JP2022099831A - Culture member and applications thereof - Google Patents

Abstract

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

Description

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

Since cells, tissues, and organs can only be cultured under conditions suitable for growth, they should be placed in a culture vessel such as a dish, plate, or flask together with a medium containing appropriate nutrients, and the temperature, humidity, and gas concentration of the culture vessel should be adjusted. It is necessary to stand in an incubator that can be maintained at a predetermined level.

Furthermore, in order to efficiently realize the above culture, sufficient and appropriate oxygen supply must be performed.
In order to supply oxygen to cells and carbon dioxide for adjusting the pH of the medium, it is necessary to supply gas such as oxygen and carbon dioxide into the culture vessel. The culture container made of the material is provided with an opening at the upper part of the culture container such as a cap and a lid to secure gas supply from the inside of the incubator to the inside of the container. However, the cultured cells are usually adhered to the bottom surface of the culture vessel, or are often suspended near the bottom surface, and the upper surface is covered with the medium, so that the oxygen diffusion rate in the medium becomes rate-determining. In particular, it has long been known that the oxygen supply to the cultured cells at the bottom is insufficient and the proliferation and growth of the cells are hindered. It is also known that when cells are cultured by increasing the cell density in order to bring the cells closer to in vivo, the oxygen supply to the cultured cells is insufficient and the proliferation and growth of the cells are hindered.

In order to promote the supply of oxygen to cells, there are means such as increasing the oxygen partial pressure in the incubator, but a dedicated incubator for controlling the oxygen partial pressure is required, and generally in the atmosphere. The cost is high compared to the culture device for culturing. 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. Care must be taken when handling oxygen cylinders.
As a simple and practical method for improving oxygen supply, a method of using a material having high oxygen permeability such as polydimethylsiloxane (PDMS) (Non-Patent Document 1) and polybutadiene (Patent Document 1) for the culture surface is known. .. However, such a material having high oxygen permeability may have low strength and may be easily torn, and may be easily bent when a medium is added, so that the shape may become unstable. When the culture device is bent, the cells attached to the inner wall of the culture device are peeled off due to the deformation of the container or the impact caused by the deformation, and the cells being cultured gather in the bent place. Cannot be cultivated. In addition, depending on the material used for the culture device, the sorption of the drug is likely to occur, which limits its use in drug discovery screening and diagnostic purposes.

Jikkenhei 1-112697 Gazette

Yasuyuki Sakai, "Improvement of Oxygen Supply in Liver Culture", [online], Hepatocyte Study Group, [Searched April 9, 2019], Internet <http://hepato.umin.jp/kouryu/kouryu28.html >

The present invention has been made in view of the above circumstances, and provides a culture member and a culture device having excellent shape stability and oxygen supply property and capable of culturing at high density while maintaining cell functions. The task is to do.

The present inventors have diligently studied to solve the above problems. As a result, it has been found that the above-mentioned problems can be solved by a culture member having the following constitution, and the present invention has been completed. The present invention is, for example, the following [1] to [11].
[1] A culture member for culturing cells, tissues, or organs on the culture surface thereof, wherein the culture member contains a resin, and the culture surface has 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 polystyrene-based resin, polyethylene terephthalate, and 4-methyl-1-pentene polymer.
[4] The culture member according to any one of [1] to [3], wherein the average pore diameter of the pores is 0.1 to 100 μm.
[5] The culture member according to any one of [1] to [4], wherein the number of the pores per unit area is 0.01 to 250 per cm ² .
[6] The culture member according to any one of [1] to [5], wherein the culture member has a film-like shape or a sheet-like shape.
[7] A culture instrument in which at least the culture surface is formed of the culture member according to any one of [1] to [6].
[8] The incubator according to [7], which has at least one well.
[9] The method according to [7] or [8], wherein the culture surface of the culture member has a coating layer of at least one material selected from the group consisting of a natural polymer material, a synthetic polymer material, and an inorganic material. Incubator.
[10] A step of contacting a cell, tissue or organ with the culture surface of the culture member according to any one of [1] to [6]; and supplying oxygen to the cell, tissue or organ in contact with the culture surface. Step; A method of culturing a cell, tissue, or organ comprising.
[11] The resin is a thermoplastic resin, the average pore diameter of the pores is 0.1 to 100 μm, and the number of the pores per unit area is 0.01 to 250 per cm ² . The culture method according to [10].

According to the present invention, it is possible to provide a culture member and a culture device having excellent shape stability and oxygen supply property and capable of culturing at a high density while maintaining the function of cells.

FIG. 1A is a diagram showing an example of 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 is an image of cells observed under a microscope one day after cell seeding in Examples 1 to 3 and Comparative Examples 4 and 5.

The description of "A to B" regarding the numerical range indicates that it is A or more and B or less unless otherwise specified. For example, the description of "1 to 5%" means 1% or more and 5% or less.

[Culture member]
The culture member according to the present invention (obtained in) is a member for culturing cells, tissues or organs (hereinafter, also referred to as cells) on the culture surface, the culture member contains a resin, and the culture surface is at least. It is characterized by having one pore.
Here, the culture member means a member that constitutes at least a part of a culture instrument used for culturing cells and the like. When the culture member is a part of the culture device, at least the culture surface for culturing cells and the like is composed of the culture member of the present invention. Here, the culture surface means a surface on which a medium is formed, a surface on which cells or the like are seeded, or a surface on which a medium is formed and cells or the like are seeded when the cells or the like are cultured. That is, the culture surface is a concept including a medium formation schedule surface and a seeding schedule surface such as cells.

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

The culture member of the present invention means that the culture surface is not coated with a natural polymer material, a synthetic polymer material, or an inorganic material for serving as a scaffold for cells or the like.

The thickness of the culture member of the present invention is not particularly limited. 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, the pores are easily perforated, the strength tends to be excellent, and the oxygen supply property tends to be excellent.

The thickness of the culture member when the culture member of the present invention is arranged on the bottom surface of the container to prepare a culture device such as a dish (also referred to as a petri dish), a flask, an insert or a plate is not particularly limited, but is preferably 20 μm to 400 μm. The thickness is preferably 20 μm to 300 μm, more preferably 20 μm to 200 μm. It is selected in a timely manner according to the morphology of the incubator, but by adjusting it to the above upper and lower limit range, it is easy to obtain an appropriate oxygen concentration in the medium necessary for cell growth, and it is suitable because it has sufficient strength. It will be easier to make culture equipment.

The surface of the culture member of the present invention may be microfabricated other than pores in order to produce spheroids and improve the scaffolding function of cells. As a method for performing microfabrication, self-assembly of fine particles such as cutting, optical lithography, direct electron beam drawing, particle beam beam processing, scanning probe processing, or nanoimprint from a master formed by these methods. It can be appropriately selected from a method, a casting method, a molding processing method typified by an injection molding method, a plating method, and the like. The shape of the microfabrication is not particularly limited, but the height from the groove portion to the mountain portion is preferably 20 nm to 500 μm. Further, the thickness of the thinnest portion of the culture member can be reduced to 20 μm as compared with the case where the surface is not microfabricated.

The microfabricated culture member may be used as a microchannel device (also referred to as a microchannel chip). Microchannel device is a general term for devices for creating microchannels and reaction vessels by microfabrication on the surface of culture members and applying them to bio-research and chemical engineering. For example, devices called microTAS (micro Total Analysis Systems), Lab-on-a-Chip, and the like are mentioned, and their application as next-generation culture devices is being promoted. As one aspect of the present invention, there is a microchannel device including the culture member of the present invention.

The culture member of the present invention may be subjected to surface modification treatment. The method used for the surface modification treatment is not particularly limited, but for example, hydrophilization treatment such as corona treatment, plasma treatment, ozone treatment, ultraviolet treatment, chemical vapor deposition, etching, or hydroxyl group, amino group, sulfone group, thiol group, Examples thereof include addition of a specific functional group such as a carboxyl group, treatment with a specific functional group such as silane coupling, and surface roughening with an oxidizing agent or the like. These surface modification treatments may be performed alone or in combination of two or more. When the surface modification treatment is performed, it is preferable to perform the surface modification treatment 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 preferable in order to improve the wettability of the surface of the culture member and enable efficient cell culture. By hydrophilizing the surface of the culture member, for example, the adhesion between the culture member and the cells is improved, and the cells can grow uniformly on the surface of the culture member. Further, by hydrophilizing the surface of the culture member, it becomes easy to coat the culture surface of the culture instrument with a natural polymer material, a synthetic polymer material, or an inorganic material. In particular, collagen is evenly loaded on the surface of the culture member, making it easier to adhere to it, and even after the collagen loading treatment, collagen does not come off in the environment of washing with physiological saline or cell culture, and a stable initial state is maintained. Can be used for cell culture. When the plasma treatment is performed, nitrogen, hydrogen, helium, oxygen, argon or the like is used as the accompanying gas, and at least one gas selected from nitrogen, helium and argon is preferably selected.

