JP7627566B2 - Culture material and its uses - Google Patents

Description

The present invention relates to a culture member and its use.

Cells, tissues, and organs can only be cultured under conditions suitable for their growth, so they must be placed in a culture vessel such as a dish, plate, or flask along with a culture medium containing the appropriate nutrients, and the culture vessel must be placed in an incubator that can maintain the temperature, humidity, and gas concentration at specified levels.

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 culture medium, it is necessary to supply gases such as oxygen and carbon dioxide into the culture vessel, so that the culture vessel made of a material such as glass or polystyrene with low gas permeability has an opening at the top of the culture vessel, such as a cap or a lid, to ensure gas supply from inside the incubator to the inside of the vessel. However, since 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 the culture medium, the oxygen diffusion rate in the culture medium becomes the rate limiting factor, and the supply of oxygen 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 order to achieve a state closer to in vivo, the supply of oxygen to the cultured cells is insufficient, which has also been known to hinder cell proliferation and growth.

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 have low strength and may be easily broken, and may be easily bent when a culture medium is placed in 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 deformation of the container or 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.

Japanese Utility Model Application Publication No. 1-112697

Yasuyuki Sakai, "Improvement of oxygen supply in liver culture", [online], Hepatocyte Research Society, [searched April 9, 2019], Internet <http://hepato.umin.jp/kouryu/kouryu28.html>

The present invention was made in consideration of the above circumstances, and aims to provide a culture component and culture device that have excellent shape stability and oxygen supply properties, and that enable high-density culture while maintaining cellular function.

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 the group consisting of polystyrene-based resins, polyethylene terephthalate, and 4-methyl-1-pentene polymers.
[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 culture member according to any one of [1] to [4], wherein the number of pores per unit area is 0.01 to 250 pores/cm ² .
[6] The culture member according to any one of [1] to [5], wherein the culture member has a film-like or sheet-like shape.
[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 instrument 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 method for culturing cells, tissues, or organs, comprising: 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 step of supplying oxygen to the cell, tissue, or organ in contact with the culture surface.
[11] The culture method according to [10], wherein the resin is a thermoplastic resin, the average pore size of the pores is 0.1 to 100 μm, and the number of the pores per unit area is 0.01 to 250 pores/cm ² .

The present invention provides a culture component and culture device that have excellent shape stability and oxygen supply properties, and that enable high-density culture while maintaining cellular function.

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. 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.

When referring to a numerical range, "A-B" means that the range is greater than or equal to A and less than or equal to B, unless otherwise specified. For example, "1-5%" means greater than or equal to 1% and less than or equal to 5%.

[Culture material]
The culture member of the present invention (obtained by) is a member for culturing cells, tissues or organs (hereinafter also referred to as cells, etc.) on its culture surface, and is characterized in that the culture member 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.

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 the form of a film or sheet, at least one surface of the film or sheet culture member can be suitably used as a culture device as a culture surface.

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

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

When the thickness of the culture member is within the above range, it tends to be easier to drill fine holes, has excellent strength, and has excellent oxygen supply properties.

When the culture member of the present invention is placed on the bottom surface of a container to produce a culture device such as a dish (also called a petri dish), flask, insert or plate, the thickness of the culture member 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 device, by adjusting it to the above upper and lower limit ranges, it is easy to obtain an appropriate oxygen concentration in the medium necessary for cell proliferation, and it is easy to produce a suitable culture device with sufficient strength.

The culture member of the present invention may have a surface that is microfabricated with other features than pores in order to create spheroids or improve the scaffolding function of cells. The method of microfabrication may be appropriately selected from cutting, photolithography, direct electron beam 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 other molding methods, plating, etc. 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.

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 the surface of a culture member is microfabricated to create microchannels or reaction vessels for application in bioresearch or chemical engineering. For example, devices called microTAS (micro Total Analysis Systems) or Lab on a Chip are being used, and their application as next-generation culture devices is being promoted. One aspect of the present invention is a microchannel device including the culture member of the present invention.

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 include hydrophilization treatments 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.

