WO2019146732A1 - 細胞培養モジュール - Google Patents
細胞培養モジュール Download PDFInfo
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- WO2019146732A1 WO2019146732A1 PCT/JP2019/002375 JP2019002375W WO2019146732A1 WO 2019146732 A1 WO2019146732 A1 WO 2019146732A1 JP 2019002375 W JP2019002375 W JP 2019002375W WO 2019146732 A1 WO2019146732 A1 WO 2019146732A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/42—Integrated assemblies, e.g. cassettes or cartridges
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
- C08G73/10—Polyimides; Polyester-imides; Polyamide-imides; Polyamide acids or similar polyimide precursors
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G75/00—Macromolecular compounds obtained by reactions forming a linkage containing sulfur with or without nitrogen, oxygen, or carbon in the main chain of the macromolecule
- C08G75/20—Polysulfones
- C08G75/23—Polyethersulfones
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/34—Internal compartments or partitions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/44—Multiple separable units; Modules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/50—Fuel cells
Definitions
- the present invention relates to cell culture modules.
- proteins such as enzymes, hormones, antibodies, cytokines, viruses (virus proteins) and the like used for treatment and vaccines have been industrially produced using cultured cells.
- protein production techniques have problems in terms of efficiency, which affects the timely and stable supply of biopharmaceuticals for which a sustainable and wide supply is essential. Therefore, in order to establish efficient, stable, and rapid protein production methods, innovative and simple techniques such as increasing the amount of protein production, such as cell culture techniques at high density and high-efficiency continuous production methods, Technology was required.
- anchorage-dependent adherent cells that adhere to the culture substrate may be used. Such cells need to adhere and culture on the surface of a petri dish, plate or chamber in order to proliferate in a scaffold-dependent manner.
- adherent cells it was necessary to increase the surface area for adhesion.
- a culture method using a microporous carrier in particular, a microcarrier
- a culture method using a microporous carrier has been developed (for example, Patent Document 1).
- Cell culture systems using microcarriers need to be well agitated and diffused in order to prevent microcarriers from aggregating each other. Therefore, since the volume which can fully stir and spread the culture solution which disperse
- Polyimide porous membranes have been used for applications such as filters, low dielectric constant films, electrolyte membranes for fuel cells, etc., in particular, focusing on cells, before the present application.
- Patent documents 2 to 4 are particularly excellent in permeability to substances such as gas, high in porosity, excellent in smoothness on both surfaces, relatively high in strength, and in spite of high porosity, in the film thickness direction
- the polyimide porous membrane which has many macro voids which are excellent in the resistance with respect to the compressive stress to it is described. Each of these is a polyimide porous membrane produced via an amic acid.
- Patent Document 5 A method for culturing cells has been reported, including applying the cells to a polyimide porous membrane and culturing.
- the present invention aims to provide a means capable of culturing cells stably and in large amounts by applying homogeneous culture conditions to a plurality of porous polymer membranes.
- the present invention includes the following ⁇ 1> to ⁇ 15>.
- ⁇ 1> At the top, At the bottom, A side with a culture fluid outlet and A plurality of compartments having a culture fluid inlet / outlet which separates the space formed by the top, bottom and sides; Two or more selected from a gap space between the top and the adjacent partition, a gap space between the bottom and the adjacent partition, and a plurality of gap spaces between the adjacent partitions A porous polymer membrane fixed to each of the interstitial spaces;
- a cell culture module comprising
- the polymer porous membrane has a three-layer 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.
- Porous film wherein the average pore diameter of the pores present in the surface layer A is smaller than the average pore diameter of the pores present 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.
- ⁇ 3> Two or more clearances selected from a clearance space between the top and the adjacent partition, a clearance space between the bottom and the adjacent partition, and a plurality of clearances between the adjacent partitions
- the polymer porous membrane is a polyimide porous membrane.
- the polymer porous membrane is a polyethersulfone porous membrane.
- ⁇ 6> The cell culture module according to any one of ⁇ 1> to ⁇ 5>, wherein the top and the bottom have a culture fluid outlet / inlet.
- ⁇ 7> The cell culture module according to any one of ⁇ 1> to ⁇ 6>, which has a rectangular parallelepiped shape.
- ⁇ 8> The cell culture module according to any one of ⁇ 1> to ⁇ 6>, which has a cubic shape.
- ⁇ 9> The cell culture module according to any one of ⁇ 1> to ⁇ 6>, which has an egg shape.
- ⁇ 11> Multiple cell culture submodules, What is claimed is: 1.
- a cell culture module comprising: a cell culture submodule housing casing having a culture solution outlet and inlet for stacking and housing the plurality of cell culture submodules,
- the cell culture submodule is Polymer porous membrane, A polymer porous membrane-containing casing having a culture fluid outlet / inlet, wherein the polymer porous membrane-containing casing is fixed and accommodated;
- the polymer porous membrane is a three-layered polymer porous membrane 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 diameter of the pores present in the surface layer A is smaller than the average pore diameter of the pores present in the surface layer B
- the macrovoid layer is a partition 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 holes in the surface layers A and B communicate with the macrovoids, Cell culture module.
- Cell culture module as described in ⁇ 11> which has a clearance space between the laminated
- ⁇ 13> The cell culture module according to ⁇ 11> or ⁇ 12>, wherein 3 to 100 sheets of the polymer porous membrane are stacked and accommodated in the polymer porous membrane accommodating casing.
- ⁇ 14> The cell culture module according to any one of ⁇ 11> to ⁇ 13>, wherein the polymer porous membrane is a polyimide porous membrane.
- ⁇ 15> The cell culture module according to any one of ⁇ 11> to ⁇ 13>, wherein the polymer porous membrane is a polyethersulfone porous membrane.
- homogeneous culture conditions can be applied to a plurality of polymer porous membranes, and cells can be cultured stably and in large amounts.
- FIG. 1 is a perspective view of a cell culture module 1. However, illustration of the porous polymer membrane in the cell culture module is omitted.
- FIG. 2 is a front view for explaining the inside of the cell culture module 1.
- FIG. 3 is a perspective view for explaining the inside of the cell culture module 1. It is a perspective view for demonstrating the inside of the cell culture module 1 cut
- FIG. 5 is a perspective view of a cell culture module complex 10 produced by preparing two cell culture modules 1 shown in FIGS. 1 to 3 and connecting one top to the other. However, illustration of the porous polymer membrane in the cell culture module is omitted.
- 6 is a front view of cell culture module complex 10.
- FIG. FIG. 7 is a perspective view of the cell culture module 20.
- FIG. 8 is a front view of the cell culture module 20.
- FIG. 9 is a top view of the cell culture submodule 30 used for the cell culture module 20 shown in FIGS. 7 and 8.
- FIG. 10 is a cross-sectional view of the cell culture submodule 30 taken along line BB in FIG.
- FIG. 11 shows the time course of the amount of antibody produced by cell culture using the cell culture module 20.
- 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, and for example, 0.01 ⁇ m Or more and less than 200 ⁇ m, 0.01 to 150 ⁇ m, 0.01 to 100 ⁇ m, 0.01 to 50 ⁇ m, 0.01 to 40 ⁇ m, 0.01 to 30 ⁇ m, 0.01 to 25 ⁇ m, 0.01 to 20 ⁇ m, or 0.01 ⁇ m It is ⁇ 15 ⁇ m, preferably 0.01 ⁇ m ⁇ 25 ⁇ m.
