JP2018014898A - Cell culture method and cell culture apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture method using a polymer porous membrane and a cell culture apparatus using the same.SOLUTION: According to the present invention, there is provided a cell culture method. The method comprises: (1) applying cells to a polymer porous membrane; and (2) culturing in a gas phase while applying a medium formed into droplets by droplet medium supply means to the polymer porous membrane to which the cells are applied. According to the present invention, there is provided a cell culture apparatus comprising a polymer porous membrane, a polymer porous membrane mount portion on which the polymer porous membrane is placed; a casing accommodating the polymer porous membrane mount portion; a droplet medium supply unit disposed inside the casing; a medium supply line communicating with the droplet medium supply unit; a medium reservoir communicating with the medium supply line; and a pump provided in a part of the medium supply line. Here, the polymer porous membrane mount portion comprises a plurality of slit-shaped or mesh-shaped medium outlets.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture method using a polymer porous membrane. The present invention also relates to a cell culture apparatus provided with a polymer porous membrane.

  In recent years, proteins such as enzymes, hormones, antibodies, cytokines, viruses (virus proteins) used for treatment and vaccines are industrially produced using cultured cells. However, these protein production techniques are costly, which raises medical costs. Therefore, with the aim of drastically reducing costs, a technology for culturing cells at high density and an innovative technology for increasing the amount of protein production have been demanded.

  As cells that produce proteins, anchorage-dependent adherent cells that adhere to a culture substrate may be used. Since such cells proliferate in a scaffold-dependent manner, it is necessary to culture them while adhering to the surface of a petri dish, plate or chamber. Conventionally, in order to culture such adherent cells in large quantities, 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, which is a factor that increases the cost.

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

<Polyimide porous membrane>
The polyimide porous membrane has been used for applications such as filters, low dielectric constant films, fuel cell electrolyte membranes, etc., particularly for battery-related applications. Patent Documents 2 to 4 are particularly excellent in material permeability such as gas, high porosity, excellent smoothness of both surfaces, relatively high in strength, and in the direction of film thickness despite high porosity. A polyimide porous membrane having a large number of macrovoids having excellent proof stress against compressive stress is described. These are all polyimide porous membranes prepared via an amic acid.

  A method for culturing cells has been reported, which includes culturing cells by applying them to a polyimide porous membrane (Patent Document 5).

International Publication No. 2003/054174 International Publication No. 2010/038873 JP 2011-219585 A JP 2011-219586 A International Publication No. 2015/012415

  An object of the present invention is to provide a cell culture method using a polymer porous membrane. Moreover, an object of this invention is to provide the cell culture apparatus provided with the polymer porous membrane.

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

[1] A method for culturing cells,
(1) applying cells to the polymer porous membrane;
(2) culturing in a gas phase while applying the medium formed into droplets by the droplet formation medium supply means to the polymer porous membrane to which the cells are applied;
Including
Here, the polymer porous membrane has a three-layered polymer porous 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 Here, 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. And a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and 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 pieces are connected by a thread-like structure,
iv) The method according to [1], wherein the polymer porous membrane is tied in a rope shape and / or v) two or more are laminated.
[3] The polymer porous membrane is a modular polymer porous membrane having a casing,
Wherein the casing has two or more cell culture medium outlets;
Here, the modular polymer porous membrane is
(I) Two or more independent polymer porous membranes are aggregated,
(Ii) the polymer porous membrane is folded;
(Iii) The polymer porous membrane is wound into a roll 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 device according to any one of [1] to [4], wherein the average pore diameter of the surface layer A is 0.01 to 50 μm.
[6] The method according to any one of [1] to [5], wherein the average pore diameter of the surface layer B is 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 film is a polyimide porous film containing polyimide obtained from tetracarboxylic dianhydride and diamine.
[10] By forming a polyamic acid solution composition containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a colored precursor, and then heat-treating at 250 ° C. or higher. The method according to [8] or [9], which is a colored polyimide porous membrane to be obtained.
[11] The method according to any one of [1] to [7], wherein the polymer porous membrane is a polyethersulfone porous membrane.

[12] a polymer porous membrane;
A polymer porous membrane placement section on which the polymer porous membrane is placed;
A housing in which the polymer porous membrane mounting portion is accommodated;
A droplet culture medium supply unit disposed inside the housing;
A medium supply line in communication with the dropletized medium supply unit;
A medium storage section communicating with the medium supply line;
A pump provided in a part of the medium supply line;
With
Here, the polymer porous membrane has a three-layered polymer porous 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 Here, 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. And a plurality of macro voids surrounded by the partition wall and the surface layers A and B,
Here, the polymer porous membrane placement part is a cell culture device provided with a plurality of slit-like or mesh-like medium outlets.
[13] The cell culture device according to [12], wherein at least one droplet-forming medium supply unit is disposed on an upper part, a lower part, and / or a side part of the polymer porous membrane placement unit.
[14] The cell culture device according to [12] or [13], wherein the polymer porous membrane placement section has two or more stages.
[15] In the above [12] to [14], the culture medium storage section is disposed inside the casing and is disposed below the polymer porous membrane placement section, and the culture medium circulates. The cell culture device according to any one of the above.
[16] The porous polymer membrane is
i) folded,
ii) rolled up,
iii) Sheets or pieces are connected by a thread-like structure,
iv) The cell culture device according to any one of [12] to [15], which is a polymer porous membrane tied in a rope shape and / or v) two or more laminated.
[17] The polymer porous membrane is a modular polymer porous membrane having a casing,
Wherein the casing has two or more cell culture medium outlets;
Here, the modular polymer porous membrane is
(I) Two or more independent polymer porous membranes are aggregated,
(Ii) the polymer porous membrane is folded;
(Iii) The polymer porous membrane is wound into a roll and / or
(Iv) The polymer porous membrane is tied in a rope shape,
The cell culture device according to any one of [12] to [15], which is housed in the casing.
[18] The cell culture device 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 device according to any one of [12] to [18], wherein the average pore diameter of the surface layer A is 0.01 to 50 μm.
[20] The cell culture device according to any one of [12] to [19], wherein the average pore diameter of the surface layer B is 20 to 100 μm.
[21] The cell culture device according to any one of [12] to [20], wherein the total thickness of the polymer porous membrane is 5 to 500 μm.
[22] The cell culture device according to any one of [12] to [21], wherein the polymer porous membrane is a polyimide porous membrane.
[23] The cell culture device according to [22], wherein the polyimide porous membrane is a polyimide porous membrane containing polyimide obtained from tetracarboxylic dianhydride and diamine.
[24] By forming a polyamic acid solution composition containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a colored precursor, and then heat-treating the polyimide porous membrane at 250 ° C. or higher. The cell culture device according to [22] or [23], which is a colored polyimide porous membrane to be obtained.
[25] The cell culture device according to any one of [12] to [21], wherein the polymer porous membrane is a polyethersulfone porous membrane.