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 subjecting the surface of the culture member to hydrophilization treatment, 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 subjecting the surface of the culture member to hydrophilization treatment, it becomes easier to coat the culture surface of the culture device with natural polymer materials, synthetic polymer materials, or inorganic materials. In particular, it becomes easier to uniformly load and adhere collagen 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.

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 including a resin is formed, and the film or sheet is molded as necessary to form a molded product having a desired shape, and pores are formed in this film, sheet or molded product as described above to produce the culture member. The film, sheet or other molded product that will become the culture member can also be obtained by direct molding using methods such as extrusion molding, solution casting molding, injection molding, and blow molding.

Specific methods for forming the film or sheet include, for example, the usual inflation method and T-die extrusion method. The production is usually carried out under heating. When the T-die extrusion method is used, the extrusion temperature differs 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 also preferably 20°C to 100°C, and more preferably 30°C to 90°C.

The film or sheet may also be produced by a solution casting method in which the resin is dissolved in a solvent and 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 hydrocarbon solvents such as cyclohexane, hexane, decane, and toluene may be used. Two or more types of solvents may also be mixed, taking into consideration the solubility of the resin and the drying efficiency. The polymer solution can be 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 process into a film or sheet.

The culture member of the present invention is preferably a cell culture member, and more preferably a hepatic cell culture member.

<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.

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

<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.

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 with other monomers are also collectively referred to as "4-methyl-1-pentene polymers."

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.

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, or oxycarboxylic acids (e.g., p-oxybenzoic acid), and 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.

Among these, polyolefin resins, polystyrene resins, and polyester resins are preferred because of their 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 resins, and polyethylene terephthalate are more preferred.

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

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 2 mm thick injection test piece made of a thermoplastic resin, the total amount of transmitted light is measured in accordance with JIS K7361 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 x (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.

<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.
A copolymer of 4-methyl-1-pentene and another monomer, 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 a copolymer of 4-methyl-1-pentene and another monomer, 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) is preferable because it has high strength, is unlikely to break or crack, and has little deflection even when used as a member.

The 4-methyl-1-pentene polymer is preferably at least one polymer selected from 4-methyl-1-pentene homopolymers and copolymers of 4-methyl-1-pentene with at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), and more preferably a copolymer of 4-methyl-1-pentene with at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

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 end breakage tends to be less likely to occur. In addition, the rate of defective products of the culture member is reduced.

The olefins may be used alone or in combination of two 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.

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 an α-olefin 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 an α-olefin 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 based on the total amount of repeating structural units in the 4-methyl-1-pentene polymer being 100 mol%. 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.

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.

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 that can be used include the compounds described in paragraphs [0035] to [0041] of JP 2013-169685 A.

The 4-methyl-1-pentene polymer may be used alone or in combination of two or more.

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. 4-methyl-1-pentene polymers that satisfy the above requirements from other manufacturers can also be used preferably. These commercially available products can be used alone or in combination of two or more.

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 culture components such as culture equipment 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 culture equipment containing 4-methyl-1-pentene polymers makes it easy to observe cultured cells. In addition, they show excellent chemical resistance to most chemicals and are not prone to sorbing drugs, so they are also suitable for use in drug discovery screening and diagnostic applications. 4-methyl-1-pentene polymers can be heat sealed, and are easy to heat-seal not only between themselves but also with other materials. In addition, they can be thermoformed, so they can be easily molded into culture equipment of any shape, and can also be easily molded using the imprint method or insert method, for example.

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. 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. Orthodichlorobenzene is preferably used as the solvent for GPC. Examples of measurement conditions include those shown in the examples described below, but are not limited to these measurement conditions.

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 it is easy to produce a film with a uniform surface. In addition, when producing by a solution casting method, the solubility in the solvent is improved, and defects such as gels in the film are easily suppressed, and it is easy to produce a film with a uniform surface.

In addition, by setting the weight-average molecular weight (Mw) to the above lower limit or higher, the culture member tends to have sufficient strength. Furthermore, by setting the molecular weight distribution within the above range, 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 bending during molding and cracks during cutting.

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. When two or more types of 4-methyl-1-pentene polymers are used, the Mw and Mw/Mn of each resin may be within the above range.