- the average pore diameter of the pores present in the surface layer B (hereinafter also referred to as “B surface” or “large pore surface”) in the polymer porous membrane used in the present invention is the average pore diameter of the pores present in the surface layer A It is not particularly limited as long as it is larger, for example, more than 5 ⁇ m to 200 ⁇ m or less, 20 ⁇ m to 100 ⁇ m, 25 ⁇ m to 100 ⁇ m, 30 ⁇ m to 100 ⁇ m, 35 ⁇ m to 100 ⁇ m, 40 ⁇ m to 100 ⁇ m, 50 ⁇ m to 100 ⁇ m, or 60 ⁇ m to 100 ⁇ m, preferably 30 ⁇ m to 100 ⁇ m.
- the average pore size of the polymer porous membrane surface is an area average pore size.
- the area average pore size can be determined according to the following (1) and (2).
- parts other than the polymer porous membrane surface can be calculated
- All pore sizes determined by the above-mentioned formula I are applied to the following formula II, and the area average pore size da when the shape of the pores is a true circle is determined.
- the thickness of the surface layers A and B is not particularly limited, and is, for example, 0.01 to 50 ⁇ m, preferably 0.01 to 20 ⁇ m.
- the average pore diameter of the macrovoids in the macrovoid layer in the macrovoid layer in the polymer porous membrane is not particularly limited, but is, for example, 10 to 500 ⁇ m, preferably 10 to 100 ⁇ m, and more preferably 10 to 80 ⁇ m.
- the thickness of the partition wall in the macro void layer is not particularly limited, but is, for example, 0.01 to 50 ⁇ m, preferably 0.01 to 20 ⁇ m.
- at least one partition in the macrovoid layer communicates between adjacent macrovoids, one or more with an average pore diameter of 0.01 to 100 ⁇ m, preferably 0.01 to 50 ⁇ m. It has a hole.
- the partition walls in the macrovoid layer have no pores.
- the total film thickness of the polymer porous membrane surface used in the present invention is not particularly limited, it may be 5 ⁇ m or more, 10 ⁇ m or more, 20 ⁇ m or more, or 25 ⁇ m or more, 500 ⁇ m or less, 300 ⁇ m or less, 100 ⁇ m or less, 75 ⁇ m or less Or you may be 50 micrometers or less. Preferably, it is 5 to 500 ⁇ m, more preferably 25 to 75 ⁇ m.
- the film thickness of the polymer porous membrane used in the present invention can be measured by a contact-type thickness meter.
- the porosity of the polymer porous membrane used in the present invention is not particularly limited, and 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 III from the coated weight by measuring the film thickness and mass of the porous film cut to a predetermined size. (Wherein, 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 a polyimide, the density is 1.34 g / cm 3 And)
- 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.
- the average pore diameter of the pores present in the surface layer A is 0.01 ⁇ m to 25 ⁇ m, and the average pore diameter of the pores present in the surface layer B is 30 ⁇ m to 100 ⁇ m.
- the macrovoid layer has a partition coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition and the surface layers A and B, and the partition of the macrovoid layer and the surface
- the thickness of the layers A and B is 0.01 to 20 ⁇ m, and the pores in the surface layers A and B communicate with the macrovoids, the total film thickness is 5 to 500 ⁇ m, and the porosity is 40% or more Less than 95%, Is mer porous membrane.
- at least one partition in the macrovoid layer communicates adjacent macrovoids with one or more pores, preferably having an average pore diameter of 0.01 to 100 ⁇ m, preferably 0.01 to 50 ⁇ m. Have.
- the septum does not have such holes.
- the polymeric porous membrane used in the present invention is preferably sterile.
- 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 structural features, but is preferably a polyimide porous membrane or a polyethersulfone (PES) porous membrane.
- PES polyethersulfone
- polyimide is a generic term for a polymer containing imide bond in the repeating unit, and usually means an aromatic polyimide in which an aromatic compound is directly linked by imide bond.
- Aromatic polyimides have a conjugated structure between an aromatic and an aromatic group via an imide bond, so that they have a rigid and rigid molecular structure and a very high level of heat because the imide bond has a strong intermolecular force. Have mechanical, mechanical and chemical properties.
- the polyimide porous membrane which can be used in the present invention is preferably a polyimide porous membrane containing (as a main component) a polyimide obtained from tetracarboxylic acid dianhydride and diamine, more preferably tetracarboxylic acid dianhydride. It is a polyimide porous membrane which consists of polyimide obtained from a substance and diamine. “Contained as a main component” means that components other than the polyimide obtained from tetracarboxylic acid dianhydride and diamine may be essentially not contained or contained as a component of the polyimide porous membrane. It is an additional component that does not affect the properties of the polyimide obtained from tetracarboxylic acid dianhydride and diamine.
- the polyimide porous membrane which can be used in the present invention is formed at 250 ° C. after forming 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 membrane obtained by heat processing above is also included.
- the polyamic acid is obtained by polymerizing a tetracarboxylic acid component and a diamine component.
- Polyamic acid is a polyimide precursor that can be closed to a polyimide by thermal imidization or chemical imidization.
- the polyamic acid 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.
- fine particles such as an imidation catalyst, an organic phosphorus-containing compound, inorganic fine particles, organic fine particles and the like can be added to the polyamic acid solution, as necessary.
- fine particles such as a chemical imidization agent, a dehydrating agent, inorganic fine particles, organic fine particles and the like can be added to the polyamic acid solution, as necessary. It is preferable to carry out on the conditions which a coloring precursor does not precipitate, even if it mix
- colored precursor means a precursor that is partially or wholly carbonized by heat treatment at 250 ° C. or higher to form a colored product.
- the coloring precursor which 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, and is 250 ° C. or more, preferably 260 ° C. or more, more preferably 280 ° C. or more, more preferably Is thermally decomposed and carbonized by heat treatment at 300 ° C. or higher, preferably 250 ° C. or higher, preferably 260 ° C. or higher, more preferably 280 ° C. or higher, more preferably 300 ° C. or higher in the presence of oxygen such as air.
- Those which produce a colored product are preferable, those producing a black colored product are more preferable, and the carbon-based colored precursor is more preferred.
- the colored precursor appears to be carbonized at first glance when heated, but systematically contains foreign elements other than carbon and has a layered structure, an aromatic crosslinked structure, and a disordered structure including tetrahedral carbon Including.
- the carbon-based coloring precursor is not particularly limited, and examples thereof include tars such as petroleum tar, petroleum pitch, coal tar, coal pitch and the like, pitch, coke, polymers obtained from monomers including acrylonitrile, ferrocene compounds (ferrocene and ferrocene derivatives) Etc.
- tars such as petroleum tar, petroleum pitch, coal tar, coal pitch and the like
- pitch coke
- polymers obtained from monomers including acrylonitrile, ferrocene compounds (ferrocene and ferrocene derivatives) Etc are preferable, and as polymers obtained from monomers containing acrylonitrile, polyacrylonitrile is preferable.
- the polyimide porous membrane that can be used in the present invention is formed from a polyamic acid solution obtained from the tetracarboxylic acid component and the diamine component without using the above-mentioned colored precursor.
- a polyimide porous membrane obtained by heat treatment is also included.
- the polyimide porous membrane produced without using a coloring precursor is, for example, composed of 3 to 60% by mass of polyamic acid having an intrinsic viscosity of 1.0 to 3.0 and 40 to 97% by mass of an organic polar solvent.
- the resulting polyamic acid solution is cast into a film and dipped or brought into contact with a coagulation solvent containing water as an essential component to prepare a porous film of polyamic acid, and then the porous film of polyamic acid is heat-treated to form an imide
- the coagulation solvent containing water as an essential component is water, or a mixed liquid of 5% by mass or more and less than 100% by mass water and an organic polar solvent of more than 0% by mass and 95% by mass or less May be
- at least one surface of the obtained porous polyimide film may be subjected to plasma treatment.
- Arbitrary tetracarboxylic acid dianhydride can be used for tetracarboxylic acid dianhydride which may be used in manufacture of the said polyimide porous membrane, According to a desired characteristic etc., it can select suitably.