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

Specifically, as a method for forming the film or sheet, for example, a normal inflation method, a T-die extrusion method, or the like is adopted. Manufacture is usually carried out by heating. When the T-die extrusion method is adopted, the extrusion temperature varies depending on the type of resin, but is preferably 100 ° C to 400 ° C, and particularly preferably 150 ° C to 300 ° C. The roll temperature is preferably 20 ° C to 100 ° C, particularly preferably 30 ° C to 90 ° C.

Further, the film or sheet may be produced by a solution casting method in which the resin is dissolved in a solvent, poured onto the resin or metal, and slowly dried while leveling to form a film (sheet). The solvent used is not particularly limited, but a hydrocarbon solvent such as cyclohexane, hexane, decane, or toluene may be used. Further, two or more kinds of solvents may be mixed in consideration of the solubility of the resin and the drying efficiency. The polymer solution can be applied by a method such as table coat, spin coat, dip coat, die coat, spray coat, bar coat, roll coat, curtain flow coat, etc., dried and peeled off to form a film or sheet.

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

<resin>
The material for forming the culture member of the present invention may contain a resin, may be composed of only the resin, or may be composed of a composition containing the resin.
It is desirable that the resin used as the material of the culture member has low toxicity to cells and the like, and as such a material, either a natural resin or a synthetic resin can be used. Among these, synthetic resin is preferable from the viewpoint of moldability and chemical resistance. The synthetic resin may be a thermoplastic resin or a thermosetting resin, but a thermoplastic resin is preferable because it is easy to mold.

As the resin used as the material of the culture member, an appropriate material can be selected from the viewpoints of shape stability, drug sorption, autofluorescence, transparency, oxygen permeability and the like. From this point of view, by selecting a culture material, it is possible to obtain a culture member which can efficiently culture cells, can be used for drug discovery screening and diagnostic purposes, and can easily observe cells.

<Thermoplastic resin>
The thermoplastic resin that can be used as a material for the culture member is not particularly limited, and is, for example, a polyolefin resin; a polymethacrylic resin such as polymethylmethacrylate resin; a polyacrylic resin such as methylpolyacrylate resin; a polystyrene resin. Polyvinyl acetal resin; Polyvinyl butyral resin; Polypolyformal resin; Polymethylpentene resin; Polycarbonate resin; Polyether ether ketone resin; Polyether resin such as polyether ketone resin; Polyester resin; Nylon-6, Nylon-66, Polyamide resin such as polymethoxylen adipamide; Polyamideimide resin; Polyimide resin; Polyetherimide resin; Stylium elastomer; Polyformation elastomer; Polyurethane-based elastomer; Polyester-based elastomer; Polyamide-based elastomer; Norbornen resin; Polytetrafluoro Ethylene resin; ethylene tetrafluoroethylene copolymer; polyvinylidene fluoride resin; polyvinyl fluoride resin; thermoplastic polyimide resin; vinylidene chloride resin; polyvinyl chloride resin; vinyl acetate resin; polysulfone resin; polyphenylene oxide resin, polyphenylene sulfide resin, etc. Polyphenylene resin; polysulfone resin; polylactic acid resin; polyethersulfone resin; polyacrylonitrile resin, styrene-acrylonitrile copolymer resin and the like can be mentioned.

Examples of the polyolefin resin 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 copolymer of propylene and α-olefin having 2 or more and 10 or less carbon atoms; Ethylene-vinyl acetate copolymer (EVA); Ionomer resin, etc. Can be mentioned.
Among these, 4-methyl-1-pentene polymer (4-methyl-1-pentene polymer) is excellent in the balance of molding processability, transparency, mechanical properties, breathability, light weight, and low drug adhesion. Pentene homopolymers and copolymers) are preferred. In the present invention, 4-methyl-1-pentene homopolymer and copolymer of 4-methyl-1-pentene and other monomers are collectively referred to as "4-methyl-1-pentene polymer". Refer to.

Examples of the polystyrene-based resin include homopolymers of styrene-based monomers (for example, styrene, methylstyrene, ethylstyrene, isopropylstyrene, dimethylstyrene, paramethylstyrene, chlorostyrene, bromostyrene, vinyltoluene, vinylxylene). ; Examples thereof include a copolymer of a styrene-based monomer and a monomer copolymerizable with the styrene-based monomer (hereinafter, also referred to as “modified polystyrene”).
Examples of the monomer copolymerizable with the styrene-based monomer include vinyl monomers (for example, acrylonitrile, methacrylonitrile, acrylic acid, methacrylic acid, methyl methacrylate, maleic anhydride, butadiene). ..
Examples of the modified polystyrene include acrylonitrile-styrene copolymer (AS), methyl methacrylate-styrene copolymer, acrylonitrile-methyl methacrylate-styrene copolymer, acrylonitrile-butadiene-styrene copolymer (ABS), and acrylonitrile. -Acrylonitrile-styrene styrene copolymer (AAS), acrylonitrile-ethylenepropylene diene rubber-styrene copolymer (AES) can be mentioned.
Among these, a styrene homopolymer is preferable from the viewpoint of excellent balance of molding processability, transparency, mechanical properties, breathability, light weight, and low chemical adhesion.

Examples of the polyester resin include dicarboxylic acids such as isophthalic acid, phthalic acid, terephthalic acid, 2,6-naphthalenedicarboxylic acid, adipic acid and sebacic acid, oxycarboxylic acids (eg, p-oxybenzoic acid) and ethylene. Examples thereof include a resin obtained by polycondensing with an aliphatic glycol such as glycol, propylene glycol, butanediol, diethylene glycol, 1,4-cyclohexanedimethanol and neopentyl glycol. As the dicarboxylic acid, oxycarboxylic acid and aliphatic glycol, one kind or two or more kinds can be used respectively.
Typical examples of the polyester resin include polyethylene terephthalate (PET), polyethylene-2,6-naphthalenedicarboxylate (PEN), and polybutylene terephthalate (PBT). Further, the resin may be a copolymer containing 30 mol% or less of a third component in addition to the homopolymer.
Among these, polyethylene terephthalate is preferable because it has an excellent balance of molding processability, transparency, mechanical properties, breathability, light weight, and low chemical adhesion.

Among these, polyolefin-based resins, polystyrene-based resins, and polyester-based resins are preferable because they have an excellent balance of molding processability, transparency, mechanical properties, breathability, light weight, and low chemical adhesion. Methyl-1-pentene polymers (homogenes and copolymers of 4-methyl-1-pentene), polystyrene resins and polyethylene terephthalates are more preferred.

The resin 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, still 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 thermoplastic resin, using a haze / transmittance meter HM-150 (D65 light source) manufactured by Murakami Color Technology Research Institute Co., Ltd. in accordance with JIS K7361. The amount of transmitted light is measured, and the total light transmittance is calculated by the following formula.
Total light transmittance (%) = 100 x (total transmitted light amount) / (incident light amount)
When 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, the 4-methyl-1-pentene polymer, which is a suitable example of the material of the culture member, will be described in more detail.
Examples of the copolymer of 4-methyl-1-pentene, which is an example of 4-methyl-1-pentene polymer, and other monomers include random copolymers, alternating copolymers, block copolymers, and grafts. It may be any of the copolymers. The copolymers of 4-methyl-1-pentene and other monomers include 4-methyl-1-pentene, ethylene, and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene). ), A copolymer with at least one olefin selected from) is preferable because it has high strength, is hard to tear even when used as a member, is hard to crack, and has little bending.

Examples of the 4-methyl-1-pentene polymer include 4-methyl-1-pentene homopolymer, 4-methyl-1-pentene, ethylene and α-olefin having 3 to 20 carbon atoms (4-methyl-1). -Preferably, it is at least one polymer selected from a polymer selected from a polymer with at least one olefin selected from (excluding penten), preferably 4-methyl-1-pentene, ethylene and 3 to 20 carbon atoms. It is more preferable that the polymer is a copolymer with at least one olefin selected from α-olefin (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-eikosen. Can be mentioned. The olefin can be appropriately selected depending on the physical characteristics required for the culture member. For example, as the olefin, α-olefin having 8 to 18 carbon atoms is preferable from the viewpoint of appropriate oxygen permeability and excellent rigidity, and 1-octene, 1-decene, 1-dodecene, 1-tetradecene, At least one selected from 1-hexadecene, 1-heptadecene and 1-octadecene is more preferred. When the carbon number of the olefin is in the above range, the processability of the polymer becomes better, and the appearance of the culture member is less likely to be deteriorated due to cracks or cracks at the ends. In addition, the rate of defective products in the culture member is low.

