JP6907477B2 - Cell culture method and cell culture device - Google Patents

Description

The present invention relates to a method for culturing cells using a polymer porous membrane. The present invention also relates to a cell culture device provided with a polymer porous membrane.

In recent years, proteins such as enzymes, hormones, antibodies, cytokines, and viruses (viral proteins) used for treatment and vaccines have been industrially produced using cultured cells. However, the cost of producing these proteins was high, which raised medical costs. Therefore, with the aim of drastically reducing costs, there has been a demand for a technique for culturing cells at high density and an innovative technique for increasing the amount of protein produced.

As the cells that produce proteins, scaffold-dependent adherent cells that adhere to the culture medium may be used. Since these cells proliferate in a scaffold-dependent manner, they need to be adhered to the surface of a petri dish, plate or chamber for culturing. Conventionally, in order to culture a large amount of such adherent cells, it has been necessary to increase the surface area for adhesion. However, in order to increase the culture area, it is necessary to increase the space inevitably, which has been a factor of increasing the cost.

As a method for culturing a large amount of adherent cells while reducing the culture space, a culture method using a carrier having microporous properties, particularly a microcarrier, has been developed (for example, Patent Document 1). The cell culture system using microcarriers needs to be sufficiently stirred and diffused so that the microcarriers do not aggregate with each other. Therefore, there is an upper limit to the density of cells that can be cultured because a volume that is sufficient to sufficiently stir and diffuse the medium in which microcarriers are dispersed is required. Further, in order to separate the microcarrier and the medium, it is necessary to separate the fine particles with a filter capable of separating them, which has been a cause of increasing the cost. Under these circumstances, an innovative cell culture methodology for culturing high-density cells has been sought.

<Polyimide porous membrane>
Prior to this application, polyimide porous membranes have been used for applications such as filters, low dielectric constant films, and electrolyte membranes for fuel cells, especially for battery-related applications. Patent Documents 2 to 4 particularly have excellent permeability to substances such as gas, high porosity, excellent smoothness on both surfaces, relatively high strength, and despite the high porosity, the film thickness direction. Describes a polyimide porous membrane having a large number of macrovoids having excellent resistance to compressive stress. All of these are polyimide porous membranes prepared via amic acid.

A method for culturing cells including applying the cells to a polyimide porous membrane and culturing the cells has been reported (Patent Document 5).

International Publication No. 2003/054174 International Publication No. 2010/0388873 Japanese Unexamined Patent Publication No. 2011-219585 Japanese Unexamined Patent Publication No. 2011-219586 International Publication No. 2015/012415

An object of the present invention is to provide a method for culturing cells using a polymer porous membrane. Another object of the present invention is to provide a cell culture apparatus provided with a polymer porous membrane.

The present inventors have found that a polymer porous membrane having a predetermined structure provides a moist environment resistant to drying, and by applying a droplet medium, a large amount of cells can be efficiently supplied while supplying oxygen. The method and apparatus for culturing were completed. That is, the present invention preferably includes, but is not limited to, the following aspects.

[1] A method for culturing cells.
(1) Applying cells to a polymer porous membrane and
(2) Culturing in the gas phase while applying the medium dropleted by the droplet medium supply means to the polymer porous membrane to which the cells are applied.
Including
Here, the polymer porous membrane has a three-layer structure polymer porous having a surface layer A and a surface layer B having a plurality of pores and a macrovoid layer sandwiched between the surface layer A and the surface layer B. In the quality film, the average pore diameter of the pores existing in the surface layer A is smaller than the average pore diameter of the pores existing in the surface layer B, and the macrovoid layer is bonded to the surface layers A and B. It has the partition wall, the partition wall, and a plurality of macrovoids surrounded by the surface layers A and B, and the holes in the surface layers A and B communicate with the macrovoids.
Method.
[2] The polymer porous membrane is
i) Folded,
ii) Rolled up,
iii) Sheets or small pieces connected by a thread-like structure,
iv) The method according to [1], wherein the method is a rope-shaped and / or v) polymer porous membrane in which two or more are laminated.
[3] The polymer porous membrane is a modularized polymer porous membrane provided with a casing.
Here, the casing has two or more cell culture outlets.
Here, the modularized polymer porous membrane is
(I) Two or more independent polymer porous membranes are aggregated and
(Ii) The polymer porous membrane is folded and
(Iii) The polymer porous membrane is rolled up and / or
(Iv) The polymer porous membrane is tied in a rope shape,
The method according to [1], which is housed in the casing.
[4] The method according to any one of [1] to [3], wherein the polymer porous membrane has a plurality of pores having an average pore diameter of 0.01 to 100 μm.
[5] The cell culture apparatus according to any one of [1] to [4], wherein the surface layer A has an average pore size of 0.01 to 50 μm.
[6] The method according to any one of [1] to [5], wherein the surface layer B has an average pore diameter of 20 to 100 μm.
[7] The method according to any one of [1] to [6], wherein the total film thickness of the polymer porous membrane is 5 to 500 μm.
[8] The method according to any one of [1] to [7], wherein the polymer porous membrane is a polyimide porous membrane.
[9] The method according to [8], wherein the polyimide porous membrane is a polyimide porous membrane containing a polyimide obtained from tetracarboxylic dianhydride and diamine.
[10] The polyimide porous film is formed by molding a polyamic acid solution composition containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a coloring precursor, and then heat-treated at 250 ° C. or higher. The method according to [8] or [9], which is the obtained colored polyimide porous film.
[11] The method according to any one of [1] to [7], wherein the polymer porous membrane is a polyether sulfone porous membrane.

[12] With a polymer porous membrane,
The polymer porous membrane mounting portion on which the polymer porous membrane is mounted and the polymer porous membrane mounting portion
A housing in which the polymer porous membrane mounting portion is housed, and
A droplet medium supply unit arranged inside the housing and
A medium supply line communicating with the droplet medium supply unit and
A medium storage unit that communicates with the medium supply line and
A pump provided in a part of the medium supply line and
With
Here, the polymer porous membrane has a three-layer structure polymer porous having a surface layer A and a surface layer B having a plurality of pores and a macrovoid layer sandwiched between the surface layer A and the surface layer B. In the quality film, the average pore diameter of the pores existing in the surface layer A is smaller than the average pore diameter of the pores existing in the surface layer B, and the macrovoid layer is bonded to the surface layers A and B. It has a partition wall, the partition wall, and a plurality of macrovoids surrounded by the surface layers A and B.
Here, a cell culture apparatus in which the polymer porous membrane mounting portion is provided with a plurality of slit-shaped or mesh-shaped medium outlets.
[13] The cell culture apparatus according to [12], wherein one or more of the droplet medium supply parts are arranged on the upper part, the lower part and / or the side part of the polymer porous membrane mounting part.
[14] The cell culture apparatus according to [12] or [13], wherein the polymer porous membrane mounting portion has two or more stages.
[15] The medium storage portion is arranged inside the housing and below the polymer porous membrane mounting portion, and the culture medium circulates. The cell culture apparatus according to any one item.
[16] The polymer porous membrane is
i) Folded,
ii) Rolled up,
iii) Sheets or small pieces connected by a thread-like structure,
iv) The cell culture apparatus according to any one of [12] to [15], which is a rope-shaped and / or v) polymer porous membrane in which two or more are laminated.
[17] The polymer porous membrane is a modularized polymer porous membrane provided with a casing.
Here, the casing has two or more cell culture outlets.
Here, the modularized polymer porous membrane is
(I) Two or more independent polymer porous membranes are aggregated and
(Ii) The polymer porous membrane is folded and
(Iii) The polymer porous membrane is rolled up and / or
(Iv) The polymer porous membrane is tied in a rope shape,
The cell culture apparatus according to any one of [12] to [15], which is housed in the casing.
[18] The cell culture apparatus according to any one of [12] to [17], wherein the polymer porous membrane has a plurality of pores having an average pore diameter of 0.01 to 100 μm.
[19] The cell culture apparatus according to any one of [12] to [18], wherein the surface layer A has an average pore size of 0.01 to 50 μm.
[20] The cell culture apparatus according to any one of [12] to [19], wherein the surface layer B has an average pore size of 20 to 100 μm.
[21] The cell culture apparatus according to any one of [12] to [20], wherein the total film thickness of the polymer porous membrane is 5 to 500 μm.
[22] The cell culture apparatus according to any one of [12] to [21], wherein the polymer porous membrane is a polyimide porous membrane.
[23] The cell culture apparatus according to [22], wherein the polyimide porous membrane is a polyimide porous membrane containing a polyimide obtained from tetracarboxylic dianhydride and diamine.
[24] The polyimide porous film is formed by molding a polyamic acid solution composition containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a coloring precursor, and then heat-treated at 250 ° C. or higher. The cell culture apparatus according to [22] or [23], which is the obtained colored polyimide porous film.
[25] The cell culture apparatus according to any one of [12] to [21], wherein the polymer porous membrane is a polyether sulfone porous membrane.