- tetracarboxylic acid dianhydride pyromellitic acid dianhydride, 3,3 ′, 4,4′-biphenyltetracarboxylic acid dianhydride (s-BPDA), 2,3,3 ′, 4 ′ -Biphenyltetracarboxylic acid dianhydride (a-BPDA) and the like
- biphenyltetracarboxylic acid dianhydride such as oxydiphthalic acid dianhydride, diphenyl sulfone-3,4,3 ', 4'-tetracarboxylic acid dianhydride, bis (3,4-Dicarboxyphenyl) sulfide dianhydride, 2,2-bis (3,4-
- At least one aromatic tetracarboxylic acid dianhydride selected from the group consisting of biphenyltetracarboxylic acid dianhydride and pyromellitic acid dianhydride is particularly preferable.
- the biphenyltetracarboxylic acid dianhydride 3,3 ', 4,4'-biphenyltetracarboxylic acid dianhydride can be suitably used.
- any diamine can be used as diamine which may be used in manufacture of the above-mentioned polyimide porous membrane.
- the following can be mentioned as specific examples of the diamine.
- 1) benzene benzene with one benzene nucleus such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, 2,6-diaminotoluene, etc .
- Diaminodiphenyl ether 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'-diaminobi
- the diamine to be used can be suitably selected according to a desired characteristic etc.
- aromatic diamine compounds are preferable, and 3,3′-diaminodiphenyl ether, 3,4′-diaminodiphenyl ether, 4,4′-diaminodiphenyl ether and paraphenylene diamine, 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 suitably used.
- at least one diamine selected from the group consisting of benzenediamine, diaminodiphenylether and bis (aminophenoxy) phenyl is preferable.
- the polyimide porous membrane that can be used in the present invention has a glass transition temperature of 240 ° C. or higher, or a tetracarboxylic acid having no clear transition point at 300 ° C. or higher, from the viewpoint of heat resistance and dimensional stability at high temperatures. It is preferable that it is formed from the polyimide obtained by combining acid dianhydride and diamine.
- the polyimide porous membrane which can be used in the present invention is preferably a polyimide porous membrane made of the following aromatic polyimide from the viewpoint of heat resistance and dimensional stability under high temperature.
- An aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and an aromatic diamine unit
- 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 diaminodiphenylether unit and a bis (aminophenoxy) phenyl unit, And / or (Iii) at least one group 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, a benzenediamine unit
- the polyimide 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.
- the average pore diameter of the pores present in the surface layer A is 0.01 ⁇ m to 25 ⁇ m, and the average pore diameter of the pores present in the surface layer B is 30 ⁇ m to 100 ⁇ m.
- the macrovoid layer has a partition coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition and the surface layers A and B, and the partition of the macrovoid layer and the surface
- the thickness of the layers A and B is 0.01 to 20 ⁇ m, and the pores in the surface layers A and B communicate with the macrovoids, the total film thickness is 5 to 500 ⁇ m, and the porosity is 40% or more Less than 95% A polyimide porous film.
- at least one partition wall in the macrovoid layer has one or more pores having an average pore diameter of 0.01 to 100 ⁇ m, preferably 0.01 to 50 ⁇ m, which communicate adjacent macrovoids with each other.
- polyimide porous membrane described in WO 2010/038873, JP 2011-219585, or JP 2011-219586 can also be used in the present invention.
- PES porous membranes that can be used in the present invention include polyethersulfone, and typically consist essentially of polyethersulfone.
- the polyether sulfone may be one synthesized by a method known to those skilled in the art, for example, a method of subjecting a dihydric phenol, an alkali metal compound and a dihalogeno diphenyl compound to a polycondensation reaction in an organic polar solvent, a dihydric phenol
- the alkali metal disalt can be prepared beforehand by a method of polycondensation reaction in an organic polar solvent with a dihalogeno diphenyl compound and the like.
- alkali metal compound examples include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides and alkali metal alkoxides.
- sodium carbonate and potassium carbonate are preferred.
- dihydric phenol compounds examples include hydroquinone, catechol, resorcin, 4,4'-biphenol, bis (hydroxyphenyl) alkanes (eg, 2,2-bis (hydroxyphenyl) propane, and 2,2-bis (hydroxyphenyl) Methane), dihydroxydiphenyl sulfones, dihydroxydiphenyl ethers, or at least one hydrogen of their benzene rings is a lower alkyl group such as methyl, ethyl or propyl, or a lower alkoxy group such as methoxy or ethoxy What is substituted is mentioned.
- the above-mentioned compound can be used in mixture of 2 or more types.
- the polyether sulfone may be a commercially available product.
- 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 polyethersulfone is preferably 0.5 or more, more preferably 0.55 or more from the viewpoint of favorably forming the macrovoids of the porous polyethersulfone membrane, and the production 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.
- the PES porous membrane or 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. Preferably not observed.
- the manufacturing method of the PES porous membrane which can be used in the present invention is not particularly limited, for example, Polyether sulfone solution containing 0.3% by mass to 60% by mass of polyethersulfone with a logarithmic viscosity of 0.5 to 1.0 and 40% by mass to 99.7% by mass of organic polar solvent is cast as a film And immersing or bringing into contact with a coagulating solvent comprising a poor solvent or non-solvent of polyether sulfone as an essential component to produce a coagulated membrane having pores, and the coagulated membrane having pores obtained in the above step The heat treatment is carried out to coarsen the pores to obtain a PES porous film, and the heat treatment is carried out until the coagulation film having the pores is higher than the glass transition temperature of the polyether sulfone or 240 ° C. or higher. It may be manufactured by a method including heating.
- the PES porous membrane that can be used in the present invention is preferably a PES porous membrane having a surface layer A, a surface layer B, and a macrovoid layer sandwiched between the surface layer A and the surface layer B.
- the macrovoid layer is composed of partition walls 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 having an average pore diameter in the film plane direction of 10 ⁇ m to 500 ⁇ m.
- the partition wall of the macro void layer has a thickness of 0.1 ⁇ m to 50 ⁇ m
- the surface layers A and B each have a thickness of 0.1 ⁇ m to 50 ⁇ m
- One of the surface layers A and B has a plurality of pores with an average pore diameter of 5 ⁇ m to 200 ⁇ m and the other has a plurality of pores with an average pore diameter of 0.01 ⁇ m or more and less than 200 ⁇ m.
- One surface aperture ratio of the surface layer A and the surface layer B is 15% or more, and the surface aperture ratio of the other surface layer is 10% or more.
- the pores of the surface layer A and the surface layer B are in communication with the macrovoids
- the PES porous membrane has a total film thickness of 5 ⁇ m to 500 ⁇ m and a porosity of 50% to 95%. PES porous membrane.
- Cell culture module refers to a cell culture substrate applicable to a cell culture vessel and a cell culture apparatus.
- the cell culture vessel and the cell culture apparatus which can use the cell culture module of the present invention are not particularly limited, and can be used, for example, in commercially available cell culture vessels and cell culture apparatuses.
- it can be used for a culture apparatus provided with a culture container consisting of a flexible bag, and can be used while being suspended in the culture container.
- a culture container can be applied to a stirred culture type container such as a spinner flask and cultured in cells.
- a culture container it is applicable also to an open container and a closed container. For example, petri dishes for cell culture, flasks, plastic bags, test tubes, and large tanks can be suitably used.
- a sterile bottle for example, a simple columnar container such as a storage bottle manufactured by Corning Co., can be efficiently used as a culture container by using a shaking device such as an orbital shaker or a program shaker.
- a shaking device such as an orbital shaker or a program shaker.
- petri dishes and flasks can also be used as culture vessels by using a shaker.
- the components of the cell culture module of the present invention except the polymer porous membrane preferably have a strength not to be deformed by the movement of the culture solution under normal culture conditions, for example, stirring culture and shaking culture conditions.