Because 4-methyl-1-pentene-1 polymer has the above-mentioned excellent properties, a culture device 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 excellent material for culture components.

<Method for producing 4-methyl-1-pentene polymer>
The method for producing the 4-methyl-1-pentene polymer may be any method capable of polymerizing 4-methyl-1-pentene, olefin, and other monomers. In addition, a chain transfer agent, such as hydrogen, may be present 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. From the viewpoint of polymerization efficiency, it is preferable to carry out the polymerization at 20°C to 80°C, and it is 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.

<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, for example, 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., and 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.

Examples of reactors include complete mixing reactors, laminar flow reactors, and loop reactors in which a portion of the polymerization liquid is withdrawn while the polymerization is proceeding. There are no particular limitations on the order in which these reactors are arranged.

When polymerizing the polystyrene 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 the molecular weight, and the like, 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.

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 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.

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

The conditions for the devolatilization step are not particularly limited. For example, when the polymerization of 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% by mass or less, more preferably 40% by mass or less. The devolatilization process can remove volatile matters such as unreacted materials (styrene monomers) and/or solvents.

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

<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 terephthalic acid ester-forming derivatives include alkyl esters of terephthalic acid such as dimethyl terephthalate. Examples of ethylene glycol ester-forming derivatives include fatty acid esters such as ethylene glycol fatty acid esters.

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 glycols 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.

<Method for producing polyethylene terephthalate>
Polyethylene terephthalate can be produced by known polycondensation methods, ring-opening polymerization methods, and the like. Either a batch polymerization method or a continuous polymerization method may be applied, but the 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 an ester exchange reaction or a direct polymerization reaction may be applied, but the direct polymerization reaction is preferred in terms of cost. In order to efficiently proceed with the esterification reaction, the ester exchange reaction, and/or the 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 per 100 parts by mass of polyethylene terephthalate.

There are no particular limitations on the type of polyethylene terephthalate as long as it does not impede the effects of the present invention, but crystalline polyethylene terephthalate (C-PET) is preferred from the standpoint of mechanical strength and moldability.

When a polyethylene terephthalate film is used as a culture material, the polyethylene terephthalate film may or may not be stretched. When a polyethylene terephthalate film is stretched, it undergoes crystal orientation, which is preferable from the standpoint of mechanical properties. The preferred stretching method is biaxial stretching.

When the culture component is formed from 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 component. If a large amount of components other than resin are contained, not only will the oxygen permeability decrease, but the transparency and strength will also decrease.

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

<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 and penetrating the culture member, and is different from the unevenness generated by the surface processing of the culture member.

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, or a cross. 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.

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

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.

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 measuring the pore size by observing the pores with a microscope, electron microscope, etc., 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 minor axis of the rectangle. The method for estimating the average pore size by the diameter of the instrument used for perforation, such as a needle or laser beam, is preferred because it is simple.

The culture member of the present invention preferably has pores with an average pore size of 0.1 to 100 μm at a pore density of 0.01 to 250 pores/cm ² , more preferably has pores with an average pore size of 1 to 90 μm at a pore density of 0.1 to 230 pores/cm ² , and even more preferably has pores with an average pore size of 3 to 85 μm at 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.

The opening area of the pores is preferably 0.005 to 8000 μm ² , more preferably 0.1 to 7000 μm ² , and even 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.

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 x 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.

When the culture member of the present invention has multiple pores, the pore sizes of the multiple 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 in this way, the medium is less likely to leak, the oxygen supply is excellent, and the metabolic activity of the cells tends to be high.

When the culture member of the present invention has a plurality of pores, the arrangement of the pores is not particularly limited, and they may be arranged in a fixed pattern or randomly. Examples of an arrangement in a fixed pattern include 60° staggered, 45° staggered, parallel, herringbone, etc., and it is also possible to draw multiple polygons or circles of different sizes that share a common center and arrange the pores at equal intervals on their sides or circumferences. Arranging in a fixed pattern is preferable because it tends to reduce medium leakage, provide excellent oxygen supply, and increase metabolic activity of cells.