[26] A method for culturing cells using the cell culture apparatus according to any one of [12] to [24].

Since the polymer porous membrane used in the present invention has a slightly hydrophilic porous property, stable liquid retention is achieved in the polymer porous membrane, and a moist environment resistant to drying is provided. Therefore, even when a part or all of the polymer porous membrane is exposed to air, the supported cells can be cultured without drying. Further, by culturing while applying the droplet-dropped medium to the polymer porous membrane, the amount of the medium used is extremely reduced. Furthermore, by culturing while applying the dropletized medium, it becomes possible to cultivate a large amount of cells while efficiently supplying oxygen. Therefore, the present invention does not particularly require an oxygen supply device. In addition, by using small droplets, it is possible to supply the medium from many directions in any direction, so that it is homogeneous with respect to the polymer porous membrane containing cells existing at each location and the modularized polymer porous membrane. It will be possible to supply the medium. (Figs. 1 and 2)

FIG. 1 is a diagram showing a configuration example of a cell culture device according to an embodiment. FIG. 2 is a diagram showing a configuration example of the cell culture device according to the embodiment. FIG. 3 shows a model diagram of cell culture using a polymer porous membrane. FIG. 4 is a diagram showing a cell culture apparatus used in the examples. (A) Overall view, (B) (A) Enlarged view of the housing portion

Hereinafter, embodiments of the present invention will be described with reference to the drawings as necessary. The configuration of the embodiment is an example, and the configuration of the present invention is not limited to the specific configuration of the embodiment.

1. 1. Polymer Porous Membrane The average pore diameter of the pores existing in the surface layer A (hereinafter, also referred to as “A surface” or “mesh surface”) in the polymer porous film used in the present invention is not particularly limited, but is, for example. , 0.01 μm or more and less than 200 μm, 0.01 to 150 μm, 0.01 to 100 μm, 0.01 to 50 μm, 0.01 μm to 40 μm, 0.01 μm to 30 μm, 0.01 μm to 20 μm, or 0.01 μm to 15 μm It is preferably 0.01 μm to 15 μm.

The average pore diameter of the pores existing in the surface layer B (hereinafter, also referred to as “B surface” or “large hole surface”) in the polymer porous membrane used in the present invention is the average pore diameter of the pores existing in the surface layer A. It is not particularly limited as long as it is larger than, but is, for example, more than 5 μm and 200 μm or less, 20 μm to 100 μm, 30 μm to 100 μm, 40 μm to 100 μm, 50 μm to 100 μm, or 60 μm to 100 μm, preferably 20 μm to 100 μm.

（式中、Ｓａは孔面積の平均値を意味する。） The average pore size of the surface of the polymer porous film is determined by measuring the pore area of 200 or more openings from the scanning electron micrograph of the surface of the porous film and using the average value of the pore area according to the following formula (1). The average diameter when the shape of is a perfect circle can be obtained by calculation.

(In the formula, Sa means the average value of the hole area.)

The thicknesses of the surface layers A and B are not particularly limited, but are, for example, 0.01 to 50 μm, preferably 0.01 to 20 μm.

The average pore size of the macrovoids in the macrovoid layer in the polymer porous membrane in the film plane direction is not particularly limited, but is, for example, 10 to 500 μm, preferably 10 to 100 μm, and more preferably 10 to 80 μm. The thickness of the partition wall in the macrovoid layer is not particularly limited, but is, for example, 0.01 to 50 μm, preferably 0.01 to 20 μm. In one embodiment, at least one partition wall in the macrovoid layer communicates with adjacent macrovoids and has an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm, one or more. Has a hole. In another embodiment, the bulkhead in the macrovoid layer is non-perforated.

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

The film thickness of the polymer porous membrane used in the present invention can be measured with a contact-type thickness gauge.

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

（式中、Ｓは多孔質フィルムの面積、ｄは総膜厚、ｗは測定した質量、Ｄはポリマーの密度をそれぞれ意味する。ポリマーがポリイミドである場合は、密度は１．３４ｇ／ｃｍ^３とする。） The porosity of the polymer porous film used in the present invention can be determined from the grain mass according to the following formula (2) by measuring the film thickness and mass of the porous film cut to a predetermined size.

(In the formula, S means the area of the porous film, d means the total film thickness, w means the measured mass, and D means the density of the polymer. When the polymer is polyimide, the density is 1.34 g / cm ³ (.)

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

The polymer porous membrane used in the present invention is preferably sterilized. The sterilization treatment is not particularly limited, and examples thereof include dry heat sterilization, steam sterilization, sterilization with a disinfectant such as ethanol, and arbitrary sterilization treatment such as electromagnetic wave sterilization such as ultraviolet rays and gamma rays.

The polymer porous membrane used in the present invention is not particularly limited as long as it has the above-mentioned structural characteristics, but is preferably a polyimide porous membrane or a polyether sulfone (PES) porous membrane.

1-1. Polyimide Porous film Polyimide is a general term for polymers containing an imide bond in a repeating unit, and usually means an aromatic polyimide in which an aromatic compound is directly linked by an imide bond. Aromatic polyimide has a rigid and strong molecular structure because the aromatic and the aromatic have a conjugated structure via an imide bond, and the imide bond has a strong intermolecular force, so that a very high level of heat is generated. It has physical, mechanical and chemical properties.

The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane containing (as a main component) a polyimide obtained from tetracarboxylic dianhydride and diamine, and more preferably tetracarboxylic dianhydride. It is a polyimide porous film made of polyimide obtained from a substance and a diamine. "Included as a main component" means that, as a constituent component of the polyimide porous film, a component other than the polyimide obtained from tetracarboxylic dianhydride and diamine is essentially not contained or may be contained. , Means that it is an additional component that does not affect the properties of the polyimide obtained from tetracarboxylic dianhydride and diamine.

In one embodiment, the polyimide porous membrane that can be used in the present invention is 250 ° C. after molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component and a coloring precursor. The colored polyimide porous film obtained by the above heat treatment is also included.

The polyamic acid is obtained by polymerizing a tetracarboxylic acid component and a diamine component. The polyamic acid is a polyimide precursor that can be ring-closed to form a polyimide by thermal imidization or chemical imidization.

As the polyamic acid, even if a part of the amic acid is imidized, it can be used as long as it does not affect the present invention. That is, the polyamic acid may be partially thermally imidized or chemically imidized.

When the polyamic acid is thermally imidized, an imidization catalyst, an organic phosphorus-containing compound, fine particles such as inorganic fine particles and organic fine particles, and the like can be added to the polyamic acid solution, if necessary. When the polyamic acid is chemically imidized, fine particles such as a chemical imidizing agent, a dehydrating agent, inorganic fine particles, and organic fine particles can be added to the polyamic acid solution, if necessary. Even if the above-mentioned components are mixed with the polyamic acid solution, it is preferable to carry out the process under the condition that the coloring precursor does not precipitate.

As used herein, the term "colored precursor" means a precursor that is partially or wholly carbonized by heat treatment at 250 ° C. or higher to produce a colored product.

The coloring precursor that can be used in the production of the polyimide porous film is uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution, and is 250 ° C. or higher, preferably 260 ° C. or higher, more preferably 280 ° C. or higher, more preferably. Is thermally decomposed and carbonized by heat treatment at 300 ° C. or higher, preferably 250 ° C. or higher in the presence of oxygen such as air, preferably 260 ° C. or higher, more preferably 280 ° C. or higher, more preferably 300 ° C. or higher. Those that produce colored products are preferable, those that produce black-based colored products are more preferable, and carbon-based colored precursors are more preferable.