- it is formed of a flexible material.
- the said structural member is formed with the raw material which does not affect the growth of a cell in cell culture.
- materials include, for example, polyolefin (eg, polyethylene, polypropylene), nylon, polyester, polystyrene, polycarbonate, polymethyl methacrylate, polymers such as polyethylene terephthalate, polyethylene terephthalate, metals such as stainless steel, titanium, etc. I will not.
- One aspect of the cell culture module of the present invention is At the top, At the bottom, A side with a culture fluid outlet and A plurality of compartments having a culture fluid inlet / outlet which separates the space formed by the top, bottom and sides; Two or more selected from a gap space between the top and the adjacent partition, a gap space between the bottom and the adjacent partition, and a plurality of gap spaces between the adjacent partitions A porous polymer membrane fixed to each of the interstitial spaces; A cell culture module comprising
- the polymer porous membrane has a three-layer 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.
- Porous film wherein the average pore diameter of the pores present in the surface layer A is smaller than the average pore diameter of the pores present 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.
- It is a cell culture module.
- cell culture module A it is also referred to as "cell culture module A".
- the "divider” in the cell culture module of the present invention is substantially planar.
- the “top” in the cell culture module of the present invention is a portion present at the top in the direction substantially perpendicular to the extending direction of the partition, and the shape thereof is not particularly limited.
- the “bottom portion” in the cell culture module of the present invention is a portion located at the lowermost portion in the direction substantially perpendicular to the extending direction of the partition portion, and the shape thereof is not particularly limited.
- the “side” is a part other than the top and the bottom, which constitutes the periphery of the cell culture module, and the shape thereof is not particularly limited.
- the top and bottom of the cell culture module A may have a culture fluid outlet port.
- the number and shape of the culture medium outlet located at the top and bottom are not particularly limited as long as the culture medium is well supplied to the inside of the module. Also, the size is not particularly limited as long as it does not prevent the passage of culture cells and culture solution.
- the top and bottom each have a plurality of culture medium flow inlets.
- the side of the cell culture module A has a culture fluid outlet port.
- the number and shape of the culture solution outlet ports present on the side are not particularly limited as long as the culture solution is well supplied to the inside of the module. Also, the size is not particularly limited as long as it does not prevent the passage of culture cells and culture solution.
- the side has a plurality of culture medium flow inlets.
- the number of partition parts of the cell culture module A is not particularly limited, but is preferably 2 to 50, more preferably 2 to 30, still more preferably 2 to 20, and particularly preferably 2 to 10 It is a sheet.
- the number and shape of the culture medium flow inlet / outlet which the partition has are not particularly limited as long as they do not hinder good movement of the culture medium. Also, the size is not particularly limited as long as it does not prevent the passage of culture cells and culture solution.
- the method for fixing the polymer porous membrane to the interstitial space is not particularly limited. For example, they are fixed by being held between the top and the adjacent partition, between the bottom and the adjacent partition, and between the adjacent partitions. Also, for example, at least one location of the polymer porous membrane is fixed by at least one location of the top, the bottom or the partition forming the gap space by any method (for example, adhesion with an adhesive or fixation with a fastener) Be done.
- any method for example, adhesion with an adhesive or fixation with a fastener
- the polymer porous membrane can be fixed to the interstitial space in any form.
- the polymeric porous membrane is a laminate of a plurality of polymeric porous membranes.
- the polymer porous membrane to be laminated may be small pieces, and the shape may be any shape such as, for example, circle, oval, square, triangle, polygon, or string.
- the pieces of polymeric porous membrane to be laminated are substantially square.
- the size of the pieces can be of any size. When the small pieces are substantially square, the length is not particularly limited, and for example, 80 mm or less, 50 mm or less, 30 mm or less, 20 mm or less, or 10 mm or less.
- the polymer porous membrane fixed to the gap space is a laminate of a plurality of polymer porous membranes, preferably, it is two or more, three or more, four or more or five or more, and 100 sheets
- the following is a laminate of 50 or less, 40 or less, 30 or less, 20 or less, 15 or less or 10 or less polymer porous membranes, more preferably 3 to 100, more preferably 5 to It is a laminate of 50 polymer porous membranes.
- an insole may be provided between the polymer porous membrane and the polymer porous membrane.
- the insole is not particularly limited as long as it has an arbitrary space between the laminated polymer porous membranes and has a function of efficiently supplying the culture solution, but, for example, a planar structure having a mesh structure It can be used.
- the material of the insole may be, for example, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, a mesh made of stainless steel, but is not limited thereto. When it has an insole having a mesh structure, it may be selected appropriately as long as it has an opening enough to supply the culture solution between the laminated polymer porous membranes.
- the polymer porous membrane fixed to the interstice space may be used by processing its shape into a three-dimensional shape instead of a planar shape.
- a porous polymer membrane may be used i) folded, ii) rolled up in a roll, iii) used to connect sheets or pieces with a threadlike structure, or iv) tie in a rope Good.
- the overall shape of the cell culture module A is not particularly limited. By changing the shapes of the members constituting the top, bottom, sides and partition, cell culture modules of various overall shapes can be manufactured.
- the corners of the module may be chamfered in order to soften the impact at the time of mutual collision of the modules and to avoid destruction of the module.
- At least one of the interstitial space adjacent to the interstitial space to which the polymeric porous membrane is fixed does not include the polymeric porous membrane.
- the polymer porous membrane is contained in the space because there is only one adjacent gap space. I can not.
- the porous polymer membrane is fixed in the gap space between the bottom and the adjacent partition part, there is only one adjacent gap space, so the polymer porous membrane is Not included
- the polymer porous membrane is not included in both of them?
- the polymer porous membrane is not included in one or the other.
- the interstice space not containing the polymer porous membrane functions as a culture solution passage clearance.
- the culture solution is supplied from the culture solution outlet port to the culture solution passage clearance present inside the cell culture module A.
- the culture solution supplied to the culture solution passage clearance can efficiently contact the porous polymer membrane through the culture solution outlet of the partition.
- the cell culture module A has a top, a partition and a bottom to provide a clearance space between the top and the partition adjacent thereto, between the bottom and the partition adjacent thereto, and between the adjacent partitions. May be provided with placement means for placing the at a constant distance.
- the shape of the arrangement means is not particularly limited, and may be appropriately determined according to the configuration of the cell culture module.
- it may be a mounting base of an adjacent partition part provided in the partition part as shown in FIGS. 3 and 4.
- it may be a stage provided on the side inner wall of the container formed by the bottom and the side, for placing the partition and the top.
- it may be a (for example, columnar) connecting member for connecting each of the members constituting the top, the bottom and the partition.
- the cell culture module 1 includes a top 2, a bottom 3, a side 4, a first partition 5, and a second partition 6.
- the polymer porous membrane laminates 7 a and 7 b are sandwiched between the top 2 and the first partition 5 of the cell culture module 1 and between the bottom 3 and the second partition 6.
- the top 2, bottom 3, side 4, first partition 5 and second partition 6 of the cell culture module 1 respectively have culture medium outflow ports 8 a to 8 e.
- the upper surface of the second partition 6 includes a mounting table 9 for mounting the adjacent first partition 5.
- the mounting table 9 functions as an arrangement means for arranging the first partition 5 and the second partition 6 at a constant interval.
- a gap space in which the porous polymer membrane is not disposed is formed between the second partition 6 and the first partition 5 by the mounting table 9.
- the clearance space functions as a culture solution passage clearance.
- the culture fluid supplied to the culture fluid passage clearance from the culture fluid outlet / inlet present in each of the top 2, the bottom 3 and the side 4 is a culture existing in each of the first partition 5 and the second partition 6 It is possible to pass through the liquid outlet 8 and be in efficient contact with the polymer porous membrane laminates 7a and 7b.