The spacing between multiple pores is not particularly limited, but is preferably larger than the average pore size of the pores, and more preferably is a constant spacing. The spacing between 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 spacing between multiple 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.

The multiple pores may be connected to each other inside the culture member.

The method for perforating the pores is not particularly limited, and any known method can be used. Examples of methods for perforating the pores include a method in which a very thin needle such as a microsyringe is heated and pierced into the film to melt and open the pores, and a method using a laser.

There are various types of lasers, such as carbon dioxide lasers, YAG lasers, semiconductor lasers, and argon lasers, and there are no particular limitations as long as they can drill holes. Among them, it is preferable to use a YAG laser with a wavelength range of 100 to 1100 nm, as it is possible to drill holes with a small diameter. An ultraviolet YAG laser with a wavelength range of 100 to 400 nm is more preferable, as it is absorbent and possible to drill holes even in transparent materials that transmit visible light, and a deep ultraviolet YAG laser with a wavelength range of 260 to 270 nm is even more preferable. When a deep ultraviolet YAG laser with a wavelength range of 260 to 270 nm is used, the output is preferably 10 to 200 mW. If the output range is within the above range, workability is good and the desired diameter of the hole can be achieved while avoiding excessive irradiation.

The scanning speed of the laser beam is preferably 1 to 100 mm/s. If the moving speed of the laser beam is within this range, workability is good and it is possible to achieve perforation of the desired diameter while avoiding excessive irradiation.

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 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, pores are perforated in the film or sheet, and the perforated film or sheet is molded into a culture member having a desired shape. Alternatively, preferably, the pores are perforated after a molded body having a desired shape is prepared by molding the resin-containing component.

Examples of methods for perforating pores simultaneously with the production of a molded body from a resin-containing component 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 resulting from 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 heated gas by the melt-blown method, and the thermoplastic resin is stretched by the heated gas to form a fibrous resin, thereby obtaining a culture member having pores.

<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.

In the present invention, tissue means a collection of similar cells that perform a similar function. The tissue is not particularly limited, and examples include epithelial tissue, connective tissue, muscle tissue, and nerve tissue. Because of their high oxygen requirement, the tissue is preferably hepatic lobule, cardiac muscle tissue, nerve tissue, or skeletal muscle tissue, and more preferably hepatic lobule.

In the present invention, an organ refers to a group of tissues that work together to accomplish a common purpose. The organ is not particularly limited, and may be, for example, the lungs, heart, liver, kidneys, spleen, pancreas, gallbladder, esophagus, stomach, skin, or brain. Because of their high oxygen demand, the organ is preferably the skin, kidneys, liver, or brain, and more preferably the liver.

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.

<Hepatocytes>
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.

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, but examples 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.

The hepatocytes may be derived from any mammal, but are preferably derived from humans, cows, dogs, cats, pigs, minipigs, rabbits, hamsters, rats, or mice, and more preferably from humans, rats, mice, or cows.

The hepatocytes may be a cell population that includes cell types other than hepatocytes, for example, a cell population that includes 20% or more, 30% or more, 40% or more, or 50% or more of hepatocytes.

<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.

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

Three-dimensional culture is the intentional cultivation of cells in a three-dimensional space, and may be either a scaffold type in which cells are cultivated in a scaffold material, 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 ^™ , collagen gel, laminin, alginate hydrogel, and vitrigel are preferred as scaffold materials because they allow efficient cultivation of cells.

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

When using the culture member of the present invention for culture, in other words, when culturing using the culture apparatus of the present invention, the cell culture density is preferably 0.1 x 10 ⁵ cells/cm ² to 10.0 x 10 ⁵ cells/cm ² , more preferably 0.5 x 10 ⁵ cells/cm ² to 5.0 x 10 ⁵ cells/cm ² , and even more preferably 1.0 x 10 ⁵ cells/cm ² to 4.0 x 10 ⁵ cells/cm ² .
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 culture even when the cell culture density is high. Generally, the cell density in a living body is said to be about 2.5×10 ⁵ cells/cm ² , but the culture member of the present invention is preferable because it can be used for culture at a cell culture density similar to that of a living body, and therefore it can be used for culture in vitro under conditions closer to those in vivo.