The coloring precursor looks like a carbonized product when heated, but structurally, it contains a foreign element other than carbon, and has a layered structure, an aromatic crosslinked structure, and a disordered structure containing tetrahedral carbon. include.

The carbon-based coloring precursor is not particularly limited, and for example, a polymer obtained from a tar or pitch such as petroleum tar, petroleum pitch, coal tar, coal pitch, coke, and a monomer containing acrylonitrile, and a ferrocene compound (ferrocene and ferrocene derivative). And so on. Among these, a polymer obtained from a monomer containing acrylonitrile and / or a ferrocene compound is preferable, and a polymer obtained from a monomer containing acrylonitrile is preferably polyacrylic nitrile.

Further, in another embodiment, the polyimide porous film that can be used in the present invention is obtained after molding a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component without using the above-mentioned coloring precursor. A polyimide porous membrane obtained by heat treatment is also included.

The polyimide porous film produced without using a coloring precursor is composed of, for example, 3 to 60% by mass of a polyamic acid having an ultimate viscosity of 1.0 to 3.0 and 40 to 97% by mass of an organic polar solvent. A polyamic acid solution is cast into a film and immersed or brought into contact with a coagulation solvent containing water as an essential component to prepare a porous film of the polyamic acid, and then the porous film of the polyamic acid is heat-treated to imide. It may be manufactured by converting. In this method, the coagulation solvent containing water as an essential component is water, or a mixed solution of water of 5% by mass or more and less than 100% by mass and an organic polar solvent of more than 0% by mass and 95% by mass or less. You may. Further, after the imidization, at least one surface of the obtained porous polyimide film may be subjected to plasma treatment.

As the tetracarboxylic dianhydride that can be used in the production of the polyimide porous membrane, any tetracarboxylic dianhydride can be used, and it can be appropriately selected according to desired properties and the like. Specific examples of tetracarboxylic dianhydrides are pyromellitic dianhydrides, 3,3', 4,4'-biphenyltetracarboxylic dianhydrides (s-BPDA), 2,3,3', 4'. Biphenyltetracarboxylic dianhydride such as -biphenyltetracarboxylic dianhydride (a-BPDA), oxydiphthalic acid dianhydride, diphenylsulfone-3,4,3', 4'-tetracarboxylic dianhydride, bis (3,4-dicarboxyphenyl) sulfide dianhydride, 2,2-bis (3,4-dicarboxyphenyl) -1,1,1,3,3,3-hexafluoropropanedianhydride, 2, 3,3', 4'-benzophenonetetracarboxylic dianhydride, 3,3', 4,4'-benzophenonetetracarboxylic dianhydride, bis (3,4-dicarboxyphenyl) methanedianhydride, 2 , 2-Bis (3,4-dicarboxyphenyl) propanedianhydride, p-phenylenebis (trimellitic acid monoesteric acid anhydride), p-biphenylenebis (trimellitic acid monoesteric acid anhydride), m- Tarphenyl-3,4,3', 4'-tetracarboxylic dianhydride, p-terphenyl-3,4,3', 4'-tetracarboxylic dianhydride, 1,3-bis (3, 4-dicarboxyphenoxy) benzene dianhydride, 1,4-bis (3,4-dicarboxyphenoxy) benzene dianhydride, 1,4-bis (3,4-dicarboxyphenoxy) biphenyl dianhydride, 2 , 2-Bis [(3,4-dicarboxyphenoxy) phenyl] propanedianhydride, 2,3,6,7-naphthalenetetracarboxylic dianhydride, 1,4,5,8-naphthalenetetracarboxylic dianhydride Examples thereof include anhydrides and 4,4'-(2,2-hexafluoroisopropyridene) diphthalic acid dianhydrides. It is also preferable to use an aromatic tetracarboxylic acid such as 2,3,3', 4'-diphenylsulfonetetracarboxylic acid. These can be used alone or in combination of two or more.

Among these, at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydride and pyromellitic dianhydride is particularly preferable. As the biphenyltetracarboxylic dianhydride, 3,3', 4,4'-biphenyltetracarboxylic dianhydride can be preferably used.

Any diamine can be used as the diamine that can be used in the production of the polyimide porous membrane. Specific examples of the diamine include the following.
1) Benzenediamine with one benzene nucleus such as 1,4-diaminobenzene (para-phenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, 2,6-diaminotoluene;
2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenylmethane, 3,3'-dimethyl-4,4'-diaminobiphenyl, 2,2'- Dimethyl-4,4'-diaminobiphenyl, 2,2'-bis (trifluoromethyl) -4,4'-diaminobiphenyl, 3,3'-dimethyl-4,4'-diaminodiphenylmethane, 3,3'- Dicarboxy-4,4'-diaminodiphenylmethane, 3,3', 5,5'-tetramethyl-4,4'-diaminodiphenylmethane, bis (4-aminophenyl) sulfide, 4,4'-diaminobenzanilide, 3,3'-dichlorobenzidine, 3,3'-dimethylbenzidine, 2,2'-dimethylbenzidine, 3,3'-dimethoxybenzidine, 2,2'-dimethoxybenzidine, 3,3'-diaminodiphenyl ether, 3, 4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, 3,3'-diaminodiphenyl sulfide, 3,4'-diaminodiphenyl sulfide, 4,4'-diaminodiphenyl sulfide, 3,3'-diaminodiphenyl sulfone, 3,4'-diaminodiphenylsulfone, 4,4′-diaminodiphenylsulfone, 3,3′-diaminobenzophenone, 3,3′-diamino-4,4′-dichlorobenzophenone, 3,3′-diamino-4, 4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-diaminodiphenylmethane, 2,2-bis (3-aminophenyl) propane, 2,2-bis (4) -Aminophenyl) propane, 2,2-bis (3-aminophenyl) -1,1,1,3,3,3-hexafluoropropane, 2,2-bis (4-aminophenyl) -1,1, Two diamines of benzene nuclei such as 1,3,3,3-hexafluoropropane, 3,3'-diaminodiphenylsulfoxide, 3,4'-diaminodiphenylsulfoxide, 4,4'-diaminodiphenylsulfoxide;
3) 1,3-bis (3-aminophenyl) benzene, 1,3-bis (4-aminophenyl) benzene, 1,4-bis (3-aminophenyl) benzene, 1,4-bis (4-amino) Phenyl) benzene, 1,3-bis (4-aminophenoxy) benzene, 1,4-bis (3-aminophenoxy) benzene, 1,4-bis (4-aminophenoxy) benzene, 1,3-bis (3) -Aminophenoxy) -4-trifluoromethylbenzene, 3,3'-diamino-4- (4-phenyl) phenoxybenzophenone, 3,3'-diamino-4,4'-di (4-phenylphenoxy) benzophenone, 1,3-bis (3-aminophenyl sulfide) benzene, 1,3-bis (4-aminophenyl sulfide) benzene, 1,4-bis (4-aminophenyl sulfide) benzene, 1,3-bis (3-) Aminophenylsulfone) Benzene, 1,3-bis (4-aminophenylsulfone) benzene, 1,4-bis (4-aminophenylsulfone) benzene, 1,3-bis [2- (4-aminophenyl) isopropyl] Benzene, 1,4-bis [2- (3-aminophenyl) isopropyl] benzene, 1,4-bis [2- (4-aminophenyl) isopropyl] benzene and other benzene nuclei 3 diamines;
4) 3,3'-bis (3-aminophenoxy) biphenyl, 3,3'-bis (4-aminophenoxy) biphenyl, 4,4'-bis (3-aminophenoxy) biphenyl, 4,4'-bis (4-Aminophenoxy) biphenyl, bis [3- (3-aminophenoxy) phenyl] ether, bis [3- (4-aminophenoxy) phenyl] ether, bis [4- (3-aminophenoxy) phenyl] ether, Bis [4- (4-aminophenoxy) phenyl] ether, bis [3- (3-aminophenoxy) phenyl] ketone, bis [3- (4-aminophenoxy) phenyl] ketone, bis [4- (3-amino) phenyl] Phenoxy) phenyl] ketone, bis [4- (4-aminophenoxy) phenyl] ketone, bis [3- (3-aminophenoxy) phenyl] sulfide, bis [3- (4-aminophenoxy) phenyl] sulfide, bis [ 4- (3-Aminophenoxy) phenyl] sulfide, bis [4- (4-aminophenoxy) phenyl] sulfide, bis [3- (3-aminophenoxy) phenyl] sulfone, bis [3- (4-aminophenoxy) Phenyl] sulfone, bis [4- (3-aminophenoxy) phenyl] sulfone, bis [4- (4-aminophenoxy) phenyl] sulfone, bis [3- (3-aminophenoxy) phenyl] methane, bis [3- (4-Aminophenoxy) phenyl] methane, bis [4- (3-aminophenoxy) phenyl] methane, bis [4- (4-aminophenoxy) phenyl] methane, 2,2-bis [3- (3-amino) Phenoxy) phenyl] propane, 2,2-bis [3- (4-aminophenoxy) phenyl] propane, 2,2-bis [4- (3-aminophenoxy) phenyl] propane, 2,2-bis [4- (4-Aminophenoxy) phenyl] propane, 2,2-bis [3- (3-aminophenoxy) phenyl] -1,1,1,3,3,3-hexafluoropropane, 2,2-bis [3 -(4-Aminophenoxy) phenyl] -1,1,1,3,3,3-hexafluoropropane, 2,2-bis [4- (3-aminophenoxy) phenyl] -1,1,1,3 , 3,3-Hexafluoropropane, 2,2-bis [4- (4-aminophenoxy) phenyl] -1,1,1,3,3,3-hexafluoropropane and other four benzene nuclei diamines.