- the number, position and shape of the mounting tables are not particularly limited.
- the mounting table may be provided on the lower surface of the adjacent first partition 5 instead of the upper surface of the second partition 6.
- the mounting table may be provided on the side inner wall of the container formed by the bottom 3 and the side 4.
- FIGS. 5 and 6 show a configuration example of a cell culture module complex in which two rectangular parallelepiped cell culture modules are linked.
- the cell culture module complex 10 of FIGS. 5 and 6 is manufactured by preparing two cell culture modules 1 shown in FIGS. 1 to 4 and connecting one top to the other, and has a cubic shape. .
- Four clearance spaces selected from a clearance space between the top and the adjacent division, a clearance space between the bottom and the adjacent division, and a plurality of clearance spaces between the adjacent divisions A porous polymer membrane, fixed to each of Equipped with At least one of the interstitial spaces adjacent to the interstitial space to which the polymeric porous membrane is fixed does not include the polymeric porous membrane, It can be considered as a cube-shaped cell culture module. Therefore, the cell culture module complex can be regarded as one embodiment of the cell culture module A.
- a cell culture module comprising: a cell culture submodule housing casing having a culture solution outlet and inlet for stacking and housing the plurality of cell culture submodules,
- the cell culture submodule is Polymer porous membrane, A polymer porous membrane-containing casing having a culture fluid outlet / inlet, wherein the polymer porous membrane-containing casing is fixed and accommodated;
- the polymer porous membrane is a three-layered polymer porous membrane 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 diameter of the pores present in the surface layer A is smaller than the average pore diameter of the pores present in the surface layer B
- the macrovoid layer is a partition bonded to the surface layers A and B;
- cell culture module B it is also referred to as "cell culture module B".
- the method of fixing the polymer porous membrane in the polymer porous membrane-containing casing is not particularly limited. For example, it is fixed by being pinched between the top and the bottom of the casing. Also, for example, at least one location of the polymer porous membrane is fixed to at least one location in the casing by any method (for example, adhesion with an adhesive or fixation with a fastener).
- the form of the porous polymer membrane accommodated in the casing is not particularly limited.
- the polymeric porous membrane is a laminate of a plurality of polymeric porous membranes.
- the polymer porous membrane to be laminated may be small pieces, and the shape may be any shape such as, for example, circle, oval, square, triangle, polygon, or string.
- the pieces of polymeric porous membrane to be laminated are substantially square.
- the size of the pieces can be of any size.
- the length is not particularly limited, and for example, 80 mm or less, 50 mm or less, 30 mm or less, 20 mm or less, or 10 mm or less.
- the polymer porous membrane housed in the casing is a laminate of a plurality of polymer porous membranes, preferably, it is 2 or more, 3 or more, 4 or more or 5 or more, and 100 or less 50 or less, 40 or less, 30 or less, 20 or less, 15 or less or 10 or less polymer porous membranes, more preferably 3 to 100, particularly preferably 5 to It is a laminate of 50 polymer porous membranes.
- an insole may be provided between the polymer porous membrane and the polymer porous membrane. By providing the insole, the culture solution can be efficiently supplied between the laminated polymer porous membranes.
- the insole is not particularly limited as long as it has an arbitrary space between the laminated polymer porous membranes and has a function of efficiently supplying the culture solution, but, for example, a planar structure having a mesh structure It can be used.
- the material of the insole may be, for example, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, a mesh made of stainless steel, but is not limited thereto. When it has an insole having a mesh structure, it may be selected appropriately as long as it has an opening enough to supply the culture solution between the laminated polymer porous membranes.
- the polymer porous membrane housed in the casing may be used by processing its shape into a three-dimensional shape instead of a planar shape.
- a porous polymer membrane may be used i) folded, ii) rolled up in a roll, iii) used to connect sheets or pieces with a threadlike structure, or iv) tie in a rope Good.
- the polymer porous membrane-containing casing for containing the polymer porous membrane has a culture fluid outlet port.
- the cell culture fluid is supplied to the inside of the casing and discharged to the outside by the outflow port.
- the number and shape of the culture medium outflow inlet are not particularly limited. Also, the size is not particularly limited as long as it does not prevent the passage of culture cells and culture solution.
- the culture medium inflow port in the polymer porous membrane-containing casing may have a mesh-like structure.
- the casing itself containing the polymer porous membrane may be mesh-like.
- the mesh-shaped structure for example, has a vertical, horizontal, and / or diagonal lattice-like structure, and each opening forms a culture fluid outflow inlet to the extent that fluid can pass through.
- it is not limited to this.
- a plurality of cell culture submodules are accommodated in a cell culture submodule accommodating casing.
- the number of cell culture submodules is preferably 2 or more, 3 or more, 4 or more or 5 or more, and 30 or less, 15 or less, 10 or less, and more preferably 2 to 15, Particularly preferably, 3 to 10 are stacked.
- the cell culture submodule housing casing has a culture fluid outlet port.
- the cell culture fluid is supplied to the inside of the casing and discharged to the outside by the outflow port.
- the number and shape of the culture medium outflow inlet are not particularly limited.
- the size is not particularly limited as long as it does not prevent the passage of culture cells and culture solution.
- the cell culture submodule housing casing has a plurality of culture medium flow inlets and outlets.
- the cell culture module B preferably has a clearance space between the stacked plurality of cell culture submodules and the cell culture submodule housing casing.
- the clearance space functions as a culture solution passage clearance.
- the culture solution is supplied from the culture solution outlet / inlet present in the cell culture submodule housing casing to the culture solution passage clearance present in the cell culture module B.
- the culture solution supplied to the culture solution passage clearance can efficiently contact the polymer porous membrane by passing through the culture solution outlet port present in the polymer porous membrane-containing casing.
- FIG. 7 and 8 show a configuration example of the cell culture module B.
- cell culture submodules 30 are stacked.
- the laminate is accommodated in the cell culture submodule accommodating casing 21.
- the laminate is sandwiched between the top and the bottom of the cell culture submodule housing casing 21.
- the cell culture submodule housing casing 21 has culture medium flow inlets 8f and 8g on the top and sides thereof.
- the cell culture submodule housing casing 21 also has a culture solution outflow port at its bottom.
- a clearance space 22 exists between the stacked plurality of cell culture submodules 30 and the cell culture submodule housing casing 21.
- the clearance space functions as a culture solution passage clearance.
- FIGS. 9 and 10 show a configuration example of a cell culture submodule which is a component of the cell culture module 20 shown in FIGS. 7 and 8.
- FIG. Six pieces of one set of square polymer porous membrane laminates 7c, 7d and 7e, between the polymer porous membrane laminates 7c and 7d, and between the polymer porous membrane laminates 7d and 7e
- the insoles 32a and 32b are accommodated in the polymer porous membrane accommodating casing 31, respectively.
- the upper surface of the polymer porous membrane-containing casing 31 has a plurality of culture solution outflow inlets 8 h.
- the lower surface also has a culture solution outlet port.
- the cell culture submodules shown in FIGS. 9 and 10 can be manufactured by sandwiching a stack of polymer films between two flat plate substrates and then fixing the peripheral edge of the flat substrate.
- the cell culture module of the present invention shown in FIGS. 1 to 8 has excellent liquid flowability inside the module, and can supply oxygen and nutrients well to the polymer porous membrane accommodated in the module.
- the entire module has a high floating force and can efficiently stir a plurality of modules.
- homogeneous culture conditions can be applied to the polymer porous membrane inside the module.
- the polymer porous since the polymer porous is fixed, it is prevented that stress is applied to the cells grown in the polymer porous membrane, apoptosis and the like are suppressed, and stable and large amounts of cells can be cultured. It is possible.