<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.

The culture device is typically used in an apparatus such as an incubator, mass culture apparatus, or perfusion culture apparatus.

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 like, preferably culture vessels.

The culture device is preferably a culture device 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 of 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells, etc. In general, a culture device 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 of 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.

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, and therefore 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 via 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 retaining their functions.

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

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.

The culture device may be a culture device whose culture surface is coated with a natural polymer material, a synthetic polymer material, or an inorganic material.

The coated culture instrument may be obtained, for example, by coating the culture instrument with a natural polymer material, a synthetic polymer material, or an inorganic material by a known method, or by using an already coated culture member on at least the culture surface of the culture instrument.

The coated culture device has better adhesion and proliferation properties for cells, etc. This is thought to be because the natural polymer material, synthetic polymer 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 the culture member or culture device with a natural polymer material, synthetic polymer material, or inorganic material and use it as a culture device.

The natural polymeric materials, synthetic polymeric materials, and inorganic materials are not particularly limited, but examples of natural polymeric 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 polymeric materials include polyglucose acid, polylactic acid, polyethylene glycol, polycaprolactone, synthetic peptides, and synthetic proteins; synthetic polymeric materials include polyethylene glycol, polyhydroxyethyl methacrylate, and polyethyleneimine; and inorganic materials include β-tricalcium phosphate and calcium carbonate.

The natural polymeric material, synthetic polymeric material, or inorganic material may also include a vitrigel obtained by vitrifying a hydrogel such as a conventional extracellular matrix component and then rehydrating it. For example, a collagen vitrigel made of a high-density collagen fiber network made from collagen, which is one of the extracellular matrix components, may also be used.

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

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 examples thereof include physical disinfection methods such as steam circulation, boiling, intermittent irradiation, and ultraviolet light; chemical disinfection methods 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 these, ethanol disinfection, high-pressure steam sterilization, gamma ray sterilization, and ethylene oxide gas sterilization are preferred because they are easy to operate and can perform sufficient sterilization. These disinfection and sterilization methods may be performed alone or in combination of two or more.

The method for manufacturing 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 manufactured by the same method as the method for manufacturing the culture member. When only 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 joining method, 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.

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

Cultivation Method
The method for culturing cells or the like of the present invention is a culture component for culturing cells, tissues, or organs on its culture surface, the culture component containing a resin, and the culture surface having at least one pore, and is a method for culturing cells, tissues, or organs, the method comprising: a step of contacting the cells, tissues, or organs with the culture surface of the culture component; and a step of 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.
It is preferable that the resin is a thermoplastic resin, the average pore size of the pores is 0.1 to 100 μm, and the number of the pores per unit area is 0.01 to 250 pores/cm ² .
The method for culturing cells or the like is preferably a method for culturing cells, and more preferably a method for culturing hepatocytes.

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.

[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) was diluted 6-fold with a 0.001 M hydrochloric acid solution to prepare a 0.5 mg/mL collagen solution.

[Cell types and medium preparation]
A medium was added to a centrifuge tube (50 ml) containing a cell suspension containing rat primary frozen hepatocytes. The medium was composed of 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) solution diluted to 3.0 g/mL with water for injection (Fuso Pharmaceutical Industries), 1.5 μL of dexamethasone (for biochemistry, Fujifilm Wako Pure Chemical Industries) solution diluted to 1 × 10 ^-3 M with ethanol (for molecular biology, Fujifilm Wako Pure Chemical Industries), 21 μL of hydrocortisone (for culture, Fujifilm Wako Pure Chemical Industries) solution diluted to 36 mM with ethanol, and epidermal growth factor (EGF) diluted to 20 μg/mL using BSA solution diluted to 1.0 mg/mL with water for injection. factor, EGF, for cell biology, Fujifilm Wako Pure Chemical Industries) solution 15 μL, insulin solution (10 mg/mL in HEPESS, SIGMA) 8.7 μL, penicillin-streptomycin solution (containing 5000 units/mL penicillin and 5000 μg/mL streptomycin, for culture, Fujifilm Wako Pure Chemical Industries) 0.3 mL, and D-MEM medium (containing 4500 mg/mL D-glucose, 584 mg/mL L-glutamine, 15 mg/mL phenol red, 110 mg/mL sodium pyruvate, and 3700 mg/mL sodium bicarbonate, for culture, Fujifilm Wako Pure Chemical Industries) 13 mL were added to adjust the medium. The cell density was adjusted by adjusting the cell number of a cell suspension containing frozen rat primary hepatocytes. Unless otherwise specified, the cells were cultured at a cell density of 4.0 x ¹⁰⁵ cells/ ^cm2 , and in Example 9 and Comparative Examples 1, 3, and 5, the cells were cultured at a cell density of 1.0 x ¹⁰⁵ cells/ ^cm2 .