These can be used alone or in combination of two or more. The diamine to be used can be appropriately selected according to desired characteristics and the like.

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

From the viewpoint of heat resistance and dimensional stability at high temperatures, the polyimide porous film that can be used in the present invention has a glass transition temperature of 240 ° C. or higher, or a tetracarboxylic dianoxide having no clear transition point at 300 ° C. or higher. It is preferably formed from a polyimide obtained by combining an acid dianhydride and a diamine.

The polyimide porous film that can be used in the present invention is preferably a polyimide porous film made of the following aromatic polyimides from the viewpoint of heat resistance and dimensional stability at high temperatures.
(I) An aromatic polyimide composed of at least one tetracarboxylic acid unit selected from the group consisting of a biphenyl tetracarboxylic acid unit and a pyromellitic acid unit, and an aromatic diamine unit.
(Ii) Aromatic polyimide consisting of a tetracarboxylic acid unit and at least one aromatic diamine unit selected from the group consisting of a benzenediamine unit, a diaminodiphenyl ether unit and a bis (aminophenoxy) phenyl unit.
And / or
(Iii) At least one tetracarboxylic acid unit selected from the group consisting of a biphenyl tetracarboxylic acid unit and a polyimide acid unit, and at least one selected from the group consisting of a benzene diamine unit, a diaminodiphenyl ether unit and a bis (aminophenoxy) phenyl unit. Aromatic polyimide consisting of a kind of aromatic diamine unit.

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

For example, the polyimide porous membrane described in International Publication No. 2010/038873, JP-A-2011-219585, or JP-A-2011-219586 can also be used in the present invention.

1-2. Polyether Sulfone (PES) Porous Membrane The PES porous membrane that can be used in the present invention comprises a polyether sulfone and typically comprises substantially a polyether sulfone. The polyether sulfone may be synthesized by a method known to those skilled in the art, for example, a method of polycondensing a dihydric phenol, an alkali metal compound and a dihalogenodiphenyl compound in an organic polar solvent, a divalent phenol. It can be produced by a method in which an alkali metal dihydrate is synthesized in advance and polycondensed with a dihalogenodiphenyl compound in an organic polar solvent.

Examples of the alkali metal compound include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides, alkali metal alkoxides and the like. In particular, sodium carbonate and potassium carbonate are preferable.

Divalent phenol compounds include hydroquinone, catechol, resorcin, 4,4'-biphenol, bis (hydroxyphenyl) alkanes (eg 2,2-bis (hydroxyphenyl) propane, and 2,2-bis (hydroxyphenyl)). Methane), dihydroxydiphenylsulfones, dihydroxydiphenylethers, or at least one of the hydrogens on their benzene rings is a lower alkyl group such as a methyl group, an ethyl group, or a propyl group, or a lower alkoxy group such as a methoxy group or an ethoxy group. Examples include those that have been replaced. As the divalent phenol compound, two or more of the above compounds can be mixed and used.

The polyether sulfone may be a commercially available product. Examples of commercially available products include Sumika Excel 7600P, Sumika Excel 5900P (all manufactured by Sumitomo Chemical Co., Ltd.) and the like.

The logarithmic viscosity of the polyether sulfone is preferably 0.5 or more, more preferably 0.55 or more, from the viewpoint of satisfactorily forming macrovoids of the porous polyether sulfone film, and the production of the porous polyether sulfone film is produced. From the viewpoint of ease, it is preferably 1.0 or less, more preferably 0.9 or less, still more preferably 0.8 or less, and particularly preferably 0.75 or less.

Further, the PES porous membrane or the polyether sulfone as a raw material thereof has a glass transition temperature of 200 ° C. or higher or a clear glass transition temperature from the viewpoint of heat resistance and dimensional stability under high temperature. It is preferably not observed.

The method for producing the PES porous membrane that can be used in the present invention is not particularly limited, but for example,
A flow of a polyether sulfone solution containing 0.3% by mass to 60% by mass of a polyether sulfone having a logarithmic viscosity of 0.5 to 1.0 and 40% by mass to 99.7% by mass of an organic polar solvent in a film form. Then, a step of preparing a coagulating film having pores by immersing or contacting with a coagulating solvent containing a poor solvent or a non-solvent of polyether sulfone as an essential component, and a coagulating film having pores obtained in the above step. The heat treatment includes a step of coarsening the pores to obtain a PES porous film, and the heat treatment heats the coagulated film having the pores to the glass transition temperature of the polyether sulfone or higher, or 240 ° C. or higher. It may be manufactured by a method including raising the temperature.

The PES porous film that can be used in the present invention is preferably a PES porous film having a surface layer A, a surface layer B, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. And
The macrovoid layer includes a partition wall bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B and having an average pore diameter of 10 μm to 500 μm in the membrane plane direction. Have and
The partition wall of the macrovoid layer has a thickness of 0.1 μm to 50 μm, and has a thickness of 0.1 μm to 50 μm.
The surface layers A and B have a thickness of 0.1 μm to 50 μm, respectively, and have a thickness of 0.1 μm to 50 μm.
Of the surface layers A and B, one has a plurality of pores having an average pore diameter of more than 5 μm and 200 μm or less, and the other has a plurality of pores having an average pore diameter of 0.01 μm or more and less than 200 μm.
One of the surface layer A and the surface layer B has a surface aperture ratio of 15% or more, and the other surface layer has a surface aperture ratio of 10% or more.
The pores of the surface layer A and the surface layer B communicate with the macrovoid.
The PES porous membrane has a total film thickness of 5 μm to 500 μm and a porosity of 50% to 95%.
It is a PES porous membrane.

2. Cell culture method

One aspect of the present invention is
A method of culturing cells
(1) Applying cells to a polymer porous membrane and
(2) Culturing in the gas phase while applying the medium dropleted by the droplet medium supply means to the polymer porous membrane to which the cells are applied.
Including
Here, the polymer porous membrane has a three-layer structure polymer porous having a surface layer A and a surface layer B having a plurality of pores and a macrovoid layer sandwiched between the surface layer A and the surface layer B. In the quality film, the average pore diameter of the pores existing in the surface layer A is smaller than the average pore diameter of the pores existing in the surface layer B, and the macrovoid layer is bonded to the surface layers A and B. The present invention relates to a method in which the partition wall is provided, and the partition wall and a plurality of macrovoids surrounded by the surface layers A and B are provided, and the holes in the surface layers A and B communicate with the macrovoids. The method for culturing cells of the present invention is also hereinafter referred to as "method for culturing cells of the present invention".