As the olefin, one kind or two or more kinds can be used. From the viewpoint of the strength of the material, the number of carbon atoms is preferably 2 or more, 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 the structural unit 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%.
Further, the 4-methyl-1-pentene polymer is at least one selected from 4-methyl-1-pentene, ethylene and α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene). In the case of a copolymer with an olefin, it is derived from at least one olefin selected from ethylene in the copolymer and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene). The content of the structural unit is preferably 0 to 40 mol%, more preferably 2 to 20 mol%. The content of these structural units is 100 mol% based on the total amount of repeated structural units in the 4-methyl-1-pentene polymer. When the content of the structural unit is within the above range, a uniform culture surface having excellent processability can be obtained, and the toughness and strength of the film are well-balanced, so that the bending is reduced.

The 4-methyl-1-pentene polymer is derived from the structural unit derived from 4-methyl-1-pentene and the ethylene and α-olefin having 3 to 20 carbon atoms to the extent that the effect of the present invention is not impaired. It may have a structural unit other than the structural unit (hereinafter, also referred to as “other structural unit”). 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 kind or two or more kinds.

Examples of the monomer leading to other structural units include cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins, and halogenated olefins. Examples of the cyclic olefin, aromatic vinyl compound, conjugated diene, non-conjugated polyene, functional vinyl compound, hydroxyl group-containing olefin and halogenated olefin are described in paragraphs [0035] to [0041] of JP2013-169685A. Compounds can be used.

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

Commercially available products can also be used as the 4-methyl-1-pentene polymer. Specific examples thereof include TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31 and DX845 manufactured by Mitsui Chemicals, Inc.). Further, a 4-methyl-1-pentene polymer that meets the above requirements can be preferably used even if it is manufactured by another manufacturer. These commercially available products may be used alone or in combination of two or more.

The 4-methyl-1-pentene polymer usually has a melting point of 200 ° C. to 240 ° C. and has high heat resistance. In addition, since it does not cause hydrolysis and has excellent water resistance, boiling water resistance, and steam resistance, culture members such as culture instruments containing 4-methyl-1-pentene polymer can be subjected to high-pressure steam sterilization treatment. Since the 4-methyl-1-pentene polymer also has a high visible light transmittance (usually 90% or more) and does not emit autofluorescence, a culture instrument containing the 4-methyl-1-pentene polymer is cultured. Easy to observe cells. Furthermore, since it exhibits excellent chemical resistance to most chemicals and it is difficult for the chemicals to be collected, it is also suitably used for drug discovery screening applications and diagnostic applications. The 4-methyl-1-pentene polymer can be heat-sealed, and not only heat fusion between own materials but also heat adhesion with other materials is easy. Further, since thermoforming is possible, it is easy to mold into a culture instrument having an arbitrary shape, and for example, molding using an imprint method or an insert method is also easy.

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 2000000, more preferably 20000. It is up to 1,000,000, more preferably 30,000 to 500,000. Here, the sample concentration at the time of 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 used in GPC. Further, as an example of the measurement conditions, the conditions shown in the examples described later can be mentioned, but the measurement conditions are not limited to the measurement conditions.

By setting the weight average molecular weight (Mw) to be equal to or less than the above upper limit, the film produced by melt molding in the resin molding method described later can easily suppress the occurrence of defects such as gel, and can form a film having a uniform surface. It will be easier. Further, when the film is produced by the solution casting method, the solubility in a solvent is improved, defects such as gel of the film can be easily suppressed, and a film having a uniform surface can be easily formed.

Further, by setting the weight average molecular weight (Mw) to be equal to or higher than the above lower limit, the strength of the culture member tends to be sufficient. Furthermore, by keeping the molecular weight distribution within the above range, stickiness on the surface of the produced culture member tends to be suppressed, the toughness of the culture member tends to be sufficient, and bending during molding and cracking during cutting tend to occur. It becomes easier to suppress.

The weight average molecular weight (Mw) and molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer are each resin when two or more kinds are used as the 4-methyl-1-pentene polymer. The respective Mw and Mw / Mn may be in the above range.

Since the 4-methyl-1-penten-1 polymer has the above-mentioned excellent properties, at least the culture instrument in which the culture surface is formed of the culture member of the present invention has a bad influence on the culture. It is very excellent as a material for culture members because it has good stability, light transmission, and moldability and can be sterilized.

<Method for producing 4-methyl-1-pentene polymer>
The method for producing the 4-methyl-1-pentene polymer may be any method as long as 4-methyl-1-pentene, an olefin, or another monomer can be polymerized. Further, a chain transfer agent such as hydrogen may coexist in order to control the molecular weight and the molecular weight distribution. The equipment used for manufacturing is also not limited. The polymerization method may be a known method, or may be a vapor phase method, a slurry method, a solution method, or a bulk method. The slurry method and the solution method are preferable. Further, 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 of the single-stage and multi-stage polymerization methods, when hydrogen is used as the chain transfer agent, it may be charged all at once or dividedly, for example, at the initial stage, middle stage, or final stage of polymerization. The polymerization may be carried out at room temperature or may be heated if necessary, but from the viewpoint of polymerization efficiency, it is preferably carried out at 20 ° C to 80 ° C, and particularly preferably at 40 ° C to 60 ° C. .. The catalyst used for production is also not limited, but from the viewpoint of polymerization efficiency, it is preferable to use, for example, the solid titanium catalyst component (I) described in International Publication No. 2006/054613.

<Manufacturing method of polystyrene resin>
The polystyrene-based resin can be produced, for example, by the polymerization method shown below.
Examples of the polymerization method of the polystyrene-based resin include known styrene polymerization methods such as a bulk polymerization method, a solution polymerization method, and a suspension polymerization method. These polymerization methods may be either a batch polymerization method or a continuous polymerization method, and are preferably continuous polymerization methods from the viewpoint of productivity.
As a specific example of such a polymerization method, for example, a styrene-based monomer, a polymerization solvent, a polymerization initiator, a chain transfer agent and the like, if necessary, are added and mixed to prepare a raw material solution containing the monomers. do. The above raw materials are provided in equipment equipped with two or more reactors arranged alone or in series and / or in parallel, and a devolatilization device for a volatilization step of removing volatile components such as unreacted monomers. Examples thereof include a method in which the solution is continuously fed and the polymerization proceeds step by step.

Examples of the reactor include a complete mixture type reactor, a laminar flow type reactor, a loop type reactor in which a part of the polymerization solution is extracted while advancing the polymerization, and the like. The order of arrangement of these reactors is not particularly limited.

When polymerizing a polystyrene-based resin, a polymerization solvent, a polymerization initiator such as an organic peroxide, and a chain transfer agent can be used, if necessary, from the viewpoint of controlling the polymerization reaction.
The polymerization solvent is generally used for adjusting the polymerization rate, molecular weight and the like. 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 is usually from the viewpoint of controlling gelation, improving productivity, increasing the molecular weight, etc., with respect to 100% by mass of the entire polymerization solution in the polymerization reactor. It is preferably 1 to 50% by mass, more preferably 3 to 20% by mass.

When polymerizing a polymerization raw material to obtain a 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, but is limited to organic peroxides, for example, 2,2-bis (t-butylperoxy) butane, 1,1-bis (t-butylperoxy) cyclohexane, and n-butyl-. Peroxyketals such as 4,4-bis (t-butylperoxy) peroxide, dialkyl peroxides such as di-t-butyl peroxide, t-butylcumyl peroxide, and dicumyl peroxide, acetyl peroxide, isobutyryl peroxide, etc. Diacil peroxides, peroxydicarbonates such as diisopropylperoxydicarbonate, peroxyesters such as t-butylperoxyacetate, ketone peroxides such as acetylacetone peroxide, and hydroperoxides such as t-butylhydroperoxide. Can be done.
The polymerization initiator is preferably used in an amount of 0.005 to 0.08% by mass based on the styrene-based monomer.
The chain transfer agent is not particularly limited, and 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-based monomer.