[26] A cell culture method using the cell culture device according to any one of [12] to [24].

  Since the porous polymer membrane used in the present invention has a slightly hydrophilic porous property, it stably retains the liquid in the porous polymer membrane and provides a moist environment resistant to drying. 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-formed medium to the polymer porous membrane, the amount of the medium to be used is extremely reduced. Furthermore, by culturing while applying the droplet-formed medium, it becomes possible to culture 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 culture media from many directions to any direction, so it is homogeneous for polymer porous membranes and modular polymer porous membranes that contain cells in each location. It will be possible to supply the medium. (Figures 1 and 2)

Drawing 1 is a figure showing the example of composition of the cell culture device in an embodiment. FIG. 2 is a diagram illustrating 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 the cell culture apparatus used in the examples. (A) Overall view, (B) Enlarged view of the housing part of (A)

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

1. Polymer porous membrane The average pore diameter of the pores present in the surface layer A (hereinafter also referred to as "A surface" or "mesh surface") in the polymer porous membrane used in the present invention is not particularly limited, 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 Preferably, they are 0.01 micrometer-15 micrometers.

  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, but it 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, and preferably 20 μm to 100 μm.

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

  Although the thickness of the surface layers A and B is not specifically limited, For example, it is 0.01-50 micrometers, Preferably it is 0.01-20 micrometers.

  The average pore diameter in the film plane direction of the macrovoids in the macrovoid layer in the polymer porous membrane is not particularly limited, but is, for example, 10 to 500 μm, preferably 10 to 100 μm, and more preferably 10 to 80 μm. Moreover, the thickness of the partition in the said macrovoid layer is although it does not specifically limit, For example, it is 0.01-50 micrometers, Preferably, it is 0.01-20 micrometers. In one embodiment, the at least one partition wall in the macrovoid layer has one or more average pore diameters of 0.01 to 100 μm, preferably 0.01 to 50 μm, communicating adjacent macrovoids. Has holes. In another embodiment, the partition in the macrovoid layer has no pores.

  The total film thickness on 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, 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less. Or 50 micrometers or less may be sufficient. Preferably, it is 5-500 micrometers, More preferably, it is 25-75 micrometers.

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

  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%.

The porosity of the polymer porous membrane used in the present invention can be determined according to the following formula (2) from the basis weight by measuring the thickness and mass of the porous film cut to a predetermined size.
(In the formula, S represents the area of the porous film, d represents the total film thickness, w represents the measured mass, and D represents the density of the polymer. When the polymer is polyimide, the density is 1.34 g / cm ^3. And)

  The polymer porous membrane used in the present invention is preferably 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. A polymer porous membrane having a structure, wherein the average pore diameter of the pores present in the surface layer A is 0.01 μm to 15 μm, and the average pore diameter of the pores present in the surface layer B is 20 μm to 100 μm; The macrovoid layer includes a partition bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition and the surface layers A and B. The partition of the macrovoid layer and the surface The thicknesses of the layers A and B are 0.01 to 20 μm, the holes in the surface layers A and B communicate with the macrovoids, the total film thickness is 5 to 500 μm, and the porosity is 40% or more. Less than 95% poly Over a porous membrane. In one embodiment, at least one partition in the macrovoid layer has one or more pores having an average pore size of 0.01-100 μm, preferably 0.01-50 μm, communicating between adjacent macrovoids. Have In another embodiment, the septum does not have such holes.

  The polymer porous membrane used in the present invention is preferably sterilized. The sterilization treatment is not particularly limited, and includes any sterilization treatment such as dry heat sterilization, steam sterilization, sterilization with a disinfectant such as ethanol, and 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 structural features described above, but is preferably a polyimide porous membrane or a polyethersulfone (PES) porous membrane.

1-1. Polyimide Porous Membrane Polyimide is a general term for polymers containing imide bonds in repeating units, and usually means an aromatic polyimide in which aromatic compounds are directly linked by imide bonds. Aromatic polyimide has a conjugated structure through the imide bond between aromatic and aromatic, so it has a rigid and strong molecular structure, and the imide bond has a strong intermolecular force, so it has a very high level of heat. Has mechanical, mechanical and chemical properties.

  The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane containing (obtained as a main component) a polyimide obtained from tetracarboxylic dianhydride and diamine, more preferably tetracarboxylic dianhydride. It is a polyimide porous membrane which consists of a polyimide obtained from a thing and diamine. “Containing as a main component” means that a component other than polyimide obtained from tetracarboxylic dianhydride and diamine may be essentially not included or included as a component of the polyimide porous membrane. It 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 prepared by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component and a colored precursor, and then at 250 ° C. The colored polyimide porous membrane obtained by heat treatment as described above is also included.

  A polyamic acid is obtained by polymerizing a tetracarboxylic acid component and a diamine component. Polyamic acid is a polyimide precursor that can be ring-closed to form 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, if necessary, fine particles such as an imidization catalyst, an organic phosphorus-containing compound, inorganic fine particles, and organic fine particles can be added to the polyamic acid solution. Moreover, when chemically imidizing a polyamic acid, fine particles, such as a chemical imidating agent, a dehydrating agent, inorganic fine particles, and organic fine particles, etc. can be added to a polyamic acid solution as needed. Even when the above components are added to the polyamic acid solution, it is preferable that the coloring precursor is not precipitated.

  In the present specification, the “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 colored precursor that can be used in the production of the polyimide porous membrane is uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution, 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 preferred, those that produce black colored products are more preferred, and carbon-based colored precursors are more preferred.

  When colored precursors are heated, they appear to be carbonized at first glance, but structurally they contain foreign elements other than carbon, and have a layered structure, aromatic bridge structure, and disordered structure containing tetrahedral carbon. Including.