Since the above-mentioned polymer porous membrane as a cell culture carrier used in the present invention has a slightly hydrophilic porous property, stable liquid retention is achieved in the polymer porous membrane, and a moist environment resistant to drying can be created. Be kept. Therefore, it is possible to achieve cell survival and proliferation even with an extremely small amount of medium as compared with the conventional culture method using a cell culture carrier. In addition, since culturing is possible even when part or all of the polymer porous membrane is exposed to air, the cells are efficiently supplied with oxygen by applying a droplet medium. A culture method capable of providing a culture method.

According to the present invention, the amount of the medium used is extremely small, and the polymer porous membrane as the culture carrier can be exposed to the gas phase, so that oxygen is sufficiently supplied to the cells by diffusion. Therefore, the present invention does not particularly require an oxygen supply device.

As used herein, the term "medium" refers to a cell culture medium for culturing cells, particularly animal cells. Medium is used as a synonym for cell culture medium. Therefore, the medium used in the present invention refers to a liquid medium. The type of medium can be a commonly used medium and is appropriately determined depending on the type of cells to be cultured.

As used herein, the term "droplet medium" refers to a mist-like or water-dropped medium, which is a medium that can be sprayed or sprayed onto the polymer porous membrane used in the present invention. The diameter of the droplet medium is not limited, but for example, a mist-like droplet medium that does not freely fall due to gravity and is small enough to float in the air may be used. The diameter of the mist-like droplet medium may be, for example, about 1 μm to 100 μm, or may be a smaller diameter. Further, the droplet medium may be, for example, a water droplet-like medium that freely falls due to gravity, and may be, for example, 100 μm or more. As a method of dropletizing the medium, a method of dropletizing by a known means may be used, and for example, a mist-like nozzle, a shower nozzle, or the like may be used to dropletize the medium. However, the dropletization method must be dropletized by a method that does not change the components of the medium. For example, the method of dropletization excludes the method of evaporation.

In an embodiment of the invention, the droplet medium is applied to a polymer porous membrane carrying cells. The dropletized medium passes through the gas phase before reaching the polymer porous membrane, and oxygen dissolves in the medium. As a result, the medium having a sufficient amount of oxygen is continuously supplied, and the cells can be cultured without causing ischemia. Further, since the polymer porous membrane is constantly exposed to the gas phase, the medium adhering to the polymer porous membrane can always take in oxygen, and culture capable of efficiently supplying oxygen becomes possible.

The polymer porous membrane used in the embodiments of the present invention is, for example, i) folded, ii) rolled into a roll, iii) sheets or small pieces connected by a thread-like structure, iv. ) It may be a polymer porous membrane tied in a rope shape and / or v) 2 or more are laminated. By using the polymer porous membranes of the shapes i) to v), many cells can be cultured in a certain space.

As the polymer porous membrane used in the embodiment of the present invention, a modularized polymer porous membrane (hereinafter, referred to as “modular polymer porous membrane”) may be used. As used herein, the term "modular polymer porous membrane" refers to a polymer porous membrane housed in a casing.

The casing provided by the modularized polymer porous membrane used in the embodiment of the present invention has two or more cell culture outlets, through which the culture medium flows in and out of the casing. The diameter of the cell culture outflow port of the casing is preferably larger than the diameter of the cells so that the cells can flow into the inside of the casing. Further, it is preferable that the diameter of the cell culture outflow port is smaller than the diameter at which the polymer porous membrane flows out from the cell culture outflow port. The diameter smaller than the diameter at which the polymer porous membrane flows out can be appropriately selected depending on the shape and size of the polymer porous membrane housed in the casing. For example, when the polymer porous membrane is in the shape of a string, it is not particularly limited as long as it is smaller than the width of the short side of the polymer porous membrane and has an appropriate diameter so that the polymer porous membrane does not flow out. The number of cell culture outlets is preferably provided as large as possible so that the cell culture can be easily supplied and / or discharged into and out of the casing. It is preferably 5 or more, preferably 10 or more, preferably 20 or more, preferably 50 or more, and preferably 100 or more. The cell culture outlet may have a mesh-like structure in part or all of the casing. Further, the casing itself may have a mesh shape. In the present invention, the mesh-shaped structure has, for example, a vertical, horizontal, and / or diagonal grid-like structure, and each opening forms a cell culture outflow port to the extent that a fluid can pass through. However, it is not limited to this.

The casing of the modularized polymer porous membrane used in the embodiments of the present invention includes, for example, polymers such as polyethylene, polypropylene, nylon, polyester, polystyrene, polycarbonate, polymethylmethacrylate and polyethylene terephthalate, and metals such as stainless steel and titanium. However, it is not limited to this.

The modularized polymer porous membrane used in the embodiments of the present invention
(I) Two or more independent polymer porous membranes are aggregated and
(Ii) The polymer porous membrane is folded and
(Iii) The polymer porous membrane is rolled up and / or
(Iv) The polymer porous membrane is tied in a rope shape,
It is housed in the casing and the modularized polymer porous membrane can be applied.

In the present specification, "two or more independent polymer porous membranes are aggregated and housed in a casing" means that two or more independent polymer porous membranes are enclosed in a casing. Refers to the state in which they are aggregated and housed inside. In the present invention, the two or more independent polymer porous membranes are fixed at least one place of the polymer porous membrane and at least one place in the casing by an arbitrary method, and the polymer porous membrane is fixed in the casing. It may be fixed in a state where it does not move. Further, the two or more independent polymer porous membranes may be small pieces. The shape of the small piece can be any shape such as a circle, an ellipse, a square, a triangle, a polygon, and a string.

In the present specification, the "folded polymer porous membrane" means that the "folded polymer porous membrane" is folded in the casing, and the inside of the casing is caused by the frictional force between each surface of the polymer porous membrane and / or the surface in the casing. It is a polymer porous membrane that has become immobile. As used herein, the term "folded" may mean that the polymer porous membrane has creases or is not creases.

As used herein, the term "rolled polymer porous membrane" means that the polymer porous membrane is rolled into a roll and rubs against each surface of the polymer porous membrane and / or the surface inside the casing. A polymer porous membrane that has become immobile in the casing due to force. Further, in the present invention, the polymer porous membrane woven in a rope shape means, for example, a plurality of strip-shaped polymer porous membranes woven into a rope shape by an arbitrary method, and the frictional force between the polymer porous membranes is used. A polymer porous membrane that does not move with each other. (I) A polymer porous membrane in which two or more independent polymer porous membranes are aggregated, (ii) a folded polymer porous membrane, (iii) a polymer porous membrane rolled into a roll, and (iv). ) A polymer porous membrane tied in a rope shape may be combined and housed in a casing.

3. 3. Cell culture device

One aspect of the present invention is
Polymer porous membrane and
The polymer porous membrane mounting portion on which the polymer porous membrane is mounted and the polymer porous membrane mounting portion
A housing in which the polymer porous membrane mounting portion is housed, and
A droplet medium supply unit arranged inside the housing and
A medium supply line communicating with the droplet medium supply unit and
A medium storage unit that communicates with the medium supply line and
A pump provided in a part of the medium supply line and
With
Here, the polymer porous membrane has a three-layer structure polymer porous having a surface layer A and a surface layer B having a plurality of pores and a macrovoid layer sandwiched between the surface layer A and the surface layer B. In the quality film, the average pore diameter of the pores existing in the surface layer A is smaller than the average pore diameter of the pores existing in the surface layer B, and the macrovoid layer is bonded to the surface layers A and B. It has a partition wall, the partition wall, and a plurality of macrovoids surrounded by the surface layers A and B.
Here, the present invention relates to a cell culture apparatus in which the polymer porous membrane mounting portion is provided with a plurality of slit-shaped or mesh-shaped medium outlets.