As the devolatilization device, for example, a normal volatilization device such as a flash drum, a twin-screw volatilizer, a thin film evaporator, an extruder, etc. can be used, and generally, a vacuum devolatilization tank equipped with a heater or a devolatilization device can be used. A volatile extruder or the like is used. As the arrangement of the devolatilization device, for example, a vacuum devolatilization tank with a heater is used in only one stage, a vacuum devolatilization tank with a heater is connected in two stages in series, and a vacuum devastation tank with a heater is used. Examples thereof include a vacuum tank and a devolatilization extruder connected in series. In order to reduce the volatile components as much as possible, it is preferable to connect a vacuum devolatilization tank with a heater in two stages in series, or a vacuum devolatilization tank with a heater and a devolatilization extruder connected in series. ..

The conditions of the devolatile step are not particularly limited. For example, when the polystyrene-based resin is polymerized by bulk polymerization, the unreacted styrene-based monomer is preferably 50% by mass or less in the polystyrene-based resin. , More preferably, the polymerization can proceed until it becomes 40% by mass or less. By the devolatile treatment, unreacted substances (styrene-based monomers) and / or volatile components such as solvents can be removed.

The temperature of the devolatile treatment is usually about 190 to 280 ° C. The pressure of the devolatile treatment is preferably 0.1 to 50 kPa, more preferably 0.13 to 13 kPa, still more preferably 0.13 to 7 kPa, and particularly preferably 0.13 to 1.3 kPa. As the devolatile method, for example, it is desirable to devolatile by reducing the pressure under heating, or through an extruder designed to remove volatile components.

<polyethylene terephthalate>
Polyethylene terephthalate (PET) is a polymer obtained by polycondensing at least one selected from terephthalic acid and an ester-forming derivative thereof and at least one selected from ethylene glycol and an ester-forming derivative thereof. Examples of the ester-forming derivative of terephthalic acid include alkyl esters of terephthalic acid such as dimethyl terephthalate. Examples of the ester-forming derivative of ethylene glycol include fatty acid esters such as ethylene glycol fatty acid ester.

Polyethylene terephthalate is obtained by copolymerizing at least one selected from terephthalic acid and an ester-forming derivative thereof and at least one selected from another dicarboxylic acid and an ester-forming derivative thereof, as long as the properties are not impaired. It may be a copolymer of at least one selected from ethylene glycol and an ester-forming derivative thereof, and at least one selected from another diol and an ester-forming derivative thereof. Examples of the dicarboxylic acid and its ester-forming derivative used as the copolymerization component include isophthalic acid, adipic acid, oxalic acid, sebacic acid, decandicarboxylic acid, naphthalenedicarboxylic acid and alkyl esters thereof. Examples of the diol used as a copolymerization component and its ester-forming derivative include ethylene glycol, propylene glycol, neopentyl glycol, 1,5-pentanediol, 1,6-hexanediol, decamethylene glycol, and cyclohexanedi. Examples thereof include methanol, cyclohexanediol, long-chain glycols such as polyethylene glycol having a molecular weight of 400 to 6000, poly 1,3-propylene glycol, 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 material forming polyethylene terephthalate.

<Manufacturing method of polyethylene terephthalate>
Polyethylene terephthalate can be produced by a known polycondensation method, ring-opening polymerization method, or the like. Either the batch polymerization method or the continuous polymerization method may be applied, but the continuous polymerization method is preferable in that the amount of the carboxyl-terminal group can be reduced and the effect of improving the fluidity is further increased. Further, either a transesterification reaction or a direct polymerization reaction may be applied, but the direct polymerization reaction is preferable in terms of cost. In order to efficiently proceed with the esterification reaction, transesterification reaction and / or polycondensation reaction, it is preferable to add a catalyst during these reactions. Specific examples of the catalyst include methyl ester of titanium acid, tetra-n-propyl ester, tetra-n-butyl ester, tetraisopropyl ester, tetraisobutyl ester, tetra-tert-butyl ester, cyclohexyl ester, phenyl ester and benzyl ester. , Trill ester, or organic titanium compounds such as mixed esters of these, dibutyltin oxide, methylphenyltin oxide, tetraethyltin, hexaethylditin oxide, cyclohexahexyldistin oxide, didodecyltin oxide, triethyltin hydrooxide, tri. Phenyltin Hydrooxide, Triisobutyltin Acetate, Dibutyltin Diacetate, Diphenyltin Dilaurate, Monobutyltin Trichloride, Dibutyltin Dichloride, Tributyltin Chloride, Dibutyltin Sulfide and Butylhydroxytin Oxide, Methylstannoic Acid, Ethylstannoic Acid, Butylstannoneic Acid Examples thereof include tin compounds such as alkylstannoic acid, zirconium compounds such as zirconium tetra-n-butoxide, antimony compounds such as antimony trioxide and antimony acetate. The catalyst may be used alone or in combination of two or more. Among these, an organic titanium compound and a tin compound are preferable, and a tetra-n-butyl ester of titanium acid is more preferable. The amount of the catalyst added is generally 0.01 to 0.2 parts by mass with respect to 100 parts by mass of polyethylene terephthalate.

The type of polyethylene terephthalate is not particularly limited as long as it does not interfere with the effects obtained in the present invention, but crystalline polyethylene terephthalate (C-PET) is preferable from the viewpoint of mechanical strength and moldability.

When the film-shaped polyethylene terephthalate is used as a culture member, the film-shaped polyethylene terephthalate may or may not be stretched. Film-shaped polyethylene terephthalate is preferable from the viewpoint of mechanical properties because it is crystallized when stretched. Biaxial stretching is preferable as the stretching method.

When the culture member is formed from a composition containing a resin, the resin is preferably 90% by mass or more and less than 100% by mass, and more preferably 95% by mass or more and 100% by mass in 100% by mass of the culture member. It is less than mass%, and particularly preferably 99% by mass or more and less than 100% by mass. If a large amount of a component other than the resin is contained, not only the oxygen permeability is lowered, but also the transparency and the strength are lowered.

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

<pore>
The culture member of the present invention has at least one pore on the culture surface thereof. Here, the pores in the culture member mean fine pores contained in the culture member and penetrate the culture member, and are different from the irregularities 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 circular shape, an elliptical shape, a square shape, a rectangular shape, a hexagonal shape, a cloud shape, and a cross shape. From the viewpoint of workability, among these, a circular shape or an elliptical shape 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) is preferably 0.01 to 250 cells / cm ² , preferably 0.1 to 230 cells / cm ² . More preferably, it is more preferably 0.3 to 200 pieces / cm ² . When the pore density of the pores is within the above range, the oxygen supply property is excellent and the metabolic activity of the cells tends to be high.

The average pore diameter 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 diameter of the pores is within the above range, the medium is less likely to leak, the oxygen supply property 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 a known method can be used. As a method for measuring the average pore diameter, for example, a method for obtaining the average pore diameter by a gas adsorption method, a mercury intrusion method, a bubble point method, etc., a method of observing the pores with a microscope, an electron microscope, etc., and measuring the pore diameter, and the average value thereof. Is used as the average pore diameter, an instrument used for drilling, for example, a method of estimating the diameter of a needle or a laser beam as the average pore diameter, and the like can be mentioned. When measuring the pore diameter by observation and when estimating the diameter of the instrument used for drilling as the average pore diameter, the pore diameter refers to the diameter of the maximum inscribed circle with respect to the opening shape of the pore, for example, the opening shape of the pore. When it 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, and when it is substantially square, it refers to the length of the side of the square. It refers to the length of the short side of the square when it is substantially rectangular. Since it is simple, a method of estimating the diameter of an instrument used for drilling, for example, a needle or a laser beam as an average pore diameter is preferable.

The culture member of the present invention preferably has pores having an average pore diameter of 0.1 to 100 μm at a pore density of 0.01 to 250 cells / cm ² , and more preferably pores having an average pore diameter of 1 to 90 μm. It has a pore density of 1 to 230 pores / cm ² , and more preferably has pores with an average pore diameter of 3 to 85 μm at a pore density of 0.3 to 200 pores / cm ² . When the average pore diameter and pore density of the pores are within the above ranges, the oxygen supply property 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 property 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 × 100 (%)) is preferably 0.000001 to 0.3%, more preferably. Is 0.000005 to 0.1%, more preferably 0.00001 to 0.08%. When the ratio of the total opening area of the pores is within the above range, the medium is less likely to leak, the oxygen supply property 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 pore diameters of the plurality of pores may be substantially the same or may vary, but it is preferable that the culture members have high uniformity. The coefficient of variation can be used as an index of the uniformity of the pore diameter. The coefficient of variation of the pore diameter (standard deviation ÷ average value × 100 (%)) is preferably 30% or less, and more preferably the coefficient of variation of the pore diameter is 20% or less. When the pore size is highly uniform as described above, the medium is less likely to leak, the oxygen supply property 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 may be arranged in a constant pattern or may be randomly arranged. Examples of arranging in a fixed pattern include 60 ° staggered, 45 ° staggered, parallel, herringbone, etc., and draw multiple polygons or circles of different sizes that share the center, and on their sides or circumferences. The pores may be arranged at equal intervals. It is preferable to arrange the cells in a certain pattern because the medium does not leak easily, the oxygen supply property is excellent, and the metabolic activity of the cells tends to be high.

