any one of claims 1 to 6; and A method for culturing cells, tissues, or organs, comprising the step of: supplying oxygen to the cells, tissues, or organs in contact with the culture surface.

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