Since the polymer porous membrane used in the present invention has a slightly hydrophilic porous property, stable liquid retention is achieved in the polymer porous membrane, and a wet environment resistant to drying is maintained. Therefore, it is possible to achieve cell survival and proliferation even with an extremely small amount of medium as compared with a culture device using a conventional cell culture carrier. In addition, since culturing is possible even when part or all of the polymer porous membrane is exposed to air, the cells are efficiently supplied with oxygen by applying a droplet medium. A culture medium capable of providing a culture medium. The cell culture device will also be referred to hereinafter as "the cell culture device of the present invention". Hereinafter, embodiments of the cell culture apparatus of the present invention will be described with reference to the drawings.

FIG. 1 is a diagram showing a configuration example of the cell culture apparatus of the present invention. A polymer porous membrane is placed on the polymer porous membrane mounting portion. The polymer porous membrane applied to the polymer porous membrane mounting portion is, for example, i) folded, ii) rolled into a roll, iii) sheets or small pieces connected by a thread-like structure. , Iv) It may be a polymer porous membrane tied in a rope shape and / or v) 2 or more are laminated. By using the polymer porous membranes of the shapes i) to v), many cells can be cultured in a certain space. The polymer porous membrane mounting portion includes a plurality of slit-shaped or mesh-shaped medium outlets. By providing the medium discharge port, the medium supplied from the droplet medium supply unit can be discharged to the lower part. The shape, size, and number of the medium discharge ports may be appropriately selected so that the polymer porous membrane to be placed does not fall.

As the polymer porous membrane used in the embodiment of the present invention, a modularized polymer porous membrane may be used as described above.

In the present embodiment, the cell culture apparatus includes a housing for accommodating the polymer porous membrane mounting portion, and inside the housing, the medium is dropletized and supplied to the polymer porous membrane. It is provided with a droplet medium supply unit. For the droplet medium supply unit, for example, a known mist-like nozzle or shower nozzle can be adopted. By providing the housing, the droplet medium supplied from the droplet medium supply unit fills the inside of the housing and is efficiently supplied to the polymer porous membrane. The shape and size of the housing may be appropriately selected according to the amount of cells to be cultured, the amount of the polymer porous membrane to be applied, and the like.

A medium supply line communicates with the droplet medium supply unit, and a medium storage unit communicates with the medium supply line. In addition, a pump for sending the medium to the droplet medium supply unit is provided in a part of the medium supply line. The medium storage unit stores the medium released from the droplet medium supply unit. As shown in the figure, the medium storage portion is arranged directly under the polymer porous membrane mounting portion, and has a configuration in which the medium that has fallen after being applied to the polymer porous membrane is stored, and the medium is a cell culture. It may be configured to circulate in the device. Further, the culture medium storage portion may be provided separately and independently regardless of the position of the polymer porous membrane mounting portion as shown in the figure. In this case, it is necessary to separately provide a container or the like for storing the medium that has fallen after being applied to the polymer porous membrane immediately below the polymer porous membrane mounting portion. The speed of the medium pumped to the droplet medium supply unit can be appropriately selected.

FIG. 2 is a diagram showing another configuration example of the cell culture apparatus of the present invention. As shown in the figure, the polymer porous membrane mounting portion may be provided in two or more stages. This makes it possible to increase the number of cultured cells per unit volume. Further, one or more, preferably two or more, may be arranged on the upper part, the lower part and / or the side part of the polymer porous membrane mounting part. As a result, the medium is efficiently supplied to the polymer porous membrane, and a large amount of cell culture becomes possible. The arrangement of the droplet medium supply unit can be appropriately optimized depending on the size of the cell culture device, the arrangement of the polymer porous placement unit, and the like.

4. Cell culture method using a cell culture device

<Step of applying cells to polymer porous membrane>
The specific steps of application of the cells used in the present invention to the polymer porous membrane are not particularly limited. It is possible to employ the steps described herein, or any method suitable for applying the cells to a membranous carrier. In the method of the invention, but not limited to, application of cells to a polymeric porous membrane includes, for example, aspects such as:

(A) A mode comprising seeding cells on the surface of the polymer porous membrane;
(B) A cell suspension is placed on the dry surface of the polymer porous membrane.
The cell turbidity is sucked into the membrane by leaving it to stand, or moving the polymer porous membrane to promote the outflow of the liquid, or stimulating a part of the surface, and then
The cells in the cell turbid fluid are retained in the membrane and water is drained.
Aspects comprising steps;
(C) One side or both sides of the polymer porous membrane is moistened with a cell culture solution or a sterilized liquid, and then wetted.
The moist polymer porous membrane was loaded with a cell suspension and then
The cells in the cell turbid fluid are retained in the membrane and water is drained.
Aspects comprising steps.

The aspect (A) includes directly seeding cells and cell clusters on the surface of the polymer porous membrane. Alternatively, it also includes an embodiment in which a polymer porous membrane is placed in a cell suspension and the cell culture solution is infiltrated from the surface of the membrane.

The cells seeded on the surface of the polymer porous membrane adhere to the polymer porous membrane and enter the inside of the porous membrane. Preferably, the cells adhere to the polymeric porous membrane without the need for any external physical or chemical force. The cells seeded on the surface of the polymer porous membrane can grow and proliferate stably on the surface and / or inside of the membrane. Cells can take a variety of different morphologies, depending on the location of the growing and proliferating membrane.

In the aspect (B), the cell suspension is placed on the dry surface of the polymer porous membrane. By leaving the polymer porous membrane unattended, moving the polymer porous membrane to promote the outflow of the liquid, or stimulating a part of the surface to suck the cell turbid liquid into the membrane. The cell turbid solution penetrates into the membrane. Although not bound by theory, it is considered that this is due to the property derived from each surface shape of the polymer porous membrane. According to this aspect, the cells are sucked and seeded at the place where the cell turbid solution of the membrane is loaded.

Alternatively, as in the aspect of (C), one or both sides of the polymer porous membrane is moistened with a cell culture solution or a sterilized liquid, and then cell turbidity is formed on the wet polymer porous membrane. The liquid may be loaded. In this case, the passage rate of the cell suspension is greatly improved.

For example, a method of wetting a very small part of the film (hereinafter, this will be referred to as a "one-point wet method") can be used for the main purpose of preventing the film from scattering. The one-point wet method is substantially similar to the dry method (aspect of (B)) in which the film is not wetted. However, it is considered that the membrane permeation of the cell fluid is rapid for the moistened small part. Alternatively, a method of loading the cell suspension into a polymer porous membrane in which one or both sides are sufficiently moistened (hereinafter referred to as "wet membrane") can also be used (hereinafter referred to as "wet membrane"). Is described as "wet film method"). In this case, the passage rate of the cell suspension is greatly improved in the entire polymer porous membrane.

In the embodiments (B) and (C), the cells in the cell suspension are retained in the membrane and water is allowed to flow out. This enables treatments such as concentrating the concentration of cells in the cell suspension and causing unnecessary components other than cells to flow out together with water.

The aspect of (A) may be referred to as "spontaneous sowing" and the aspects of (B) and (C) may be referred to as "suction sowing".

Preferably, but not limited to, living cells selectively remain in the polymer porous membrane. Therefore, in a preferred embodiment of the method of the present invention, live cells remain in the polymer porous membrane and dead cells preferentially flow out with water.

The sterilized liquid used in the aspect (C) is not particularly limited, but is a sterilized buffer solution or sterilized water. The buffers are, for example, (+) and (−) Dulbecco's PBS, (+) and (−) Hank's Balanced Salt Solution, and the like. Examples of buffer solutions are shown in Table 1 below.

Further, in the method of the present invention, the application of the cells to the polymer porous membrane is an embodiment in which the floating adhesive cells coexist with the polymer porous membrane in a turbid manner to attach the cells to the membrane (entanglement). ) Is also included. For example, in the method of the present invention, a cell culture medium, cells and one or more of the polymer porous membranes may be placed in a cell culture vessel in order to apply the cells to the polymer porous membrane. When the cell culture medium is liquid, the polymer porous membrane is in a state of being suspended in the cell culture medium. Due to the nature of the polymer porous membrane, cells can adhere to the polymer porous membrane. Therefore, even if the cells are not suitable for suspension culture by nature, the polymer porous membrane can be cultured in a state of being suspended in the cell culture medium. Preferably, the cells adhere to the polymeric porous membrane. By "spontaneously adhering" is meant that the cells remain on the surface or inside of the polymeric porous membrane without the need for any external physical or chemical force.