The interval between the plurality of pores is not particularly limited, but it is preferably larger than the average pore diameter of the pores, and more preferably a constant interval. The spacing between the plurality of 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 distance between the plurality of pores is within the above range, the medium is less likely to leak, the oxygen supply property is excellent, and the metabolic activity of the cells tends to be high.

The plurality of pores may communicate with each other inside the culture member.

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

The laser includes various lasers such as a carbon dioxide gas laser, a YAG laser, a semiconductor laser, and an argon laser, and is not particularly limited as long as it can be pierced. Above all, since it is possible to perforate pores having a small pore diameter, it is preferable to use a YAG laser having a wavelength region of 100 to 1100 nm, and even a transparent material that transmits visible light can be absorbed and perforated. Therefore, an ultraviolet YAG laser having a wavelength region of 100 to 400 m is more preferable, and a deep ultraviolet YAG laser having a wavelength region of 260 to 270 nm is further preferable. When a deep ultraviolet YAG laser having a wavelength region of 260 to 270 nm is used, the output is preferably 10 to 200 mW. When the output range is within the above range, workability is good, and it is possible to achieve drilling with a desired diameter while avoiding excessive irradiation.

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

The perforation of the pores may be performed after obtaining the molded product from the component containing the resin or at the same time as producing the molded product from the component containing the resin. A molded product having perforated pores may be used as it is as a culture member, or the molded product may be stretched and then used. Preferably, a film or sheet is formed from a component containing a resin, pores are formed in the film or sheet, and then the perforated film or sheet is formed into a culture member having a desired shape. Alternatively, preferably, after molding from a component containing a resin to produce a molded product having a desired shape, pores are perforated.

As a method of perforating the pores at the same time as producing a molded product from a component containing a resin, for example, an organic solvent solution of the resin is cast on a substrate, the organic solvent is evaporated, and dew condensation occurs on the surface of the cast liquid. A method for obtaining a culture member having pores by evaporating minute water droplets generated by the dew condensation, and a melt blown method, in which a molten thermoplastic resin is discharged from a spinneret together with a heating gas, and the heating gas is used. A method of stretching the thermoplastic resin to obtain a fibrous resin and obtaining a culture member having pores can be mentioned.

<Cell, tissue, or organ>
The cell in the present invention is not particularly limited, and in the case of an animal cell, it may be a floating cell or an adhesive cell, for example, a fibroblast, a mesenchymal stem cell, a hematopoietic stem cell, or a nerve. Stem cells, nerve cells, corneal epithelial cells, oral mucosal cells, retinal pigment supracells, root membrane stem cells, myofibroblasts, myocardial cells, hepatocytes, splenic endocrine cells, skin keratinized cells, skin fibroblasts, subcutaneous fat Origin precursor cells, kidney cells, bottom root sheath cells, nasal mucosal epithelial cells, vascular endothelial precursor cells, vascular endothelial cells, vascular smooth muscle cells, osteoblasts, cartilage cells, skeletal muscle cells, immortalized cells, cancer cells, Examples thereof include keratinized cells, embryonic stem cells (ES cells), EBV-transformed B cells, and artificial pluripotent stem cells (iPS cells). It may be either a primary cultured cell or a cell that has been passaged to a strain. The present invention is based on the fact that skin, kidney, liver, brain, nerve tissue, myocardial tissue, skeletal muscle tissue, cancer stem cells, etc. have high oxygen requirement, and the cells constituting them are also cells with high oxygen requirement. The cells in the above are preferably skin, kidney, liver, brain, nerve tissue, myocardial tissue, cells constituting skeletal muscle tissue, or cancer stem cells. The cells, tissues, or organs are preferably hepatocytes, renal cells, myocardial cells, nerve cells, or cancer stem cells, more preferably hepatocytes.

The tissue in the present invention means a tissue in which similar cells gather and perform the same function. The tissue is not particularly limited, and examples thereof include epithelial tissue, connective tissue, muscle tissue, and nervous tissue. Due to the high oxygen requirement, the tissue is preferably hepatic lobule, myocardial tissue, nervous tissue, or skeletal muscle tissue, more preferably hepatic lobule.

The organ in the present invention means an organ in which the tissues gather and perform a joint work with a purpose. The organ is not particularly limited, and is, for example, lung, heart, liver, kidney, spleen, pancreas, gallbladder, esophagus, stomach, skin, brain and the like. Due to the high oxygen requirement, the organ is preferably skin, kidney, liver, brain, more preferably liver.

It is preferable that the cells or the like are cells because they are suitable for culturing on the culture surface of the culturing instrument. In the present invention, the cells and the like are preferably aerobic, and more preferably do not contain anaerobic cells. The origin of cells and the like is not particularly limited and may be any organism such as animals, plants, fungi, protists and bacteria, but animals or plants are preferable, animals are more preferable, and mammals are particularly preferable. Since the culture device in the present invention is preferably oxygen-supplied and retains cell adhesion, the cells and the like are preferably adherent, and more preferably adherent cells.

<Hepatocytes>
The hepatocytes in the present invention may be any cells including hepatocyte, as long as they are cells in the liver, specifically, vascular endothelial cells, vascular smooth muscle cells, fat cells, blood cells, and the like. Includes hepatic mononuclear cells, hepatic macrophages (including Kupper cells), hepatic stellate cells, intrahepatic bile duct epithelial cells, embryonic sac fibroblasts, and the like. The hepatocyte is a cell population containing, for example, 20% or more, 30% or more, 40% or more, or 50% or more of hepatocyte parenchymal cells.

The hepatocytes may be primary cultured cells or established passage cells, but it is preferable to use primary cultured cells as the hepatocytes. The type of passaged cells is not particularly limited, but for example, 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 And so on.

The origin of the hepatocytes may be any mammal, but human, bovine, dog, cat, pig, mini pig, rabbit, hamster, rat, or mouse cells are particularly preferable, and human, rat, mouse. , Or bovine cells are more preferred.

The hepatocyte may be a cell population containing a cell type other than the hepatocyte, for example, a cell population containing 20% or more, 30% or more, 40% or more, or 50% or more of hepatocytes. ..

<culture>
In the present invention, culturing is used in a broad sense including not only the proliferation and maintenance of cells and the like, but also processes such as seeding, passage, differentiation induction and self-organization induction of cells and the like. The medium or the like used for culturing is not limited, and a medium or the like may be selected according to the characteristics of the cells or the like.

<Cell culture>
The cell culture may be a two-dimensional (including the case where the cells spontaneously stratify) culture or a three-dimensional culture. Since the culture member of the present invention is suitable for culturing because of its oxygen supply property, it is possible to sufficiently supply oxygen to cells not only in two-dimensional culture but also in three-dimensional culture in which cells are three-dimensionally stacked. , Cells proliferate and differentiate, and highly self-organizing phenomena of cells are also likely to occur.

Three-dimensional culture is intentionally culturing cells three-dimensionally, and is divided into two types: Scaffold type, in which cells are cultured in a scaffold material, and Scaffold-free type, in which cells are cultured in a floating state as a mass (spheroid). Either may be used, but the Scaffold type is preferable. In the case of the Scaffold type, as the scaffolding material, Matrigel ^TM , collagen gel, laminin, hydrogel of alginate, and Vitrigel are preferable because cells can be efficiently cultured.

The medium used for culturing is not limited, but in order to efficiently cultivate the cells, it is preferable to cultivate the cells in the presence of serum (for example, fetal bovine serum).

When the culture member of the present invention is used for culturing, in other words, when culturing using the culturing instrument of the present invention, the cell culture density is preferably 0.1 × 10 ⁵ cells / cm ² to 10. It is 0 × 10 ⁵ cells / cm ² , more preferably 0.5 × 10 ⁵ cells / cm ² to 5.0 × 10 ⁵ cells / cm ² , and even more preferably 1.0 × 10 ⁵ cells / cm. ² to 4.0 × 10 ⁵ cells / cm ² .
The above range is preferable because the drug metabolism activity is further enhanced as compared with the case where the cell culture density is outside the above range.
Since the culture member of the present invention has excellent oxygen supply, it can be suitably cultured even when the cell culture density is high. Generally, it is said that the cell density of a living body is about 2.5 × 10 ⁵ cells / cm ² , but the culture member of the present invention can be cultured at the same cell culture density as that of a living body. Therefore, it is preferable because the culture can be performed in vitro in a state closer to in vivo.