  The carbon-based coloring precursor is not particularly limited. For example, polymers such as petroleum tar, petroleum pitch, coal tar, coal pitch, or polymers obtained from monomers including pitch, coke, and acrylonitrile, ferrocene compounds (ferrocene and ferrocene derivatives). Etc. In these, the polymer and / or ferrocene compound obtained from the monomer containing acrylonitrile are preferable, and polyacrylonitrile is preferable as a polymer obtained from the monomer containing acrylonitrile.

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

  The polyimide porous membrane produced without using a colored precursor is composed of, for example, 3 to 60% by mass of a polyamic acid having an intrinsic viscosity of 1.0 to 3.0 and 40 to 97% by mass of an organic polar solvent. The polyamic acid solution is cast into a film and immersed in or contacted with a coagulation solvent containing water as an essential component to produce a polyamic acid porous film, and then the polyamic acid porous film is heat-treated to form an imide. May be manufactured. In this method, the coagulation solvent containing water as an essential component is water or a mixed solution of 5% by mass or more and less than 100% by mass of water and more than 0% by mass and 95% by mass or less of an organic polar solvent. May be. Further, after the imidization, plasma treatment may be performed on at least one surface of the obtained porous polyimide film.

  As the tetracarboxylic dianhydride that can be used in the production of the polyimide porous membrane, any tetracarboxylic dianhydride can be used, and can be appropriately selected according to desired characteristics. Specific examples of tetracarboxylic dianhydride include pyromellitic dianhydride, 3,3 ′, 4,4′-biphenyltetracarboxylic dianhydride (s-BPDA), 2,3,3 ′, 4 ′. -Biphenyltetracarboxylic dianhydride such as biphenyltetracarboxylic dianhydride (a-BPDA), oxydiphthalic dianhydride, diphenylsulfone-3,4,3 ', 4'-tetracarboxylic dianhydride, bis (3,4-dicarboxyphenyl) sulfide dianhydride, 2,2-bis (3,4-dicarboxyphenyl) -1,1,1,3,3,3-hexafluoropropane dianhydride, 2, 3,3 ′, 4′-benzophenone tetracarboxylic dianhydride, 3,3 ′, 4,4′-benzophenone tetracarboxylic dianhydride, bis (3,4-dicarboxyphenyl) methane dianhydride 2,2-bis (3,4-dicarboxyphenyl) propane dianhydride, p-phenylenebis (trimellitic acid monoester acid anhydride), p-biphenylenebis (trimellitic acid monoester acid anhydride), m-terphenyl-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] propane dianhydride, 2,3,6,7-naphthalenetetracarboxylic dianhydride, 1,4,5,8-naphthalenetetra Carboxylic acid dianhydride, 4,4 '- (2,2-hexafluoroisopropylidene) may be mentioned diphthalic dianhydride and the like. 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, in particular, at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydride and pyromellitic dianhydride is preferable. As the biphenyltetracarboxylic dianhydride, 3,3 ′, 4,4′-biphenyltetracarboxylic dianhydride can be suitably used.

Arbitrary diamine can be used for the diamine which can be used in manufacture of the said polyimide porous membrane. Specific examples of diamines include the following.
1) One benzene diamine such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, 2,6-diaminotoluene;
2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether and 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenylmethane, 3,3'-dimethyl-4,4'-diaminobiphenyl, 2,2'- Dimethyl-4,4′-diaminobiphenyl, 2,2′-bis (trifluoromethyl) -4,4′-diaminobiphenyl, 3,3′-dimethyl-4,4′-diaminodiphenylmethane, 3,3′- Dicarboxy-4,4′-diaminodiphenylmethane, 3,3 ′, 5,5′-tetramethyl-4,4′-diaminodiphenylmethane, bis (4-aminophenyl) sulfide, 4,4′-diaminobenzanilide, 3,3′-dichlorobenzidine, 3,3′-dimethylbenzidine, 2,2′-dimethylbenzidine, 3,3′-dimethoxybenzidine 2,2′-dimethoxybenzidine, 3,3′-diaminodiphenyl ether, 3,4′-diaminodiphenyl ether, 4,4′-diaminodiphenyl ether, 3,3′-diaminodiphenyl sulfide, 3,4′-diaminodiphenyl sulfide, 4,4'-diaminodiphenyl sulfide, 3,3'-diaminodiphenyl sulfone, 3,4'-diaminodiphenyl sulfone, 4,4'-diaminodiphenyl sulfone, 3,3'-diaminobenzophenone, 3,3'-diamino -4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-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 benzene nucleus diamines such as 1,3,3,3-hexafluoropropane, 3,3′-diaminodiphenyl sulfoxide, 3,4′-diaminodiphenyl sulfoxide, 4,4′-diaminodiphenyl sulfoxide;
3) 1,3-bis (3-aminophenyl) benzene, 1,3-bis (4-aminophenyl) benzene, 1,4-bis (3-aminophenyl) benzene, 1,4-bis (4-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-aminophenyls) Fido) 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, etc. Three diamines of benzene nucleus;
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 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-amino Phenoxy) phenyl] methane, 2,2-bis [3- (3-aminophenoxy) phenyl] propane, , 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-hexafluoro Four diamine diamines such as propane and 2,2-bis [4- (4-aminophenoxy) phenyl] -1,1,1,3,3,3-hexafluoropropane.

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

  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 preferred.

  The polyimide porous membrane that can be used in the present invention has a glass transition temperature of 240 ° C. or higher or a clear transition point at 300 ° C. or higher from the viewpoint of heat resistance and dimensional stability at high temperatures. It is preferably formed from a polyimide obtained by combining acid dianhydride and diamine.

The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane made of the following aromatic polyimide from the viewpoints of heat resistance and dimensional stability at high temperatures.
(I) an aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of a biphenyltetracarboxylic acid unit and a pyromellitic acid unit, and an aromatic diamine unit,
(Ii) an aromatic polyimide comprising 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 selected from the group consisting of at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and a benzenediamine unit, diaminodiphenyl ether unit and bis (aminophenoxy) phenyl unit. An aromatic polyimide composed of a kind of aromatic diamine unit.