[Oxygen permeability measurement]
The oxygen transmission rate of the film before perforation was measured at a temperature of 23°C and a humidity of 0% RH using a differential pressure gas transmission rate measuring device MT-C3 manufactured by Toyo Seiki Seisakusho Co., Ltd. The diameter of the measurement area was 70 mm (transmission area: 38.46 ^cm2 ). An aluminum mask was applied to the sample in advance, and the actual transmission area was set to 5.0 ^cm2 .

[Presence or absence of deflection]
One day after cell seeding, the culture vessels were removed from the incubator, and the bottoms of the four culture vessels were 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 vessels were made and no sagging of the film was observed, the result was rated as "no bending," and if there was a change in at least one of the four culture vessels compared to when the vessels were made and sagging of the film was observed, the result was rated as "bending."

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

[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 with a jack, and measured for 1 hour. The oxygen partial pressure (hPa) in the medium 1 hour after the start of measurement was divided by 1013 hPa (1 atmospheric pressure) and multiplied by 100 to calculate the value (%), which was used as the oxygen concentration in the medium. Measurements were performed in three representative wells, and the average value was calculated.

[Measurement of metabolic activity value]
After removing the medium from the culture vessel 24 hours after cell seeding, Luciferin-CEE diluted in the medium was added and cultured for another 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 Reagent and Reconstitution Buffer was added and reacted for 1 hour at room temperature in the dark. After 1 hour, the luminescence amount (Relative Light Unit, RLU) was measured with a luminometer. Measurements were performed in three representative wells, and the average value was calculated.

The amount of protein 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, 22000 x g, 10 minutes). The supernatant was then removed, 100 μL of 0.1 M sodium hydroxide solution was added, and the amount of protein was measured using Pierce ^TM BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). The absorbance at a wavelength of 450 nm was measured using a plate reader (SPECTRA max PLUS384, manufactured by Molecular Devices).

The metabolic activity (pmol/L) of the Luciferin-CEE solution obtained with a luminometer was measured using a P450-Glo ^™ CYP1A1 Assay kit (Promega), and the metabolic activity value (pmol/min/mg protein) was calculated by dividing the metabolic activity by the protein amount obtained from the absorbance and the reaction time of the Luciferin-CEE solution.

[Example 1]
A 50 μm thick unstretched film of polystyrene (hereinafter referred to as PS) homopolymer cut into a 12 cm x 8 cm rectangle (manufactured by Okura Kogyo Co., Ltd., product name: Ceromer S-2, oxygen permeability 3310 cm ³ /(m ² x 24 h x atm)) was laser processed using a UV laser processing machine (manufactured by Takano Co., Ltd.) under conditions of wavelength 266 nm, output 50 mW, scanning speed 10 mm/sec., and beam diameter 5 μm, to form 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 a normal 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 an open bottom of each well via a medical adhesive (manufactured by 3M). One pore was formed per well (circular shape with a diameter of 16 mm, area approximately 2 cm ² ) to create a 24-well cell culture vessel with a pore density of 0.5 pores/cm ² . The pores were designed in advance to be arranged in each well as shown in FIG. 1B when the perforated film was attached to the 24-well culture plate frame, and perforated. Specifically, one pore was arranged 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, after which 0.5 mL of 0.5 mg/mL collagen solution was added to the culture surface of the culture vessel, and then excess collagen solution was 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.