<Culturing equipment>
In the present invention, the culture instrument means all instruments used for culturing cells and the like. 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 thereof may be composed of the culture member. When only a part of the culture device is composed of the culture member, the culture surface for culturing at least cells and the like is composed of the culture member of the present invention.

The culture device is usually used in an device such as an incubator, a mass culture device, or a perfusion culture device.

As the culture instrument, various known culture instruments can be used, and the shape and size are not particularly limited. Examples of the culture instrument include culture containers such as dishes, flasks, plates, bottles, bags, and tubes, as well as inserts, cups, inlays, slides, and the like, and are 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, still more preferably a plate having at least one well, and 6 wells. , 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells and the like. Generally, in a culture device having a hollow shape like a well on the bottom surface, it is necessary to make the bottom surface thick in order to stabilize the complicated shape of the bottom surface, and it is difficult to sufficiently supply oxygen to cells and the like. When the culture member of the present invention is used, the shape is stable even in a plate having wells such as 1 well, 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, and 1536 wells. Sufficient oxygen supply to cells and the like.

Since the culture device holds or stores the medium, it is preferable that the culture device has a bottom surface as a culture surface. When the culture instrument is a dish, a flask, an insert, or a plate, the bottom surface is the culture surface, so that the culture member of the present invention constitutes at least a part or all of the bottom surface, the side surface, and the top surface. It is preferable to do so. When at least the bottom surface (culture surface) is composed of the culture member of the present invention, oxygen can be more efficiently supplied to the medium through the culture member, and cells and the like in the medium can be proliferated more efficiently. be able to. In addition, it can be cultured at a higher density while maintaining the function of the cells.

The shape of the bottom surface of the incubator 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), and a flat bottom + curved edge. When processing into a round bottom (U bottom), flat bottom (F bottom), conical bottom (V bottom), flat bottom + curved edge, etc., it may be processed at once by general injection molding or press molding, or a film. Alternatively, it is also possible to create a sheet and perform secondary processing by vacuum forming, pneumatic forming, or the like. The shape of the bottom surface is selected according to the purpose of the culture, but when culturing cells or the like in two dimensions, it is usually desirable to have a flat bottom, and when culturing in three dimensions, a round bottom (U bottom) or a cone. It is usually desirable to have a bottom (V bottom).

The portion of the incubator 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 a known material can be used. Examples of such materials include polydimethylsiloxane (PDMS), thermosetting resins, cyclic olefin polymers, cyclic olefin copolymers, glass and the like.

The culture device may be a culture device in which a natural polymer material, a synthetic polymer material, or an inorganic material is coated on the culture surface.

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

The coated culture device is more excellent in adhesion and proliferation of cells and the like. It is considered that this is because the natural polymer material, the synthetic polymer material, or the inorganic material coated on the culture surface serves as a scaffold for cells and the like. Therefore, when culturing adherent cells or the like, it is a preferable form to coat a culture member or a culture instrument with a natural polymer material, a synthetic polymer material, or an inorganic material and use it as a culture instrument.

The natural polymer material, synthetic polymer material, or inorganic material is not particularly limited, but examples of the natural polymer material include glycosaminoglycans such as collagen, gelatin, alginic acid, hyaluronic acid and chondroitin sulfate, fibronectin, laminin and fibrinogen. Osteopontin, tenesin, vitronectin, thrombosbodin, agarose, elastin, keratin, chitosan, fibrin, fibroin, saccharides, polygluconic acid, polylactic acid, polyethylene glycol, polycaprolactone, synthetic peptides, synthetic proteins as synthetic polymer materials Examples of the synthetic polymer material include polyethylene glycol, polyhydroxyethylmethacrylate and polyethyleneimine, and examples of the inorganic material include β-tricalcium phosphate and calcium carbonate.

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

From the viewpoint of improving cell adhesion and cell proliferation, and maintaining cell function for a longer period of time, coating with proteins or peptides such as collagen, gelatin, laminin, and polylysine is preferable, and collagen or polylysine is used. A coating treatment is more preferred. These coatings may be performed individually by 1 type or in combination of 2 or more types.

The incubator of the present invention may be disinfected and sterilized in order to prevent contamination. The method of disinfection / sterilization is not particularly limited, and is a physical disinfection method such as a circulating steam method, a boiling method, an intermittent method, an ultraviolet method, a gas such as ozone, or a chemical disinfection method using a disinfectant such as ethanol; Heat sterilization methods such as high-pressure steam method and dry heat method; irradiation sterilization methods such as gamma ray method and high-frequency method; gas sterilization methods such as ethylene oxide gas method and ozone hydrogen gas plasma method can be mentioned. Among them, the ethanol disinfection method, the high-pressure steam sterilization method, the gamma ray sterilization method, or the ethylene oxide gas sterilization method is preferable because the operation is simple and the sterilization can be sufficiently performed. These disinfection / sterilization treatments may be performed individually by 1 type or in combination of 2 or more types.

The method for producing the culture instrument of the present invention is not particularly limited, and when the entire culture instrument is composed of the culture member, it can be produced by the same method as the method for producing the culture member. When a part of the culture instrument is formed of the culture member, the culture instrument can be obtained by appropriately joining the culture member and other members. The method of joining is not particularly limited, and the culture member and other members may be integrally formed, or may be brought into close contact with each other via an adhesive or an adhesive.

The culture device of the present invention is preferably a cell culture device, and more preferably a hepatocyte culture device.

<Culture method>
The method for culturing cells and the like of the present invention is a culture member for culturing cells, tissues, or organs on the culture surface thereof, the culture member containing a resin, and the culture surface having at least one pore. A method for culturing cells, tissues, or organs, which comprises a step of bringing cells, tissues, or organs into contact with the culture surface of a member; and a step of supplying oxygen to the cells, tissues, or organs in contact with the culture surface.
The method of contacting the cells or the like with the culture surface of the culture member is not particularly limited as long as the cells or the like can be brought into contact with the culture surface of the culture member, and for example, the cells or the like are seeded on the culture surface of the culture member or the culture instrument. Can be mentioned.
The method of supplying oxygen to the cells or the like in contact with the culture surface is not particularly limited as long as the cells or the like in contact with the culture surface can be supplied with oxygen. Supplying oxygen to cells and the like via a culture member can be mentioned.
The resin is a thermoplastic resin, and it is preferable that the average pore diameter of the pores is 0.1 to 100 μm, and the number of the pores per unit area is 0.01 to 250 pieces / cm ² . ..
The method for culturing cells and the like is preferably a method for culturing cells, and more preferably a method for culturing hepatocytes.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
In the examples, a method for preparing a collagen coat solution, a method for preparing a cell type and a medium, a method for measuring oxygen permeability, a method for evaluating the presence or absence of deflection, a method for evaluating cell adhesion, a method for measuring oxygen concentration in a medium, The method for measuring the metabolic activity value is described below.

[Preparation of collagen coat solution]
A 0.1 M hydrochloric acid solution (for volumetric analysis, 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 and sterilized by filtration. A 3 mg / mL collagen solution (Cellmatrix TypeIP, derived from pig tendon, Nitta Gelatin) was diluted 6-fold with a 0.001 M hydrochloric acid solution to prepare a 0.5 mg / mL collagen solution.