  The polyimide porous membrane used in the present invention is preferably 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. The structure is a polyimide porous membrane, wherein the average pore diameter of the pores present in the surface layer A is 0.01 μm to 15 μm, and the average pore diameter of the pores present in the surface layer B is 20 μm to 100 μm, The macrovoid layer includes a partition bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition and the surface layers A and B. The partition of the macrovoid layer and the surface The thicknesses of the layers A and B are 0.01 to 20 μm, the holes in the surface layers A and B communicate with the macrovoids, the total film thickness is 5 to 500 μm, and the porosity is 40% or more. Less than 95%, Polyimide is a porous membrane. Here, at least one partition wall in the macrovoid layer has one or a plurality of holes having an average pore diameter of 0.01 to 100 μm, preferably 0.01 to 50 μm, which communicate adjacent macrovoids.

  For example, a polyimide porous film described in International Publication No. 2010/038873, Japanese Unexamined Patent Application Publication No. 2011-219585, or Japanese Unexamined Patent Application Publication No. 2011-219586 can also be used in the present invention.

1-2. Polyethersulfone (PES) Porous Membrane The PES porous membrane that can be used in the present invention comprises polyethersulfone and typically consists essentially of polyethersulfone. Polyethersulfone may be synthesized by a method known to those skilled in the art, for example, a method of polycondensation reaction of a dihydric phenol, an alkali metal compound and a dihalogenodiphenyl compound in an organic polar solvent, An alkali metal disalt can be synthesized in advance and can be produced by a polycondensation reaction 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.

  Examples of dihydric phenol compounds include hydroquinone, catechol, resorcin, 4,4′-biphenol, bis (hydroxyphenyl) alkanes (for example, 2,2-bis (hydroxyphenyl) propane, and 2,2-bis (hydroxyphenyl) Methane), dihydroxydiphenyl sulfones, dihydroxydiphenyl ethers, or at least one hydrogen of the benzene ring 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. The substituted one is mentioned. As the dihydric phenol compound, a mixture of two or more of the above compounds can be used.

  The polyethersulfone may be a commercially available product. As an example of a commercial item, Sumika Excel 7600P, Sumika Excel 5900P (above, Sumitomo Chemical Co., Ltd. product) etc. are mentioned.

  The logarithmic viscosity of the polyethersulfone is preferably 0.5 or more, more preferably 0.55 or more from the viewpoint of satisfactorily forming the macrovoids of the porous polyethersulfone membrane. 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.

  In addition, the PES porous membrane or polyethersulfone 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 at high temperatures. Preferably it is not observed.

The production method of the PES porous membrane that can be used in the present invention is not particularly limited.
A polyethersulfone solution containing 0.3% to 60% by weight of polyethersulfone having a logarithmic viscosity of 0.5 to 1.0 and 40% to 99.7% by weight of an organic polar solvent is cast into a film. And a step of producing a solidified film having pores by immersing or contacting in a coagulation solvent having a poor solvent or non-solvent of polyethersulfone as an essential component, and a solidified film having pores obtained in the step Including a step of obtaining a PES porous film by heat-treating and coarsening the pores, wherein the heat treatment comprises bringing the solidified film having the pores to a glass transition temperature of the polyethersulfone or higher, or to 240 ° C or higher. It may be manufactured by a method including raising the temperature.

The PES porous membrane that can be used in the present invention preferably has a surface layer A, a surface layer B, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. Because
The macrovoid layer includes a partition bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition and the surface layers A and B and having an average pore diameter in a film plane direction of 10 μm to 500 μm. Have
The partition wall of the macrovoid layer has a thickness of 0.1 μm to 50 μm,
The surface layers A and B each 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 not more than 200 μm, and the other has a plurality of pores having an average pore diameter of 0.01 μm or more and less than 200 μm,
The surface opening ratio of one of the surface layer A and the surface layer B is 15% or more, and the surface opening ratio of the other surface layer is 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%.
PES porous membrane.

2. Cell culture method

One embodiment of the present invention provides:
A cell culture method comprising:
(1) applying cells to the polymer porous membrane;
(2) culturing in a gas phase while applying the medium formed into droplets by the droplet formation medium supply means to the polymer porous membrane to which the cells are applied;
Including
Here, the polymer porous membrane has a three-layered polymer porous 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 Here, 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. And a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and a hole in the surface layers A and B communicates with the macrovoids. Hereinafter, the cell culture method of the present invention is also referred to as “the cell culture method 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, a stable liquid retention is made in the polymer porous membrane, and there is a moist environment resistant to drying. Kept. Therefore, cell survival and proliferation can be achieved even with a very small amount of medium as compared with a conventional culture method using a cell culture carrier. In addition, since it is possible to culture even when a part or all of the polymer porous membrane is exposed to air, an efficient oxygen supply to the cells is performed by applying a droplet-form culture medium. A culture method is provided.

  According to the present invention, the amount of medium to be used is extremely small, and the porous polymer membrane as a 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, “medium” refers to a cell culture medium for culturing cells, particularly animal cells. The medium is used as the same meaning as the cell culture solution. Therefore, the medium used in the present invention refers to a liquid medium. The type of the medium can be a commonly used medium and is appropriately determined depending on the type of cells to be cultured.

  In the present specification, the “droplet-forming medium” refers to a mist-form or water-drop medium, and refers to a medium that can be sprayed or sprayed onto the porous polymer membrane used in the present invention. The diameter of the droplet formation medium is not limited. For example, the droplet formation medium may be a mist-shaped droplet formation medium that is small enough to float in the air without falling freely due to gravity. The diameter of the mist-like droplet formation medium may be, for example, about 1 μm to 100 μm, or may be a smaller diameter. Further, the droplet-forming medium may be, for example, a water-drop-like medium that freely falls by gravity, and may be, for example, 100 μm or more. As a method for forming the medium into droplets, a method for forming droplets by a known means may be used. For example, the medium may be formed using a mist nozzle, a shower nozzle, or the like. However, the droplet formation method must be formed into droplets by a method that does not change the components of the medium. For example, the evaporation method is excluded from the droplet formation method.

  In an embodiment of the present invention, the droplet formation 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 is dissolved in the medium. As a result, a medium having a sufficient amount of oxygen is continuously supplied, and the cells can be cultured without causing ischemia. In addition, since the polymer porous membrane is always exposed to the gas phase, the medium attached to the polymer porous membrane can always take up 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 up, iii) sheets or pieces connected by a thread-like structure, iv It may be a polymer porous membrane that is tied in a rope shape and / or v) two or more are laminated. By using a polymer porous membrane having the shapes of i) to v), a large number of 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. In the present specification, the “modular polymer porous membrane” refers to a polymer porous membrane housed in a casing.