Next, the culture medium (0.5 mL) containing rat primary frozen hepatocytes was seeded on each of the culture surfaces of the five culture vessels 1 with 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 in four culture vessels. Then, the oxygen concentration in the culture medium was measured using one of the four culture vessels 1. The metabolic activity was measured in 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 7 days of culture. The results are shown in Table 1. Figure 2 shows the results of observing the cells one day after cell seeding with a phase contrast microscope.

[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. A 24-well cell culture vessel with a hole diameter of 20 μm and a hole density of 0.5 pcs/ ^cm2 was produced and evaluated. The results are shown in Table 1.

[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, and a 24-well cell culture vessel with a hole diameter of 40 μm and a hole density of 0.5 pcs/ ^cm2 was produced and evaluated. The results are shown in Table 1.

[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. A 24-well cell culture vessel with a hole diameter of 80 μm and a hole density of 0.5 pcs/ ^cm2 was produced and evaluated. The results are shown in Table 1.

[Example 5]
A cell culture vessel was prepared and evaluated in the same manner as in Example 2, except that 120 20 μm holes were formed in the PS film. Five pores (pore diameter 20 μm) were formed per well (circular shape with a diameter of 16 mm, area of about 2 cm ² ), and a 24-well cell culture vessel with a pore density of 2.5 pores/cm ² was prepared. The pores were designed in advance so that they would be arranged in each well as shown in FIG. 1C when the perforated film was attached to the frame of a 24-well culture plate, and perforated. Specifically, one pore was placed at the center of a circle along the inside of the bottom surface of a circular well, and four pores were placed at the position where a concentric circle (radius 5 mm) of the circle intersects with two diagonals that are perpendicular to the circle. The results are shown in Table 1.

[Example 6]
A cell culture vessel was prepared and evaluated in the same manner as in Example 2, except that 480 20 μm holes were formed in the PS film. 20 pores (pore diameter 20 μm) were formed per well (circular shape with a diameter of 16 mm, area of about 2 cm ² ), and a 24-well cell culture vessel with a pore density of 10 pores/cm ² was prepared. The pores were designed in advance so that they would be arranged in each well as shown in FIG. 1D when the perforated film was attached to the frame of a 24-well culture plate, and perforated. Specifically, 4 pores were arranged evenly on concentric circles with radii of 1.6 mm, 3.2 mm, and 4.8 mm, and 8 pores were arranged evenly on a concentric circle with a radius of 6.4 mm, along the inside of the bottom surface of the circular well. The results are shown in Table 1.

[Example 7]
A cell culture vessel was prepared and evaluated in the same manner as in Example 2, except that 2400 20 μm holes were formed in the PS film. A 24-well cell culture vessel was prepared with 100 pores (pore diameter 20 μm) formed per well (circular shape with a diameter of 16 mm, area of about 2 cm ² ) and a pore density of 50 pores/cm ² . The pores were designed and perforated in advance so that they would be arranged as shown in FIG. 1E in each well when the perforated film was attached to the frame of a 24-well culture plate. Specifically, pores were evenly arranged on a concentric circle with a radius of 0.8 mm, 16 on concentric circles with radii of 1.6 mm and 3.2 mm, and 32 on concentric circles with radii of 4.8 mm and 6.4 mm, respectively, along the inside of the bottom surface of the circular well. The results are shown in Table 1.

[Example 8]
A cell culture vessel was prepared and evaluated in the same manner as in Example 2, except that 9,600 20 μm holes were formed in the PS film. A 24-well cell culture vessel was prepared with 400 pores (pore diameter 20 μm) formed per well (circular shape with a diameter of 16 mm, area of approximately 2 cm ² ) and a pore density of 200 pores/cm ² . The pores were designed and perforated in advance so that they would be arranged in each well as shown in FIG. 1F when the perforated film was attached to the frame of a 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 inner side of the bottom of the circular well. The results are shown in Table 1.

[Example 9]
The evaluation was carried out in the same manner as in Example 2, except that the cell seeding concentration was changed to 1.0×10 ⁵ cells/cm ^2. The results are shown in Table 1.