- the cell culture module of the present invention has high strength and is advantageous for long-term cell culture and long-term continuous substance production by cultured cells.
- the polyimide porous membrane used in the following Examples and Comparative Examples is a tetracarboxylic acid component, 3,3 ', 4,4'-biphenyltetracarboxylic acid dianhydride (s-BPDA) and a diamine component. It is prepared by forming a polyamic acid solution composition containing a polyamic acid solution obtained from 4,4'-diaminodiphenyl ether (ODA) and polyacrylamide which is a coloring precursor, followed by heat treatment at 250 ° C. or higher.
- ODA 4,4'-diaminodiphenyl ether
- the obtained polyimide porous membrane has a three-layered 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 membrane was a membrane, the average pore size of the pores present in the surface layer A was 19 ⁇ m, the average pore size of the pores present in the surface layer B was 42 ⁇ m, the film thickness was 25 ⁇ m, and the porosity was 74% .
- Example 1 Cell Culture Using Cell Culture Module Complex 10 Cell culture was performed using cell culture module complex 10 shown in FIGS. 5 and 6. Two cell culture modules 1 shown in FIGS. 1 to 4 were prepared, the tops of the respective modules were facing each other, and they were heat-sealed and connected by soldering iron to prepare a cube-shaped cell culture module complex 10.
- the size of the polyimide porous membrane used in this example is 1.4 cm ⁇ 1.4 cm.
- the cell culture module complex 10 has 10 sheets and 4 sets of polyimide porous membrane laminates, and the total area is 80 cm 2 .
- the cell culture module complex 10 was washed with diluted Milton solution (manufactured by Kyorin Pharmaceutical Co., Ltd.), ultrapure water, water containing 70% ethanol, and used after being sterilized and dried.
- Cells conditioned and suspended with anti-human IL-8 antibody-producing CHO-DP12 cells are suspended in culture medium (BalanCDTM CHO Growth A), and the number of viable cells per 1 ml is 3 .51 ⁇ 10 6 cells / ml, the culture was continued until the (total cell number 3.83 ⁇ 10 6 cells / ml, 92% cell viability).
- Three cell culture module complexes 10 were added to a bottle-type sterilization container (manufactured by Corning) having an inner volume of 150 ml, and 53.3 ml of CHO cell monolayer culture medium KBM 270 (manufactured by Kojin Bio Inc.) was added thereto.
- the sample was immersed in a CO 2 incubator for 10 minutes at a shaking speed of 35 rpm using a program shaker (Kenith).
- CHO DP-12 suspension cell culture solution total cell number 3.83 ⁇ 10 6 cells / ml, living cell number 3.51 ⁇ 10 6 cells / ml, dead cell number 3.23 ⁇ 10 5 cells / ml, living cells Add a mixture of 4.0 mL of 92%) and 22.6 mL of culture medium for floating cells (BalanCDTM CHO Growth A), and rotate for about 14 hours using a program shaker (Kenith Co., Ltd.) under rotation conditions of 35 rpm. Cells were adsorbed (assumed average cell adsorption number per sheet 5.30 ⁇ 10 4 cells). The cell adsorption rate calculated from the recovered medium was 95%.
- CHO cell monolayer culture medium KBM270 (manufactured by Kojin Bio Co., Ltd.) was added to start culture.
- the container was transferred to an electromagnetic orbital shaker (for CO 2 incubator, manufactured by As One), and culture was continued at a shaking speed of 200 rpm.
- the medium was replaced daily, and the glucose consumption, lactate production, lactate dehydrogenase and antibody production per day in the culture medium were measured using Cedex Bio (manufactured by Roche Diagnostics). It was confirmed that glucose was consumed over time, and antibody and lactic acid were continuously produced.
- the amount of glucose consumption and the amount of lactic acid production in 3 days at the start of culture are shown in Table 1. It was confirmed that culture was stably performed.
- Example 2 Cell Culture Using Cell Culture Module 20 Cell culture was performed using cell culture module 20 shown in FIGS.
- the cell culture module 20 was used after being washed with diluted Milton solution (manufactured by Kyorin Pharmaceutical Co., Ltd.), ultrapure water, water containing 70% ethanol, and sterilized and dried.
- the size of the polyimide porous membrane used in the cell culture module 20 used in the present example is 1.0 cm ⁇ 1.0 cm.
- Three sets of the laminated body which made one set of six said polyimide porous membranes concerned were prepared, and an insole was held between each.
- the total number of polyimide porous membranes in the cell culture module 20 used in this example is 108, and the total area is 108 cm 2 .
- the total number of polyimide porous membranes is 540, and the total area is 540 cm 2 .
- the materials of the cell culture sub-module housing casing and the polymer porous membrane housing casing of the cell culture module 20 used are all polyolefin resins.
- the insole used is NBC Meshtec mesh.
- Cells conditioned and suspended with anti-human IL-8 antibody-producing CHO-DP12 cells are subjected to suspension culture using culture medium (BalanCDTM CHO Growth A), and the number of viable cells per 1 ml is The culture was continued until it reached 5.41 ⁇ 10 6 cells / ml (total cell number 6.16 ⁇ 10 6 cells / ml, 88% viable cell rate).
- sterile cell culture modules 20 Five sterile cell culture modules 20 were added to a bottle-type sterilization container (manufactured by Corning) having an inner volume of 150 ml, and 26.7 ml of CHO cell monolayer culture medium KBM 270 (manufactured by Kojin Bio Inc.) was added thereto. In a CO 2 incubator, the plate was immersed for 10 minutes at a shaking speed of 35 rpm using a program shaker (Kenith).
- CHO DP-12 floating cell culture solution (total cell number 6.16 ⁇ 10 6 cells / ml, viable cell number 5.41 ⁇ 10 6 cells / ml, dead cell number 7.51 ⁇ 10 5 cells / ml, A mixed solution of 6.0 mL of viable cell rate 88%) and 7.3 mL of suspension cell culture medium (BalanCDTM CHO Growth A) is added, and 35 rpm using a program shaker (Kenith) for about 14 hours.
- the cells were adsorbed (rotational cell number 5.30 ⁇ 10 4 cells per sheet) under the following rotation conditions.
- the cell adsorption rate calculated from the collected medium was 88%.
- CHO cell monolayer culture medium KBM270 (manufactured by Kojin Bio Co., Ltd.) was added to start culture.
- the container was transferred to an electromagnetic orbital shaker (for CO 2 incubator, manufactured by As One), and culture was continued at a shaking speed of 200 rpm.
- the medium was replaced daily, and the glucose consumption, lactate production, lactate dehydrogenase and antibody production per day in the culture medium were measured using Cedex Bio (manufactured by Roche Diagnostics). It was confirmed that glucose was consumed over time, and antibody and lactic acid were continuously produced. The time course of antibody production is shown in FIG.
- Comparative Example 1 Thirty cell culture submodules 30 shown in FIGS. 9 and 10 were prepared and sterilized. The total number of polyimide porous membranes is 540, and the total area is 540 cm 2 . Add 26.7 mL of CHO cell monolayer culture medium KBM 270 (manufactured by Kojin Bio Inc.) to a 150 mL storage bottle (manufactured by Corning) filled with the submodule and program shaker (Kenith) for 10 minutes in a CO 2 incubator ) Under the rotation conditions of 35 rpm.
- KBM 270 manufactured by Kojin Bio Inc.
- CHO DP-12 suspension cell culture solution total cell number 6.16 ⁇ 10 6 cells / ml, viable cell number 5.41 ⁇ 10 6 cells / ml, dead cell number 7.51 ⁇ 10 5 cells / ml, viable cells
- a mixture of 6.0 mL of the suspension cell and 7.3 mL of the suspension cell culture medium (BalanCDTM CHO Growth A) was added, and the cells were allowed to adsorb under rotation conditions of 35 rpm for about 14 hours (one sheet). Cell adsorption number per 5.83 ⁇ 10 4 cells). The cell adsorption rate calculated from the number of cells found from the recovery medium was 97%.