[Preparation of cell type and medium]
Medium was added to a centrifuge tube (50 ml) containing a cell suspension containing rat primary frozen hepatocytes. The medium was L-proline (for culture, Wako Pure Chemical Industries, Ltd.) diluted with 1.5 mL of bovine fetal serum (Fetal Bouline Serum, FBS, Wako Pure Chemical Industries, Ltd.) and 3.0 g / mL with water for injection (Fuso Yakuhin Kogyo). 0.15 mL of a photopure drug solution, 1.5 μL of a dexamethasone (biochemical, Fujifilm Wako Pure Chemical Industries, Ltd.) solution diluted to 1 × 10 ^-3 M with ethanol (for molecular biology, Fuji Film Wako Pure Chemical Industries, Ltd.), Epithelial growth factor diluted to 20 μg / mL using 21 μL of hydrocortisone (for culture, Fujifilm Wako Pure Chemical Industries, Ltd.) solution diluted to 36 mM with ethanol and BSA solution diluted to 1.0 mg / mL with water for injection. Epidermal glow factor, EGF, for cell biology, Fujifilm Wako Pure Chemical Industries, Ltd. 15 μL, insulin solution (10 mg / mL in HEPS, SIGMA) 8.7 μL, penicillin-streptomycin solution (5000 units / mL penicillin, 5000 μg / mL) Contains streptomycin, for culture, Wako Pure Chemical Industries, Ltd. 0.3 mL, D-MEM medium (4500 mg / mL D-glucose, 584 mg / mL L-glutamine, 15 mg / mL phenol red, 110 mg / mL sodium pyruvate, 3700 mg) / ML Containing sodium hydrogen carbonate, for culture, Fujifilm Wako Pure Chemical Industries, Ltd.) was added in an amount of 13 mL. The cell density was adjusted by adjusting the number of cells in the cell suspension containing rat primary frozen hepatocytes, and the cell density was 4.0 × 10 ⁵ cells / cm ² unless otherwise specified. In Example 9 and Comparative Examples 1, 3 and 5, the cells were cultured at a cell density of 1.0 × 10 ⁵ cells / cm ² .

[Measurement of oxygen permeability]
The oxygen permeability of the film before perforating the pores was measured in an environment of a temperature of 23 ° C. and a humidity of 0% RH using a differential pressure type gas permeability measuring device MT-C3 manufactured by Toyo Seiki Seisakusho. The diameter of the measuring part was 70 mm (transmission area was 38.46 cm ² ). An aluminum mask was applied to the sample in advance to set the actual transmission area to 5.0 cm ² .

[Presence / absence of bending]
One day after cell seeding, the culture vessel was taken out from the incubator, and the bottom surface of each of the four culture vessels was viewed from the side to observe the presence or absence of film sagging in the culture environment. In all four culture containers, there is no change compared to the time when the container was made, and the case where the film does not hang down is regarded as "no bending", and in at least one of the four culture containers, there is a change compared to the time when the culture container was made. The case where the film hangs down is evaluated as "deflection".

[Evaluation of cell adhesion]
One day after cell seeding, typical 3-wells were observed under a microscope for each of the four culture vessels, and the cells were observed to grow while adhering. In all four culture vessels, the case where the cells adhere and proliferate uniformly to the entire culture surface and maintain the morphology is defined as ◯, and in at least one of the four culture vessels, the cells adhere uniformly to the entire culture surface. The case where the cells did not grow, did not grow, or did not maintain their morphology was evaluated as x.

[Measurement of oxygen concentration in the medium]
One day after cell seeding, after removing the medium from the culture vessel, 0.5 mL of the medium was newly added and used in the medium using a fluorescent oxygen sensor (FireSting oxygen monitor, manufactured by BAS Co., Ltd.). Oxygen partial pressure (hPa) was measured. The measurement was carried out 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 (atmosphere 1 atm) and multiplied by 100 to calculate the value (%), which was used as the oxygen concentration in the medium. Measurements were made with typical 3 wells, and the average value was calculated.

[Measurement of metabolic activity value]
After removing the medium in the culture vessel 24 hours after cell seeding, Luciferin-CEE diluted with the medium was added, and the cells were further cultured for 3 hours. After culturing cells were transferred to a 96-well plate accompanied by a medium containing Luciferin-CEE, a mixed solution of Luciferin Detection Regent and Reaction Buffer was added, and the cells were reacted at room temperature in the dark for 1 hour. After 1 hour, the amount of light emitted (Relative Light Unit, RLU) was measured with a luminometer. Measurements were made with typical 3 wells, and the average value was calculated.

The amount of protein was determined by removing the Luciferin-CEE solution diluted in the medium, adding 200 μL of PBS (-) to the medium, collecting the cells in an Eppen tube using a cell scraper, and centrifuging (4 ° C., 22000 × g,). 10 minutes). Then, the supernatant was removed, 100 μL of 0.1 M sodium hydroxide solution was added, and then the amount of protein was measured using Pierce ^TM BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Absorbance at a wavelength of 450 nm was measured with 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 ^TM CYP1A1 Assay kit (manufactured by Promega), and the amount of protein obtained from the absorbance and Luciferin-CEE were measured. The metabolic activity value (pmol / min / mg protein) was calculated by dividing by the reaction time of the solution.

[Example 1]
Unstretched film of polystyrene (hereinafter referred to as PS) homopolymer with a thickness of 50 μm cut into a rectangle with a size of 12 cm × 8 cm (manufactured by Okura Kogyo Co., Ltd., trade name: Cellomer S-2, oxygen permeability 3310 cm ³ ) / (M ² x 24 h x atm)), using a UV laser processing machine (manufactured by Takano Co., Ltd.), wavelength 266 nm, output 50 mW, scanning speed 10 mm / sec. Laser processing was carried out under the condition of a beam diameter of 5 μm, and 24 pores having a diameter of 5 μm were formed on the PS film. The arrangement of the pores will be described later.
Using a digital microscope (VHX-1000 manufactured by KEYENCE CORPORATION), the perforated film was photographed from above, and the hole diameter was measured based on the photographed image. Individual measurements of the pore sizes of the 24 pores are not shown, but the pore size variability was within 10%.
The perforated film was plasma-treated by filling the inside of the chamber with a stream of nitrogen using an atmospheric pressure plasma surface treatment device (manufactured by Sekisui Chemical Co., Ltd.) (treatment speed 2 m / min, output 4.5 kW, 2 reciprocations). Then, a plasma-treated perforated film was brought into close contact with the bottom surface of the PS 24-well culture plate frame in which the bottom surface of each well was open via a medical adhesive (manufactured by 3M). One pore was formed per one well (circular shape with a diameter of 16 mm, area of about 2 cm ² ), and a 24-well cell culture vessel having a pore density of 0.5 cells / cm ² was prepared. The pores were pre-designed and perforated so that they would be arranged as shown in FIG. 1B at each well when the perforated film was adhered to the 24-well culture plate frame. Specifically, one pore was arranged at the center of the circle along the inside of the bottom surface of the circular well.
Pack the cell culture vessel in a gamma-ray resistant bag, sterilize it by irradiating it with 10 kHz gamma rays, add 0.5 mL of 0.5 mg / mL collagen solution to the culture surface of the culture vessel, and then remove the excess collagen solution. did. After allowing to stand at room temperature for 30 to 60 minutes, the cells were washed with Dulbecco PBS (−) and dried overnight at room temperature. The collagen-coated cell culture vessel prepared by the same method was used as culture vessel 1, and five of the same cells were prepared.

Next, a 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 at 37 ° C., 5%. Culture under CO ₂ was started. The presence or absence of bending and cell adhesion were evaluated in four culture vessels. Then, the oxygen concentration in the medium was measured using one of the four culture vessels 1. Metabolic activity was measured in the remaining three. The results are shown in Table 1. The metabolic activity values are shown in Table 1 with the results of the three containers as average values. Furthermore, the remaining one container was evaluated for cell adhesion after culturing for 7 days. The results are shown in Table 1. FIG. 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 prepared 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, and a 24-well cell culture vessel having a pore diameter of 20 μm and a pore density of 0.5 cells / cm ² was prepared. Was created and evaluated. The results are shown in Table 1.

[Example 3]
A cell culture vessel was prepared 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 having a pore diameter of 40 μm and a pore density of 0.5 cells / cm ² was prepared. , The evaluation was carried out. The results are shown in Table 1.

[Example 4]
A cell culture vessel was prepared in the same manner as in Example 2 except that the laser processing conditions were changed to a beam diameter of 80 μm, and a 24-well cell culture vessel having a pore diameter of 80 μm and a pore density of 0.5 cells / cm ² was prepared. , The evaluation was carried out. The results are shown in Table 1.

[Example 5]
A cell culture vessel was prepared in the same manner as in Example 2 except that 120 20 μm pores were formed on the PS film, and evaluation was carried out. Five pores (pore diameter 20 μm) were formed per one well (circular shape with a diameter of 16 mm, area of about 2 cm ² ), and a 24-well cell culture vessel having a pore density of 2.5 cells / cm ² was prepared. The pores were pre-designed and perforated so that they would be arranged as shown in FIG. 1C at each well when the perforated film was adhered to the 24-well culture plate frame. Specifically, one pore is formed in the center of the circle along the inside of the bottom surface of the circular well, and the pore is located at the intersection of the concentric circle (radius 5 mm) of the circle and the two orthogonal diagonal lines of the circle. 4 were arranged. The results are shown in Table 1.