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

[Example 11]
A cell culture vessel was prepared in the same manner as in Example 2, except that a 50 μm thick TPX film (hereinafter referred to as PMP, manufactured by Mitsui Chemicals Tocello Inc., product name: Opulent X-88B, oxygen permeability 38,240 cm ³ /(m ² × 24 h × atm)) was used instead of the PS film. A 24-well cell culture vessel with a pore size of 20 μm and a pore density of 0.5/cm ² was prepared and evaluated. The results are shown in Table 1.

[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, and a 24-well cell culture vessel with a hole diameter of 40 μm and a hole density of 0.5 pcs/ ^cm2 was produced and evaluated. The results are shown in Table 1.

[Example 13]
A cell culture vessel was prepared in the same manner as in Example 2, except that a 50 μm-thick 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 × 24 h × atm)) was used instead of the PS film. A 24-well cell culture vessel with a pore size of 20 μm and a pore density of 0.5/cm ² was prepared and evaluated. The results are shown in Table 1.

[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, and a 24-well cell culture vessel with a hole diameter of 40 μm and a hole density of 0.5 pcs/ ^cm2 was produced and evaluated. The results are shown in Table 1.

[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. A 24-well cell culture vessel with a hole diameter of 120 μm and a hole density of 0.5 cells/ ^cm2 was produced, and culture evaluation was performed. The results are shown in Table 2.

[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.

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

[Comparative Example 4]
A cell culture vessel was prepared and evaluated in the same manner as in Comparative Example 2, except that a commercially available 24-well PS culture vessel (manufactured by Corning, oxygen permeability 200 ^cm3 /( ^m2 x 24h x atm)) with a PS bottom thickness of 1000 µm was used. The results are shown in Table 2.

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

[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.

[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.

Table 1 shows that long-term culture of frozen rat primary hepatocytes is possible for up to 7 days when using a culture vessel with the culture member of the present invention 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 culture 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 adhesive properties.

Looking more closely at the effect of culturing primary frozen rat hepatocytes using the culture member of the present invention, the oxygen concentration in the medium was high after one day of culture, and oxygen was effectively supplied through the culture member. In addition, the metabolic activity value, which evaluated the metabolic activity of drugs as a function of hepatocytes, was high, and it became clear that the cell function was normally maintained. Furthermore, it was shown that the cell function could be normally maintained even in high-density culture of 4 x ¹⁰⁵ cells/ ^cm2 .

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.

On the other hand, in Comparative Example 1, since the pore size was 120 μm, liquid leakage occurred from the pores, and culture evaluation was not possible. In Comparative Examples 2 to 5, since a polystyrene container without pores was used, the saturated oxygen concentration and metabolic activity value also decreased, indicating that the cells could not express normal functions. In particular, in the high-density culture of 4×10 ⁵ cells/cm ² (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 and metabolic activity value also decreased, indicating that the cells could not express normal functions.

From Examples 1 to 9 and Comparative Example 2 as well as Examples 13, 14, and Comparative Example 6, it was shown that the use of a culture member having pores on the bottom surface of the culture vessel improves the oxygen supply capacity, improves the saturated oxygen concentration, and further improves the metabolic activity value.

From Example 10, it was revealed that the culture vessel of the present invention exhibited high saturated oxygen concentration and metabolic activity value in high-density culture of 4×10 ⁵ cells/cm ² even when not collagen-coated.

These results demonstrate that the culture material of the present invention has excellent shape stability, oxygen supply, and cell adhesion, making it possible to culture cells at high densities while retaining their functions.

Claims (7)

A culture vessel having at least one well, at least a part or the whole of a bottom surface of which is formed from the culture member described below;
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. The culture vessel according to claim 1, wherein the culture member has a thickness of 20 μm to 400 μm. The culture vessel according to claim 1 or 2 , wherein the resin is a thermoplastic resin. 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. The culture vessel according to any one of claims 1 to 4, wherein the number of pores per unit area is 0.01 to 250 pores/ ^cm2 . 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 . A method for culturing a cell, tissue, or organ, comprising: 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 step of supplying oxygen to the cell, tissue, or organ in contact with the culture surface.