  The casing included in the modular polymer porous membrane used in the embodiment of the present invention has two or more cell culture medium outlets, and the culture medium flows into and out of the casing through the cell culture medium outlets. The diameter of the cell culture medium inlet / outlet of the casing is preferably larger than the diameter of the cells so that the cells can flow into the casing. The diameter of the cell culture medium outlet is preferably smaller than the diameter of the polymer porous membrane flowing out of the cell culture medium outlet. The diameter smaller than the diameter from which the polymer porous membrane flows can be appropriately selected depending on the shape and size of the polymer porous membrane accommodated in the casing. For example, when the polymer porous membrane has a string shape, there is no particular limitation as long as it is smaller than the short side width of the polymer porous membrane and has an appropriate diameter that does not allow the polymer porous membrane to flow out. It is preferable that as many cell culture medium outlets as possible are provided so that the cell culture medium is easily supplied and / or discharged into and out of the casing. Preferably, it is 5 or more, preferably 10 or more, preferably 20 or more, preferably 50 or more, preferably 100 or more. As for the cell culture medium outflow port, a part or all of the casing may have a mesh structure. Further, the casing itself may be mesh-shaped. In the present invention, the mesh-shaped structure has, for example, a vertical, horizontal, and / or diagonal lattice structure, and each mesh opening forms a cell culture medium outlet / outlet to the extent that fluid can pass through. However, it is not limited to this.

  The modular polymer porous membrane casing used in the embodiment of the present invention is made of a polymer such as polyethylene, polypropylene, nylon, polyester, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, or a metal such as stainless steel or titanium. Although it is mentioned, it is not limited to this.

The modular polymer porous membrane used in embodiments of the present invention is:
(I) Two or more independent polymer porous membranes are aggregated,
(Ii) the polymer porous membrane is folded;
(Iii) The polymer porous membrane is wound into a roll and / or
(Iv) The polymer porous membrane is tied in a rope shape,
It is possible to apply the modularized polymer porous membrane that is housed in the casing.

  In this specification, “two or more independent polymer porous membranes are aggregated and accommodated in a casing” means that a certain space in which two or more independent polymer porous membranes are surrounded by a casing. It refers to the state of being consolidated and accommodated inside. In the present invention, two or more independent polymer porous membranes are fixed by at least one location of the polymer porous membrane and at least one location in the casing by any method, and the polymer porous membrane is fixed in the casing. It may be fixed so as not to move. The two or more independent polymer porous membranes may be small pieces. The shape of the small piece can take 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” is folded in the casing, so that the frictional force between each surface of the polymer porous membrane and / or the surface in the casing is increased in the casing. This is a porous polymer membrane that is in a state of not moving. In this specification, “folded” may be a state in which the polymer porous membrane is creased or a state in which no crease is present.

  In the present specification, the “polymer porous film wound in a roll shape” means that the polymer porous film is wound in a roll shape, and friction between each surface of the polymer porous film and / or the surface in the casing. A porous polymer membrane that has become immobile in the casing due to force. Further, in the present invention, the polymer porous membrane knitted in a rope shape is, for example, a plurality of strip-shaped polymer porous membranes knitted in a rope shape by an arbitrary method, and the friction force between the polymer porous membranes. A porous polymer membrane that does not move relative to 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 wound into a roll, and (iv) ) Polymer porous membranes tied in a rope shape may be combined and accommodated in the casing.

3. Cell culture equipment

One embodiment of the present invention provides:
A polymer porous membrane;
A polymer porous membrane placement section on which the polymer porous membrane is placed;
A housing in which the polymer porous membrane mounting portion is accommodated;
A droplet culture medium supply unit disposed inside the housing;
A medium supply line in communication with the dropletized medium supply unit;
A medium storage section communicating with the medium supply line;
A pump provided in a part of the medium supply line;
With
Here, the polymer porous membrane has a three-layered polymer porous 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 Here, 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. And a plurality of macro voids surrounded by the partition wall and the surface layers A and B,
Here, the polymeric porous membrane mounting part is related with the cell culture apparatus provided with the several culture medium discharge port of slit shape or mesh shape.

  Since the polymer porous membrane used in the present invention has a slightly hydrophilic porous property, the liquid is stably retained in the polymer porous membrane, and a moist environment resistant to drying is maintained. Therefore, cell survival and proliferation can be achieved even with a very small amount of medium as compared with a culture apparatus using a conventional cell culture carrier. In addition, since it is possible to culture even when a part or all of the polymer porous membrane is exposed to air, an efficient oxygen supply to the cells is performed by applying a droplet-form culture medium. Provided is a culture apparatus capable of Hereinafter, the cell culture device is also referred to 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 a cell culture device of the present invention. A polymer porous membrane is placed on the polymer porous membrane placement portion. The polymer porous membrane applied to the polymer porous membrane mounting part is, for example, i) folded, ii) rolled up, iii) sheets or pieces connected by a thread-like structure. Iv) A polymer porous membrane tied in a rope shape and / or v) two or more laminated. By using a polymer porous membrane having the shapes of i) to v), a large number of cells can be cultured in a fixed space. The polymer porous membrane placement part is provided with a plurality of slit-like or mesh-like medium outlets. By providing the medium discharge port, the medium supplied from the droplet-forming medium supply unit can be discharged to the lower part. What is necessary is just to select suitably the shape of a culture medium outlet, a magnitude | size, and a number so that the polymer porous membrane to mount may not fall.

  The polymer porous membrane used in the embodiment of the present invention may be a modular polymer porous membrane as described above.

  In the present embodiment, the cell culture device includes a housing that accommodates the polymer porous membrane placement portion, and the inside of the housing is used to drop the medium into droplets and supply the polymer porous membrane. A droplet forming medium supply unit is provided. For example, a known mist nozzle or shower nozzle can be used for the droplet forming medium supply unit. By providing the casing, the droplet forming medium supplied from the droplet forming medium supply unit fills the inside of the casing and is efficiently supplied to the porous polymer membrane. The shape and size of the housing may be appropriately selected according to the amount of cells to be cultured and the amount of polymer porous membrane to be applied.