- the medium was replaced daily, and the daily glucose consumption, lactate production, lactate dehydrogenase, and antibody production in the culture medium were measured using Cedex Bio (manufactured by Roche Diagnostics) ( Figure 11).
- the antibody production amount was higher in the case of using a cell culture module (Example 2).
- Comparative example 2 Cells conditioned and suspended with anti-human IL-8 antibody-producing CHO-DP12 cells (ATCC CRL-12445) were suspended in culture using a medium (BalanCDTM CHO GROWTH A), and the number of cells per 1 ml was The culture was continued to 2.0 ⁇ 10 6 .
- culture 1 the number of cells on the polyimide porous membrane in three petri dishes cultured using CCK8 (Cell Countinig Kit 8; solution reagent manufactured by Dojin Chemical Research Institute) was calculated as the number of cells per area. . The next day, one of the three petri dishes was allowed to continue the culture for another 2 days (hereinafter referred to as "culture 1").
- the polyimide porous membrane on which the cells were grown was transferred to an oxygen-permeable culture bag (manufactured by Nipro) together with the culture solution, and was sterilely sealed with a heat sealer. Thereafter, setting was made so as to cause 20 to 30 swings per minute on a shaker placed in a CO 2 incubator, and shaking culture was carried out for 2 days (hereinafter referred to as “culture 2”).
- the polyimide porous membrane contained in one remaining petri dish was sterilely cut into pieces of about 0.3 cm ⁇ 0.3 cm with scissors. Thereafter, an outer shell of 30 # nylon mesh having a size of about 1 cm ⁇ 1 cm was prepared, and the chopped polyimide porous membrane was accommodated therein, and was sterilized and sealed with a heat sealer.
- the obtained cell culture module was transferred together with the culture medium to an oxygen permeation type culture bag (manufactured by Nipro), and sterilized with a heat sealer. Thereafter, setting was made so as to cause 20 to 30 swings per minute on a shaker placed in a CO 2 incubator, and shaking culture was performed for 2 days (hereinafter referred to as “culture 3”).
- the cell density after performing culture 1 to 3 was calculated using CCK8. The results are shown in Table 2.
- a significant decrease in the number of cells was observed in culture 2 which was shake-cultured without fixing the porous polyimide membrane. This is considered to be because stress is applied to the cells grown in the polyimide porous membrane to continuously deform the morphology of the polyimide porous membrane, and the cells show by apoptosis and the like.
- culture 3 in which shake culture was performed in a state where the polyimide porous membrane was accommodated and fixed in the mantle, the decrease in the number of cells observed in culture 2 was suppressed.
- the cell culture module of the present invention can be suitably used for stable cell culture and substance production.
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Abstract
Description
ポリイミド多孔質膜は、本出願前よりフィルター、低誘電率フィルム、燃料電池用電解質膜など、特に電池関係を中心とする用途のために利用されてきた。特許文献2~4は、特に、気体などの物質透過性に優れ、空孔率の高い、両表面の平滑性が優れ、相対的に強度が高く、高空孔率にもかかわらず、膜厚み方向への圧縮応力に対する耐力に優れるマクロボイドを多数有するポリイミド多孔質膜を記載している。これらはいずれも、アミック酸を経由して作成されたポリイミド多孔質膜である。
<1>
頂部と、
底部と、
培養液流出入口を有する側部と、
前記頂部、底部及び側部によって形成される空間を仕切る、培養液流出入口を有する複数の仕切り部と、
頂部とそれに隣接する仕切り部との間の隙間空間、底部とそれに隣接する仕切り部との間の隙間空間、及び隣接する仕切り部同士の間の複数の隙間空間から選択される、2つ以上の隙間空間のそれぞれに固定される、ポリマー多孔質膜と、
を備える、細胞培養モジュールであって、
ここで、前記ポリマー多孔質膜は、複数の孔を有する表面層A及び表面層Bと、前記表面層A及び表面層Bの間に挟まれたマクロボイド層とを有する三層構造のポリマー多孔質膜であり、ここで前記表面層Aに存在する孔の平均孔径は、前記表面層Bに存在する孔の平均孔径よりも小さく、前記マクロボイド層は、前記表面層A及びBに結合した隔壁と、当該隔壁並びに前記表面層A及びBに囲まれた複数のマクロボイドとを有し、前記表面層A及びBにおける孔が前記マクロボイドに連通している、
細胞培養モジュール。
<2>
ポリマー多孔質膜が固定されている隙間空間に隣接する隙間空間の少なくとも一方は、ポリマー多孔質膜を含まない、<1>に記載の細胞培養モジュール。
<3>
頂部とそれに隣接する仕切り部との間の隙間空間、底部とそれに隣接する仕切り部との間の隙間空間、及び隣接する仕切り部同士の間の複数の隙間空間から選択される、2以上の隙間空間のそれぞれに、3~100枚のポリマー多孔質膜が積層されて配置される、<1>又は<2>に記載の細胞培養モジュール。