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

[Example 7]
A cell culture vessel was prepared in the same manner as in Example 2 except that 2400 20 μm pores were formed on the PS film, and evaluation was carried out. A 24-well cell culture vessel having 100 pores (pore diameter 20 μm) formed per well (circular shape with a diameter of 16 mm and an area of about 2 cm ² ) and a pore density of 50 cells / cm ² was prepared. The pores were pre-designed and perforated so that they would be arranged as shown in FIG. 1E at each well when the perforated film was adhered to the 24-well culture plate frame. Specifically, four circles along the inside of the bottom surface of the circular well on a concentric circle with a radius of 0.8 mm, 16 on a concentric circle with a radius of 1.6 mm and 3.2 mm, a radius of 4.8 mm, 6. Thirty-two pores were evenly arranged on a 4 mm concentric circle. The results are shown in Table 1.

[Example 8]
A cell culture vessel was prepared in the same manner as in Example 2 except that 9600 20 μm pores were formed on the PS film, and evaluation was carried out. A 24-well cell culture vessel having 400 pores (pore diameter 20 μm) formed per well (circular shape with a diameter of 16 mm and an area of about 2 cm ² ) and a pore density of 200 cells / cm ² was prepared. The pores were pre-designed and perforated so that they would be arranged as shown in FIG. 1F at each well when the perforated film was adhered to the 24-well culture plate frame. Specifically, eight circles along the inside of the bottom surface of the circular well are concentric circles with radii of 0.8 mm, 1.6 mm, 2.1 mm and 2.7 mm, and radii of 3.2 mm, 3.7 mm, 4 16 pieces on a concentric circle of .3 mm, 32 pieces on a concentric circle with a radius of 4.8 mm, 5.3 mm, 64 pieces on a concentric circle with radii of 5.9 mm, 6.4 mm, 6.9 mm, and 7.5 mm, evenly thin. A hole was placed. 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 in the same manner as in Example 3 except that collagen treatment was not carried out, and evaluation was carried out. The results are shown in Table 1.

[Example 11]
Instead of the PS film, a TPX film with a thickness of 50 μm (hereinafter referred to as PMP, manufactured by Mitsui Chemicals Tocello Co., Ltd., trade name: Opulan X-88B, oxygen permeability 38240 cm ³ / (m ² × 24 h × atm)) is used. A cell culture vessel was prepared in the same manner as in Example 2 except for the above, and a 24-well cell culture vessel having a pore size of 20 μm and a pore density of 0.5 cells / cm ² was prepared and evaluated. The results are shown in Table 1.

[Example 12]
A cell culture vessel was prepared 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 having a pore diameter of 40 μm and a pore density of 0.5 cells / cm ² was prepared. , The evaluation was carried out. The results are shown in Table 1.

[Example 13]
Biaxially oriented polyethylene terephthalate film with a thickness of 50 μm instead of PS film (hereinafter referred to as PET; manufactured by Toray Co., Ltd., trade name: Lumirror # 50-T60, oxygen permeability 30 cm ³ / (m ² × 24 h × atm)) ) Was prepared, a cell culture vessel was prepared in the same manner as in Example 2, and a 24-well cell culture vessel having a pore size of 20 μm and a pore density of 0.5 cells / cm ² was prepared and evaluated. .. The results are shown in Table 1.

[Example 14]
A cell culture vessel was prepared 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 having a pore diameter of 40 μm and a pore density of 0.5 cells / cm ² was prepared. , The evaluation was carried out. The results are shown in Table 1.

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

[Comparative Example 2]
A cell culture vessel was prepared and evaluated by the same method as in Example 1 except that a PS film having no pores formed by laser processing was used. The results are shown in Table 2.

[Comparative Example 3]
Evaluation was carried out in the same manner as in Comparative Example 2 except that the cell seeding concentration was changed to 1.0 × 10 ⁵ cells / cm ² . The results are shown in Table 2.

[Comparative Example 4]
Cells in the same manner as in Comparative Example 2 except that a commercially available 24-well PS culture vessel (Oxygen permeability 200 cm ³ / (m ² × 24 h × atm) manufactured by Corning Inc.) having a PS thickness of 1000 μm on the bottom surface was used. A culture vessel was prepared and evaluated. The results are shown in Table 2.

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

[Comparative Example 6]
A cell culture vessel was prepared and evaluated by the same method as in Example 11 except that a TPX film having no pores formed by laser processing was used. The results are shown in Table 2.

[Comparative Example 7]
A cell culture vessel was prepared and evaluated by the same method as in Example 13 except that a PET film having no pores formed by laser processing was used. The results are shown in Table 2.

From Table 1, it was shown that long-term culture for up to 7 days is possible in the culture of rat primary frozen hepatocytes using the culture vessel in which the culture member of the present invention is placed at the bottom of the culture vessel. In addition, this culture vessel had no bending of the bottom surface, excellent shape stability, and no leakage of medium components. Furthermore, the cells adhered uniformly to the entire culture surface, proliferated, and maintained their morphology, indicating that this culture vessel has excellent cell adhesion.

Looking at the effect of culturing rat primary frozen hepatocytes using the culture member of the present invention in more detail, the oxygen concentration in the medium one day after the culture was high, and oxygen was effectively supplied through the culture member. In addition, the metabolic activity value of the drug, which was evaluated as the function of hepatocytes, was high, and it was clarified that the cell function was maintained normally. Furthermore, it was shown that cell function can be maintained normally even in a high-density culture of 4 × 10 ⁵ cells / cm ² .

Furthermore, from Examples 1 to 9, it was shown that by increasing the pore size and pore density, the oxygen supply capacity from the bottom surface of the culture vessel was improved, the saturated oxygen concentration was improved, and the metabolic activity value was also improved.

On the other hand, in Comparative Example 1, since the pore diameter was 120 μm, liquid leakage from the pores occurred and the culture could not be evaluated. In Comparative Examples 2 to 5, since the polystyrene container having no pores was used, the saturated oxygen concentration was lowered and the metabolic activity value was also lowered, indicating that the cells could not express normal functions. In particular, in high-density culture of 4 × 10 ⁵ cells / cm ² (Comparative Example 2 and Comparative Example 4), it was shown that the metabolic activity value was further lowered and normal function could not be expressed. In Comparative Example 6 using a polyethylene terephthalate container having no pores formed, it was shown that the saturated oxygen concentration decreased and the metabolic activity value also decreased, and the cells could not express normal functions.

From Examples 1 to 9 and Comparative Examples 2 and Examples 13, 14 and 6, by using a culture member having pores on the bottom surface of the culture vessel, the oxygen supply capacity is improved, the saturated oxygen concentration is improved, and further. Was shown to improve metabolic activity.

From Example 10, it is clear that the culture vessel of the present invention exhibits a high saturated oxygen concentration and metabolic activity value in a high-density culture of 4 × 10 ⁵ cells / cm ² even when collagen coating is not performed. became.

From the above results, the culture member of the present invention has excellent cell adhesion in addition to excellent shape stability and oxygen supply property, and can cultivate cells and the like while maintaining the functions of the cells and the like even at high density. It became clear.

Claims (11)

A culture member for culturing cells, tissues, or organs on the culture surface, wherein the culture member contains a resin, and the culture surface has at least one pore. The culture member according to claim 1, wherein the resin is a thermoplastic resin. The culture member according to claim 1 or 2, wherein the resin is at least one selected from a polystyrene-based resin, polyethylene terephthalate, and 4-methyl-1-pentene polymer. The culture member according to any one of claims 1 to 3, wherein the average pore diameter of the pores is 0.1 to 100 μm. The culture member according to any one of claims 1 to 4, wherein the number of the pores per unit area is 0.01 to 250 per cm ² . The culture member according to any one of claims 1 to 5, wherein the shape of the culture member is a film or a sheet. A culture instrument in which at least the culture surface is formed of the culture member according to any one of claims 1 to 6. The incubator according to claim 7, which has at least one well. The culture device according to claim 7 or 8, wherein the culture surface of the culture member has a coating layer of at least one material selected from the group consisting of a natural polymer material, a synthetic polymer material, and an inorganic material. A step of bringing cells, tissues or organs into contact with the culture surface of the culture member according to any one of claims 1 to 6; and a step of supplying oxygen to the cells, tissues or organs in contact with the culture surface; A method of culturing a cell, tissue, or organ containing. 10. The resin is a thermoplastic resin, the average pore diameter of the pores is 0.1 to 100 μm, and the number of the pores per unit area is 0.01 to 250 per cm ² . The culture method described in 1.

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