  A medium supply line communicates with the droplet forming medium supply unit, and a medium storage unit communicates with the medium supply line. A part of the medium supply line is provided with a pump for sending the medium to the droplet forming medium supply unit. In the culture medium storage section, the culture medium released from the droplet formation culture medium supply section is stored. As shown in the figure, the medium storage part is arranged immediately below the polymer porous film mounting part and stores the medium that has fallen after being applied to the polymer porous film, and the medium is a cell culture. The structure which circulates in an apparatus may be sufficient. Moreover, the culture medium storage part may be provided independently independently of the position of the polymer porous membrane placement part as shown in the figure. In this case, it is necessary to separately provide a container or the like for storing the culture medium that has fallen after being applied to the polymer porous membrane, immediately below the polymer porous membrane placement portion. The speed of the medium sent to the droplet formation medium supply unit by the pump can be appropriately selected.

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

4). Cell culture method using cell culture apparatus

<Process of applying cells to polymer porous membrane>
The specific process of application to the polymer porous membrane of the cell used by this invention is not specifically limited. The steps described herein or any method suitable for applying cells to a membrane-like carrier can be employed. Although it is not necessarily limited, in the method of this invention, application to the polymer porous membrane of a cell includes the following aspects, for example.

(A) an embodiment comprising a step of seeding cells on the surface of the porous polymer membrane;
(B) A cell suspension is placed on the dried surface of the porous polymer membrane,
Leave or move the polymer porous membrane to promote fluid outflow, or stimulate a portion of the surface to draw cell suspension into the membrane; and
The cells in the cell suspension are retained in the membrane, and the water flows out.
An embodiment comprising steps; and
(C) Wetting one or both sides of the polymer porous membrane with a cell culture solution or a sterilized liquid,
Loading the wet polymer porous membrane with a cell suspension; and
The cells in the cell suspension are retained in the membrane, and the water flows out.
A mode comprising the steps.

  The embodiment of (A) includes directly seeding cells and cell clusters on the surface of the polymer porous membrane. Or the aspect which puts a polymer porous membrane in a cell suspension and infiltrates a cell culture solution from the surface of a membrane is also included.

  The cells seeded on the surface of the polymer porous membrane adhere to the polymer porous membrane and enter the inside of the pore. Preferably, the cells adhere to the polymer porous membrane without any physical or chemical force applied from the outside. Cells seeded on the surface of the polymer porous membrane can stably grow and proliferate on the surface and / or inside of the membrane. Cells can take a variety of different forms depending on the location of the membrane in which they grow and multiply.

  In the embodiment (B), the cell suspension is placed on the dried surface of the polymer porous membrane. By leaving the polymer porous membrane, or moving the polymer porous membrane to promote the outflow of the liquid, or stimulating a part of the surface, the cell suspension is sucked into the membrane, Cell suspension penetrates into the membrane. Without being bound by theory, it is thought that this is due to the properties derived from the surface shape of the polymer porous membrane. According to this embodiment, the cells are sucked and seeded at the portion of the membrane where the cell suspension is loaded.

  Alternatively, as in the embodiment (C), one or both sides or the whole of the polymer porous membrane is wetted with a cell culture medium or a sterilized liquid, and then the cell suspension is applied to the wet polymer porous membrane. The liquid may be loaded. In this case, the passage speed of the cell suspension is greatly improved.

  For example, a method of moistening part of the membrane pole for the main purpose of preventing membrane scattering (hereinafter referred to as “one-point wet method”) can be used. The one-point wet method is substantially similar to the dry method (the embodiment (B)) that does not substantially wet the film. However, it is thought that the membrane permeation of the cell fluid becomes rapid for the wetted small part. In addition, a method in which a cell suspension is loaded into a polymer porous membrane that has been sufficiently wetted on one or both sides (hereinafter referred to as “wet membrane”) can be used (hereinafter referred to as “this”). Is described as “wet film method”). In this case, the passage speed 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 the water is allowed to flow out. As a result, it is possible to perform processing such as concentrating the concentration of cells in the cell suspension or allowing unnecessary components other than cells to flow out together with moisture.

  The mode of (A) may be referred to as “natural sowing” (B) and the mode of (C) as “suction sowing”.

  Preferably, but not exclusively, living cells remain selectively in the polymeric porous membrane. Thus, 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 moisture.

  The sterilized liquid used in the embodiment (C) is not particularly limited, but is a sterilized buffer or sterilized water. Examples of the buffer include (+) and (-) Dulbecco's PBS, (+) and (-) Hank's Balanced Salt Solution. Examples of buffer solutions are shown in Table 1 below.

  Furthermore, in the method of the present invention, the cell is applied to the polymer porous membrane in such a manner that the cells adhere to the membrane by allowing the adherent cells in a suspended state to coexist with the polymer porous membrane (entanglement). ) Is also included. For example, in the method of the present invention, in order to apply cells to a polymer porous membrane, a cell culture medium, cells and one or more of the polymer porous membranes may be placed in a cell culture vessel. When the cell culture medium is liquid, the polymer porous membrane is in a suspended state 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 it is a cell which is not suitable for natural suspension culture, the polymer porous membrane can be cultured in a suspended state in the cell culture medium. Preferably, the cells adhere to the polymeric porous membrane. “Spontaneously adheres” means that the cells remain on or inside the porous polymer membrane without any physical or chemical force applied from the outside.

  The above-described application of the cell to the polymer porous membrane may be performed using a combination of two or more methods. For example, you may apply a cell to a polymer porous membrane combining 2 or more methods among aspect (A)-(C). It is possible to apply the polymer porous membrane supporting the cells to the cell culture unit 2 in the cell culture apparatus 1 and culture the cells.

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

  In the present specification, the “suspended cell” means, for example, a cell obtained by forcibly suspending an adherent cell in a medium by a proteolytic enzyme such as trypsin, or a known acclimation step. Thus, the cells contain adherent cells that can be suspended in the medium.

  The types of cells that can be used in the present invention are selected from the group consisting of animal cells, insect cells, plant cells, yeasts, and bacteria, for example. Animal cells are roughly classified into cells derived from animals belonging to the vertebrate phylum and cells derived from invertebrates (animals other than animals belonging to the vertebrate phylum). In this specification, the origin of the animal cell is not particularly limited. Preferably, it means an animal-derived cell belonging to the vertebrate phylum. Vertebrates include the maxilla and maxilla, and the maxilla includes mammals, birds, amphibians, reptiles, and the like. Preferably, it is a cell derived from an animal belonging to the mammal class generally called 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, “pluripotent stem cell” is intended to be a generic term for stem cells having the ability to differentiate into cells of any tissue (differentiated pluripotency). The pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), germ stem cells (GS cells), and the like. . Preferably, they are ES cells or iPS cells. iPS cells are particularly preferred for reasons such as no ethical problems. Any known pluripotent stem cell can be used. For example, the pluripotent stem cell described in International Publication No. 2009/123349 (PCT / JP2009 / 057041) can be used.