The application of the above-mentioned cells to the polymer porous membrane may be used in combination of two or more methods. For example, the cells may be applied to the polymer porous membrane by combining two or more methods of the aspects (A) to (C). It is possible to apply the polymer porous membrane carrying cells to the cell culture section 2 in the above-mentioned cell culture device 1 for culturing.

In addition, a medium containing suspended cells may be dropped from the cell supply means 3 and seeded in advance in the cell culture unit 2 in which the polymer porous membrane is housed.

As used herein, the term "suspended cell" refers to a cell obtained by forcibly suspending adherent cells by a proteolytic enzyme such as trypsin and suspending them in a medium, or a known acclimatization step. The medium contains adherent cells that can be suspended and cultured.

The type of cell that can be used in the present invention is selected from, for example, the group consisting of animal cells, insect cells, plant cells, yeasts and bacteria. Animal cells are roughly classified into cells derived from animals belonging to the phylum Vertebrate and cells derived from invertebrates (animals other than animals belonging to the phylum Vertebrate). The origin of animal cells in the present specification is not particularly limited. Preferably, it means a cell derived from an animal belonging to the phylum Vertebrate. The vertebrate phylum includes the jawless and gnathostomata, and the gnathostomata includes mammals, birds, amphibians, reptiles and the like. Preferably, it is a cell derived from an animal belonging to the class Mammalia, generally referred to as a mammal. Mammals are not particularly limited, but preferably include mice, rats, humans, monkeys, pigs, dogs, sheep, goats and the like.

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

As used herein, the term "pluripotent stem cell" is intended to be a general term for stem cells having the ability to differentiate into cells of any tissue (pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic reproductive stem cells (EG cells), reproductive stem cells (GS cells), and the like. .. Preferably, it is an ES cell or an iPS cell. iPS cells are particularly preferable because they have no ethical problems. Any known pluripotent stem cell can be used, and for example, the pluripotent stem cell described in International Publication No. 2009/1233349 (PCT / JP2009 / 057441) can be used.

"Tissue stem cell" means a stem cell having the ability to differentiate into various cell types (pluripotency), although the cell lineage capable of differentiating is limited to a specific tissue. For example, hematopoietic stem cells in bone marrow are the source of blood cells, and neural stem cells differentiate into nerve cells. In addition, there are various types such as hepatic stem cells that make the liver and skin stem cells that become the skin tissue. Preferably, the tissue stem cells are selected from mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, skin stem cells, or hematopoietic stem cells.

"Somatic cell" refers to a cell other than a germ cell among the cells constituting a multicellular organism. In sexual reproduction, it is not passed on to the next generation. Preferably, the somatic cells are hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, cartilage cells, fat cells, skin cells, fibroblasts, pancreatic cells, renal cells, lung cells, or , Lymphocytes, erythrocytes, leukocytes, monospheres, macrophages or macronuclear cells.

"Germ cell" means a cell that plays a role in transmitting genetic information to the next generation in reproduction. For example, it includes gametes for sexual reproduction, namely eggs, egg cells, sperms, sperms, spores for asexual reproduction, and the like.

The cells may be selected from the group consisting of sarcoma cells, cell lines and transformed cells. "Sarcoma" is a cancer that develops in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, and includes soft sarcoma, malignant bone tumor, and the like. Sarcoma cells are cells derived from sarcoma. The “cell line” means a cultured cell that has been maintained in vitro for a long period of time, has a certain stable property, and is capable of semi-permanent subculture. PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human fetal kidney), HL-60 cells (derived from human leukocyte cells), HeLa cells (derived from human cervical cancer), Vero cells (Derived from African green monkey kidney epithelial cells), MDCK cells (derived from canine renal tubule epithelial cells), HepG2 cells (cell line derived from human hepatoma), BHK cells (neonatal hamster kidney cells), NIH3T3 cells (derived from mouse fetal fibroblasts) There are cell lines derived from various tissues of various biological species including humans. "Transformed cell" means a cell in which nucleic acid (DNA or the like) is introduced from the outside of the cell to change its genetic properties.

As used herein, the term "adherent cell" is generally a cell that needs to adhere itself to an appropriate surface for proliferation, and is also referred to as an adherent cell or a scaffold-dependent cell. In some embodiments of the invention, the cells used are adherent cells. The cells used in the present invention are adherent cells, more preferably cells that can be cultured even when suspended in a medium. Suspension-culturable adherent cells can be obtained by acclimatizing the adherent cells to a state suitable for suspension culture by a known method, for example, CHO cells, HEK293 cells, Vero cells, NIH3T3 cells. And so on, cell lines derived from these cells can be mentioned.

A model diagram of cell culture using a polymer porous membrane is shown in FIG. FIG. 1 is a diagram for facilitating understanding, and each element is not the actual size. In the cell culturing method of the present invention, by applying cells to the polymer porous membrane and culturing the cells, a large amount of cells grow on the multifaceted connected porous portion and the surface of the polymer porous membrane. A large amount of cells can be easily cultured. In addition, the cell culturing method of the present invention makes it possible to cultivate a large amount of cells while significantly reducing the amount of medium used for cell culturing as compared with the conventional method. For example, a large amount of cells can be cultured for a long period of time even when a part or the whole of the polymer porous membrane is not in contact with the liquid phase of the cell culture medium. It is also possible to significantly reduce the total volume of the cell culture medium contained in the cell culture vessel with respect to the total volume of the polymer porous membrane including the cell survival area.

In the present specification, the volume occupied by the polymer porous membrane containing no cells in the space including the volume of the internal gap thereof is referred to as "apparently polymer porous membrane volume" (see FIG. 3). Then, the cells are applied to the polymer porous membrane, and in a state where the cells are supported on the surface and inside of the polymer porous membrane, the polymer porous membrane, the cells, and the medium infiltrated inside the polymer porous membrane are as a whole space. The volume occupying the inside is referred to as "the volume of the polymer porous membrane including the cell viable area" (see FIG. 3). In the case of a polymer porous membrane having a thickness of 25 μm, the volume of the polymer porous membrane including the cell survival area is apparently larger than the volume of the polymer porous membrane by about 50% at the maximum. In the method of the present invention, a plurality of polymer porous membranes can be housed and cultured in one cell culture vessel, but in that case, the cell survival area for each of the plurality of polymer porous membranes carrying cells. The total volume of the polymer porous membrane including the cell may be simply referred to as "the sum of the volume of the polymer porous membrane including the cell culture area".

By using the method of the present invention, even if the total volume of the cell culture medium contained in the cell culture vessel is 10,000 times or less than the total volume of the polymer porous membrane including the cell survival area, the cells can be subjected to a long period of time. It becomes possible to culture well. Further, even if the total volume of the cell culture medium contained in the cell culture vessel is 1000 times or less than the total volume of the polymer porous membrane including the cell survival area, the cells can be cultivated well for a long period of time. .. Further, even if the total volume of the cell culture medium contained in the cell culture vessel is 100 times or less than the total volume of the polymer porous membrane including the cell survival area, the cells can be cultivated well for a long period of time. .. Then, even if the total volume of the cell culture medium contained in the cell culture vessel is 10 times or less than the total volume of the polymer porous membrane including the cell survival area, the cells can be cultivated well for a long period of time. ..

That is, according to the present invention, the space (container) for culturing cells can be made as small as possible as compared with the conventional cell culturing apparatus for two-dimensional culturing. Further, when it is desired to increase the number of cells to be cultured, it is possible to flexibly increase the volume of cell culture by a simple operation such as increasing the number of polymer porous membranes to be laminated. With the cell culture device provided with the polymer porous membrane used in the present invention, it is possible to separate the space (container) for culturing cells from the space (container) for storing cell culture medium, and the cells to be cultured can be separated. Depending on the number, it is possible to prepare the required amount of cell culture medium. The space (container) for storing the cell culture medium may be enlarged or miniaturized according to the purpose, or may be a replaceable container, and is not particularly limited.