  “Tissue stem cell” means a stem cell that has the ability to differentiate into various cell types (differentiated pluripotency), although the cell line that can be differentiated is limited to a specific tissue. For example, hematopoietic stem cells in the bone marrow become blood cells, and neural stem cells differentiate into nerve cells. In addition, there are various types such as liver stem cells that make the liver and skin stem cells that become 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 cells” refers to cells other than germ cells among the cells that constitute multicellular organisms. 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, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, kidney cells, lung cells, or , Lymphocytes, erythrocytes, leukocytes, monocytes, macrophages or megakaryocyte blood cells.

  A “germ cell” means a cell that plays a role in transmitting genetic information to the next generation in reproduction. For example, gametes for sexual reproduction, ie eggs, egg cells, sperm, sperm cells, 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, blood, etc., and includes soft tissue sarcoma, malignant bone tumor and the like. Sarcoma cells are cells derived from sarcomas. The “established cell” means a cultured cell that has been maintained outside the body 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 white blood cells), HeLa cells (derived from human cervical cancer), Vero cells (Derived from African green monkey kidney epithelial cells), MDCK cells (derived from canine kidney tubular epithelial cells), HepG2 cells (human hepatoma-derived cell line), BHK cells (newborn hamster kidney cells), NIH3T3 cells (derived from mouse fetal fibroblasts) There are cell lines derived from various tissues of various biological species including humans. A “transformed cell” means a cell in which a nucleic acid (DNA or the like) has been introduced from the outside of the cell to change its genetic properties.

  As used herein, “adherent cells” are generally cells that need to adhere to a suitable surface for growth and are also referred to as adherent cells or anchorage-dependent cells. In some embodiments of the invention, the cells used are adherent cells. The cells used in the present invention are adherent cells, and more preferably cells that can be cultured even in a state suspended in a medium. Adherent cells capable of suspension culture can be obtained by acclimating adherent cells to a state suitable for suspension culture by a known method. For example, CHO cells, HEK293 cells, Vero cells, NIH3T3 cells And cell lines derived from these cells.

  A model diagram of cell culture using a polymer porous membrane is shown in FIG. FIG. 1 is a diagram for assisting understanding, and each element is not an actual size. In the cell culturing method of the present invention, by applying cells to the polymer porous membrane and culturing, a large number of cells grow on the multifaceted connected porous portion and the surface of the polymer porous membrane, A large amount of cells can be cultured easily. Moreover, in the cell culturing method of the present invention, a large amount of cells can be cultured while the amount of medium used for cell culturing is greatly reduced as compared with the conventional method. For example, even if a part or the whole of the polymer porous membrane is not in contact with the liquid phase of the cell culture medium, a large amount of cells can be cultured over a long period of time. In addition, the total volume of the cell culture medium contained in the cell culture container can be significantly reduced 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 not containing cells in the space including the volume of the internal gap is referred to as “apparent polymer porous membrane volume” (see FIG. 3). Then, when the cells are applied to the polymer porous membrane and the cells are supported on and inside the polymer porous membrane, the polymer porous membrane, the cells, and the medium infiltrated into the polymer porous membrane are entirely space. The volume occupied therein is referred to as “polymer porous membrane volume including cell viability zone” (see FIG. 3). In the case of a polymer porous membrane having a film thickness of 25 μm, the polymer porous membrane volume including the cell survival area is apparently about 50% larger than the polymer porous membrane volume at the maximum. In the method of the present invention, a plurality of polymer porous membranes can be accommodated and cultured in one cell culture vessel. In this 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 s is sometimes simply referred to as “the total volume of the polymer porous membrane including the cell viability zone”.

  By using the method of the present invention, even if the total volume of the cell culture medium contained in the cell culture container is 10,000 times or less than the total volume of the polymer porous membrane including the cell viability area, It becomes possible to culture well. In addition, cells can be cultured well over a long period of time even under conditions where 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. . Furthermore, even when 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 cultured well over a long period of time. . And even if the total volume of the cell culture medium contained in the cell culture container is 10 times or less than the total volume of the polymer porous membrane including the cell survival area, the cells can be cultured well over a long period of time. .

  That is, according to the present invention, the space (container) for cell culture can be miniaturized to the utmost limit as compared with a conventional cell culture apparatus that performs two-dimensional culture. Moreover, when it is desired to increase the number of cells to be cultured, it is possible to increase the volume of cell culture flexibly by a simple operation such as increasing the number of polymer porous membranes to be laminated. If it is a cell culture apparatus provided with the polymer porous membrane used for this invention, it becomes possible to isolate | separate the space (container) which culture | cultivates a cell, and the space (container) which stores a cell culture medium, and the cell to culture | cultivate Depending on the number, the required amount of cell culture medium can be prepared. The space (container) in which the cell culture medium is stored may be enlarged or reduced 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 container after the culture using the polymer porous membrane is uniformly dispersed in the cell culture medium contained in the cell culture container. 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 ⁶ per 1 ml of culture medium 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, 5.0 × 10 ⁸ or more, 1 It means culturing until it becomes 0.0 × 10 ⁹ or more, 2.0 × 10 ⁹ or more, or 5.0 × 10 ⁹ or more.

  Various known methods can be used as a method for measuring the number of cells during or after culture. For example, as a method for measuring the number of cells contained in a cell culture vessel after culturing using a polymer porous membrane as if all the cells are uniformly dispersed in the cell culture medium contained in the cell culture vessel Any known method can be used as appropriate. For example, a cell count method using CCK8 can be suitably used. Specifically, the number of cells in a normal culture without using a polymer porous membrane was measured using Cell Counting Kit 8; a solution reagent manufactured by Dojindo Laboratories (hereinafter referred to as “CCK8”). The correlation coefficient with the actual cell number is obtained. Thereafter, the polymer porous membrane, to which the cells were applied and cultured, was transferred to a medium containing CCK8, stored in an incubator for 1-3 hours, the supernatant was extracted, and the absorbance was measured at a wavelength of 480 nm. The number of cells is calculated from the obtained correlation coefficient.

From another viewpoint, the mass culture of cells means, for example, that the number of cells contained per 1 cm 2 of the polymer porous membrane after the culture using the polymer porous membrane is 1.0 × 10 5 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 per square centimeter of the polymer porous membrane can be appropriately measured using a known method such as a cell counter.

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

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

Example 1
Cell culture method using polyimide porous membrane by mist and shower type reactor

Cells conditioned and suspended from anti-human IL-8 antibody-producing CHO-DP12 cells (ATCC CRL-12445) are suspended in culture using a medium (BalanCD ™ CHO GROWTH A), and the number of living cells per ml is increased. The culture was continued until it reached 3.9 × 10 ⁶ . After each 12 ml of the above suspension culture solution was poured 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 ² per module). ) 12 sealed modules were added to the petri dish. The module was wetted with the cell suspension and then left overnight in a CO2 incubator.

The next day, the module is taken out, 12 modules are installed inside the mist and shower type reactor shown in FIG. 4, and 200 ml of medium (IMDM containing 2% FBS) is stored in the medium reservoir, and the medium is passed through the tube pump. Was circulated at a rate of 60 ml per minute. When the culture was terminated after 2 days, a total cell 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 by mist and shower type reactor

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

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

Claims (26)

A cell culture method comprising:
(1) applying cells to the polymer porous membrane;
(2) culturing in a gas phase while applying the medium formed into droplets by the droplet formation medium supply means to the polymer porous membrane to which the cells are applied;
Including
Here, the polymer porous membrane has a three-layered polymer porous 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 Here, 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. And a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and holes in the surface layers A and B communicate with the macrovoids.
Method. The polymer porous membrane is
i) folded,
ii) rolled up,
iii) Sheets or pieces are connected by a thread-like structure,
The method according to claim 1, wherein the polymer porous membrane is iv) tied in a rope shape and / or v) two or more are laminated. The polymer porous membrane is a modular polymer porous membrane having a casing,
Wherein the casing has two or more cell culture medium outlets;
Here, the modular polymer porous membrane is
(I) Two or more independent polymer porous membranes are aggregated,
(Ii) the polymer porous membrane is folded;
(Iii) The polymer porous membrane is wound into a roll and / or
(Iv) The polymer porous membrane is tied in a rope shape,
The method of claim 1, wherein the method is contained within the casing.   The method according to any one of claims 1 to 3, wherein the polymer porous membrane has a plurality of pores having an average pore diameter of 0.01 to 100 µm.   The method according to any one of claims 1 to 4, wherein an average pore diameter of the surface layer A is 0.01 to 50 µm.   The method according to claim 1, wherein the surface layer B has an average pore diameter of 20 to 100 μm.   The method according to any one of claims 1 to 6, wherein a total film thickness of the polymer porous membrane is 5 to 500 µm.   The method according to claim 1, wherein the polymer porous membrane is a polyimide porous membrane.   The method according to claim 8, wherein the polyimide porous membrane is a polyimide porous membrane containing polyimide obtained from tetracarboxylic dianhydride and diamine.   Coloring obtained by forming a polyamic acid solution composition containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a colored precursor, and then heat-treating the polyimide porous membrane at 250 ° C. or higher. The method according to claim 8 or 9, which is a polyimide porous membrane.   The method according to claim 1, wherein the polymer porous membrane is a polyethersulfone porous membrane. A polymer porous membrane;
A polymer porous membrane placement section on which the polymer porous membrane is placed;
A housing in which the polymer porous membrane mounting portion is accommodated;
A droplet culture medium supply unit disposed inside the housing;
A medium supply line in communication with the dropletized medium supply unit;
A medium storage section communicating with the medium supply line;
A pump provided in a part of the medium supply line;
With
Here, the polymer porous membrane has a three-layered polymer porous 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 Here, 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. A plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and the holes in the surface layers A and B communicate with the macrovoids,
Here, the polymer porous membrane mounting portion is provided with a plurality of slit-like or mesh-like medium outlets,
Cell culture device.   The cell culture device according to claim 12, wherein at least one droplet-forming medium supply unit is disposed at an upper part, a lower part, and / or a side part of the polymer porous membrane mounting part.   The cell culture device according to claim 12 or 13, wherein the polymer porous membrane placement part has two or more stages.   15. The medium according to any one of claims 12 to 14, wherein the medium storage part is disposed inside the casing and is disposed below the polymer porous membrane mounting part, and the medium circulates. The cell culture device described. The polymer porous membrane is
i) folded,
ii) rolled up,
iii) Sheets or pieces are connected by a thread-like structure,
The cell culture device according to any one of claims 12 to 15, which is a polymer porous membrane iv) tied in a rope shape and / or v) two or more laminated. The polymer porous membrane is a modular polymer porous membrane having a casing,
Wherein the casing has two or more cell culture medium outlets;
Here, the modular polymer porous membrane is
(I) Two or more independent polymer porous membranes are aggregated,
(Ii) the polymer porous membrane is folded;
(Iii) The polymer porous membrane is wound into a roll and / or
(Iv) The polymer porous membrane is tied in a rope shape,
The cell culture device according to any one of claims 12 to 15, which is accommodated in the casing.   The cell culture device according to any one of claims 12 to 17, wherein the polymer porous membrane has a plurality of pores having an average pore diameter of 0.01 to 100 µm.   The cell culture device according to any one of claims 12 to 18, wherein an average pore diameter of the surface layer A is 0.01 to 50 µm.   The cell culture device according to any one of claims 12 to 19, wherein an average pore diameter of the surface layer B is 20 to 100 µm.   The cell culture device according to any one of claims 12 to 20, wherein a total film thickness of the polymer porous membrane is 5 to 500 µm.   The cell culture device according to any one of claims 12 to 21, wherein the polymer porous membrane is a polyimide porous membrane.   The cell culture device according to claim 22, wherein the polyimide porous membrane is a polyimide porous membrane containing polyimide obtained from tetracarboxylic dianhydride and diamine.   Coloring obtained by forming a polyamic acid solution composition containing a polyamic acid solution obtained from tetracarboxylic dianhydride and diamine and a colored precursor, and then heat-treating the polyimide porous membrane at 250 ° C. or higher. The cell culture device according to claim 22 or 23, which is a polyimide porous membrane.   The cell culture device according to any one of claims 12 to 21, wherein the polymer porous membrane is a polyethersulfone porous membrane.   A cell culture method using the cell culture apparatus according to any one of claims 12 to 24.