In the cell culture method of the present invention, for example, the number of cells contained in the cell culture vessel after culturing using the polymer porous membrane is uniformly dispersed in the cell culture medium contained in the cell culture vessel. ^{1.0 × 10 5} or more, 1.0 × 10 ⁶ or more, 2.0 × 10 ⁶ or more, 5.0 × 10 ⁶ or more, 1.0 × 10 per 1 ml of medium ⁷ or more, 2.0 x 10 ⁷ or more, 5.0 x 10 ⁷ or more, 1.0 x 10 ⁸ or more, 2.0 x 10 ⁸ or more, 5.0 x 10 ⁸ or more, 1 It means culturing until the number is 0.0 × 10 ⁹ or more, 2.0 × 10 ⁹ or more, or 5.0 × 10 ^{9 or more.}

As a method for measuring the number of cells during or after culturing, various known methods can be used. For example, as a method of measuring the number of cells contained in a cell culture vessel after culturing using a polymer porous membrane, assuming that all the cells are uniformly dispersed in the cell culture medium contained in the cell culture vessel. , A known method can be appropriately used. For example, a cell numbering method using CCK8 can be preferably used. Specifically, using Cell Counting Kit8; a solution reagent manufactured by Dojin Chemical Laboratory (hereinafter referred to as "CCK8"), the number of cells in a normal culture without a polymer porous membrane was measured, and the absorbance was measured. Obtain the correlation coefficient with the actual number of cells. After that, the cells were applied, the cultured polymer porous membrane was transferred to a medium containing CCK8, stored in an incubator for 1 to 3 hours, the supernatant was extracted, and the absorbance was measured at a wavelength of 480 nm. Calculate the number of cells from the obtained correlation coefficient.

From another point of view, mass culture of cells means, for example, that the number of cells contained per square centimeter of the polymer porous membrane after culturing using the polymer porous membrane is 1.0 × 105 or more, 2.0. × 10 ⁵ or more, 1.0 × 10 ⁶ or more, 2.0 × 10 ⁶ or more, 5.0 × 10 ⁶ or more, 1.0 × 10 ⁷ or more, 2.0 × 10 ⁷ or more , 5.0 × 10 ⁷ or more, 1.0 × 10 ⁸ or more, 2.0 × 10 ⁸ or more, or 5.0 × 10 ⁸ or more. The number of cells contained in one square centimeter of the polymer porous membrane can be appropriately measured by using a known method such as a cell counter.

Hereinafter, the present invention will be described in more detail based on Examples. The present invention is not limited to these examples. Those skilled in the art can easily modify or modify the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

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

(Example 1)
Cell culture method using polyimide porous membrane with mist and shower type reactor

Cells conditioned and suspended from anti-human IL-8 antibody-producing CHO-DP12 cells (ATCC CRL-12445) were suspended and cultured in a medium (BalanCD ™ CHO GROWTH A), and the number of viable cells per ml was increased. , and the culture was continued until 3.9 × 10 ^6. After pouring 12 ml of each of the above suspension culture solutions into one 10 cm diameter petri dish, a mantle was formed with a nylon mesh (30 #, opening 547 μm), and a fixed amount of polyimide porous membrane was sterilized (20 cm per module ^2). ) In addition, 12 sealed modules were added to the petri dish. After wetting the module with a cell suspension, it was left overnight in a CO2 incubator.

The next day, the modules were taken out, 12 modules were installed inside the mist and shower type reactor shown in FIG. 4, and 200 ml of medium (IMDM containing 2% FBS) was stored in the medium storage section, and the medium was stored via a tube pump. It was circulated at a rate of 60 ml / min. When the culture was completed 2 days later, a total number of 8.2 × 10 ⁶ cells was observed at ^{a cell density of 3.4 × 10 4} Cells / cm ^2.

(Example 2)
Cell culture method using polyimide porous membrane with mist and shower type reactor

Cells conditioned and suspended from anti-human IL-8 antibody-producing CHO-DP12 cells (ATCC CRL-12445) were suspended and cultured in a medium (BalanCD ™ CHO GROWTH A), and the number of viable cells per ml was increased. , and the culture was continued until 9.9 × 10 ^6. Ten modules were placed in an oxygen permeation bag for shaking culture, and shaking culture was performed overnight in a CO2 incubator.

The next day, the module was taken out from the shaking bag, and cell culture was performed in a mist and shower type reactor under the same conditions as in Example 1. In the liquid reservoir, 200 ml of medium (KBM270 manufactured by Kojin Bio) was stored, and the medium was circulated at a rate of 60 ml per minute via a tube pump. When the culture was completed 4 days later, a total number of 1.8 × 10 ⁷ cells was observed at ^{a cell density of 8.8 × 10 4} Cells / cm ^2.

Claims (13)

Polymer porous membrane and
The polymer porous membrane mounting portion on which the polymer porous membrane is mounted and the polymer porous membrane mounting portion
A housing in which the polymer porous membrane mounting portion is housed, and
A droplet medium supply unit arranged inside the housing and
A medium supply line communicating with the droplet medium supply unit and
A medium storage unit that communicates with the medium supply line and
A pump provided in a part of the medium supply line and
With
Here, the polymer porous membrane has a three-layer structure polymer porous having a surface layer A and a surface layer B having a plurality of pores and a macrovoid layer sandwiched between the surface layer A and the surface layer B. In the quality film, the average pore diameter of the pores existing in the surface layer A is smaller than the average pore diameter of the pores existing in the surface layer B, and the macrovoid layer is bonded to the surface layers A and B. The partition wall is formed, and the partition wall and a plurality of macrovoids surrounded by the surface layers A and B are provided, and the holes in the surface layers A and B communicate with the macrovoids.
Here, the polymer porous membrane is a polyimide porous membrane or a polyether sulfone (PES) porous membrane.
Here, the polymer porous membrane mounting portion is provided with a plurality of slit-shaped or mesh-shaped medium outlets.
Cell culture device. The liquid droplets medium supply section, the upper portion of the polymer porous Makuno portion, disposed one or the bottom and / or sides, cell culture device according to claim 1. The cell culture apparatus according to claim 1 or 2 , wherein the polymer porous membrane mounting portion has two or more stages. The present invention according to any one of claims 1 to 3 , wherein the culture medium storage portion is arranged inside the housing and below the polymer porous membrane mounting portion, and the culture medium circulates. The cell culture apparatus according to the above. The polymer porous membrane
i) Folded,
ii) Rolled up,
iii) Sheets or small pieces connected by a thread-like structure,
iv) The cell culture apparatus according to any one of claims 1 to 4 , wherein the cell culture apparatus is tied in a rope shape and / or v) is a polymer porous membrane in which two or more are laminated. The polymer porous membrane is a modularized polymer porous membrane provided with a casing.
Here, the casing has two or more cell culture outlets.
Here, the modularized polymer porous membrane is
(I) Two or more independent polymer porous membranes are aggregated and
(Ii) The polymer porous membrane is folded and
(Iii) The polymer porous membrane is rolled up and / or
(Iv) The polymer porous membrane is tied in a rope shape,
The cell culture apparatus according to any one of claims 1 to 4 , which is housed in the casing. The cell culture apparatus according to any one of claims 1 to 6 , wherein the polymer porous membrane has a plurality of pores having an average pore diameter of 0.01 to 100 μm. The cell culture apparatus according to any one of claims 1 to 7 , wherein the surface layer A has an average pore size of 0.01 to 50 μm. The cell culture apparatus according to any one of claims 1 to 8 , wherein the surface layer B has an average pore size of 20 to 100 μm. The cell culture apparatus according to any one of claims 1 to 9 , wherein the total film thickness of the polymer porous membrane is 5 to 500 μm. The cell culture apparatus according to any one of claims 1 to 10 , wherein the polyimide porous membrane is a polyimide porous membrane containing a polyimide obtained from tetracarboxylic dianhydride and diamine. Coloring obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a coloring precursor, and then heat-treating the polyimide porous film at 250 ° C. or higher. The cell culture apparatus according to any one of claims 1 to 11 , which is a polyimide porous membrane obtained from the above. A method for culturing cells using the cell culture apparatus according to any one of claims 1 to 12.

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