<4>
前記ポリマー多孔質膜がポリイミド多孔質膜である、<1>~<3>のいずれか1つに記載の細胞培養モジュール。
<5>
前記ポリマー多孔質膜がポリエーテルスルホン多孔質膜である、<1>~<3>のいずれか1つに記載の細胞培養モジュール。
<6>
前記頂部及び底部は培養液流出入口を有する、<1>~<5>のいずれか1つに記載の細胞培養モジュール。
<7>
直方体形状を有する、<1>~<6>のいずれか1つに記載の細胞培養モジュール。
<8>
立方体形状を有する、<1>~<6>のいずれか1つに記載の細胞培養モジュール。
<9>
卵形状を有する、<1>~<6>のいずれか1つに細胞培養モジュール。
<10>
複数の<7>又は<8>に記載の細胞培養モジュールが連結されてなる、細胞培養モジュール複合体。
<11>
複数の細胞培養サブモジュールと、
前記複数の細胞培養サブモジュールを積層して収容するための、培養液流出入口を有する細胞培養サブモジュール収容用ケーシングと
を備える、細胞培養モジュールであって、
ここで、前記細胞培養サブモジュールは、
ポリマー多孔質膜と、
培養液流出入口を有するポリマー多孔質膜収容用ケーシングであって、前記ポリマー多孔質膜が固定されて収容される、前記ポリマー多孔質膜収容用ケーシングと
を備え、
前記ポリマー多孔質膜は、複数の孔を有する表面層A及び表面層Bと、前記表面層A及び表面層Bの間に挟まれたマクロボイド層とを有する三層構造のポリマー多孔質膜であり、ここで前記表面層Aに存在する孔の平均孔径は、前記表面層Bに存在する孔の平均孔径よりも小さく、前記マクロボイド層は、前記表面層A及びBに結合した隔壁と、当該隔壁並びに前記表面層A及びBに囲まれた複数のマクロボイドとを有し、前記表面層A及びBにおける孔が前記マクロボイドに連通している、
細胞培養モジュール。
<12>
積層された前記複数の細胞培養サブモジュールと、細胞培養サブモジュール収容用ケーシングとの間に隙間空間を有する、<11>に記載の細胞培養モジュール。
<13>
前記ポリマー多孔質膜収容用ケーシングに、3~100枚のポリマー多孔質膜が積層されて収容される、<11>又は<12>に記載の細胞培養モジュール。
<14>
前記ポリマー多孔質膜がポリイミド多孔質膜である、<11>~<13>のいずれか1つに記載の細胞培養モジュール。
<15>
前記ポリマー多孔質膜がポリエーテルスルホン多孔質膜である、<11>~<13>のいずれか1つに記載の細胞培養モジュール。
本発明で使用されるポリマー多孔質膜中の表面層A(以下で、「A面」又は「メッシュ面」とも呼ぶ)に存在する孔の平均孔径は、特に限定されないが、例えば、0.01μm以上200μm未満、0.01~150μm、0.01~100μm、0.01~50μm、0.01μm~40μm、0.01μm~30μm、0.01μm~25μm、0.01μm~20μm、又は0.01μm~15μmであり、好ましくは、0.01μm~25μmである。
(1)多孔質膜表面の走査型電子顕微鏡写真から、200点以上の開孔部について孔面積Sを測定し、該孔面積を真円と仮定して式Iからそれぞれの孔径dを求める。
ポリイミドとは、繰り返し単位にイミド結合を含む高分子の総称であり、通常は、芳香族化合物が直接イミド結合で連結された芳香族ポリイミドを意味する。芳香族ポリイミドは芳香族と芳香族とがイミド結合を介して共役構造を持つため、剛直で強固な分子構造を持ち、かつ、イミド結合が強い分子間力を持つために非常に高いレベルの熱的、機械的、化学的性質を有する。
1)1,4-ジアミノベンゼン(パラフェニレンジアミン)、1,3-ジアミノベンゼン、2,4-ジアミノトルエン、2,6-ジアミノトルエンなどのベンゼン核1つのべンゼンジアミン;
2)4,4’-ジアミノジフェニルエーテル、3,4’-ジアミノジフェニルエーテルなどのジアミノジフェニルエーテル、4,4’-ジアミノジフェニルメタン、3,3’-ジメチル-4,4’-ジアミノビフェニル、2,2’-ジメチル-4,4’-ジアミノビフェニル、2,2’-ビス(トリフルオロメチル)-4,4’-ジアミノビフェニル、3,3’-ジメチル-4,4’-ジアミノジフェニルメタン、3,3’-ジカルボキシ-4,4’-ジアミノジフェニルメタン、3,3’,5,5’-テトラメチル-4,4’-ジアミノジフェニルメタン、ビス(4-アミノフェニル)スルフィド、4,4’-ジアミノベンズアニリド、3,3’-ジクロロベンジジン、3,3’-ジメチルベンジジン、2,2’-ジメチルベンジジン、3,3’-ジメトキシベンジジン、2,2’-ジメトキシベンジジン、3,3’-ジアミノジフェニルエーテル、3,4’-ジアミノジフェニルエーテル、4,4’-ジアミノジフェニルエーテル、3,3’-ジアミノジフェニルスルフィド、3,4’-ジアミノジフェニルスルフィド、4,4’-ジアミノジフェニルスルフィド、3,3’-ジアミノジフェニルスルホン、3,4’-ジアミノジフェニルスルホン、4,4’-ジアミノジフェニルスルホン、3,3’-ジアミノベンゾフェノン、3,3’-ジアミノ-4,4’-ジクロロベンゾフェノン、3,3’-ジアミノ-4,4’-ジメトキシベンゾフェノン、3,3’-ジアミノジフェニルメタン、3,4’-ジアミノジフェニルメタン、4,4’-ジアミノジフェニルメタン、2,2-ビス(3-アミノフェニル)プロパン、2,2-ビス(4-アミノフェニル)プロパン、2,2-ビス(3-アミノフェニル)-1,1,1,3,3,3-ヘキサフルオロプロパン、2,2-ビス(4-アミノフェニル)-1,1,1,3,3,3-ヘキサフルオロプロパン、3,3’-ジアミノジフェニルスルホキシド、3,4’-ジアミノジフェニルスルホキシド、4,4’-ジアミノジフェニルスルホキシドなどのベンゼン核2つのジアミン;
3)1,3-ビス(3-アミノフェニル)ベンゼン、1,3-ビス(4-アミノフェニル)ベンゼン、1,4-ビス(3-アミノフェニル)ベンゼン、1,4-ビス(4-アミノフェニル)ベンゼン、1,3-ビス(4-アミノフェノキシ)ベンゼン、1,4-ビス(3-アミノフェノキシ)ベンゼン、1,4-ビス(4-アミノフェノキシ)ベンゼン、1,3-ビス(3-アミノフェノキシ)-4-トリフルオロメチルベンゼン、3,3’-ジアミノ-4-(4-フェニル)フェノキシベンゾフェノン、3,3’-ジアミノ-4,4’-ジ(4-フェニルフェノキシ)ベンゾフェノン、1,3-ビス(3-アミノフェニルスルフィド)ベンゼン、1,3-ビス(4-アミノフェニルスルフィド)ベンゼン、1,4-ビス(4-アミノフェニルスルフィド)ベンゼン、1,3-ビス(3-アミノフェニルスルホン)ベンゼン、1,3-ビス(4-アミノフェニルスルホン)ベンゼン、1,4-ビス(4-アミノフェニルスルホン)ベンゼン、1,3-ビス〔2-(4-アミノフェニル)イソプロピル〕ベンゼン、1,4-ビス〔2-(3-アミノフェニル)イソプロピル〕ベンゼン、1,4-ビス〔2-(4-アミノフェニル)イソプロピル〕ベンゼンなどのベンゼン核3つのジアミン;
4)3,3’-ビス(3-アミノフェノキシ)ビフェニル、3,3’-ビス(4-アミノフェノキシ)ビフェニル、4,4’-ビス(3-アミノフェノキシ)ビフェニル、4,4’-ビス(4-アミノフェノキシ)ビフェニル、ビス〔3-(3-アミノフェノキシ)フェニル〕エーテル、ビス〔3-(4-アミノフェノキシ)フェニル〕エーテル、ビス〔4-(3-アミノフェノキシ)フェニル〕エーテル、ビス〔4-(4-アミノフェノキシ)フェニル〕エーテル、ビス〔3-(3-アミノフェノキシ)フェニル〕ケトン、ビス〔3-(4-アミノフェノキシ)フェニル〕ケトン、ビス〔4-(3-アミノフェノキシ)フェニル〕ケトン、ビス〔4-(4-アミノフェノキシ)フェニル〕ケトン、ビス〔3-(3-アミノフェノキシ)フェニル〕スルフィド、ビス〔3-(4-アミノフェノキシ)フェニル〕スルフィド、ビス〔4-(3-アミノフェノキシ)フェニル〕スルフィド、ビス〔4-(4-アミノフェノキシ)フェニル〕スルフィド、ビス〔3-(3-アミノフェノキシ)フェニル〕スルホン、ビス〔3-(4-アミノフェノキシ)フェニル〕スルホン、ビス〔4-(3-アミノフェノキシ)フェニル〕スルホン、ビス〔4-(4-アミノフェノキシ)フェニル〕スルホン、ビス〔3-(3-アミノフェノキシ)フェニル〕メタン、ビス〔3-(4-アミノフェノキシ)フェニル〕メタン、ビス〔4-(3-アミノフェノキシ)フェニル〕メタン、ビス〔4-(4-アミノフェノキシ)フェニル〕メタン、2,2-ビス〔3-(3-アミノフェノキシ)フェニル〕プロパン、2,2-ビス〔3-(4-アミノフェノキシ)フェニル〕プロパン、2,2-ビス〔4-(3-アミノフェノキシ)フェニル〕プロパン、2,2-ビス〔4-(4-アミノフェノキシ)フェニル〕プロパン、2,2-ビス〔3-(3-アミノフェノキシ)フェニル〕-1,1,1,3,3,3-ヘキサフルオロプロパン、2,2-ビス〔3-(4-アミノフェノキシ)フェニル〕-1,1,1,3,3,3-ヘキサフルオロプロパン、2,2-ビス〔4-(3-アミノフェノキシ)フェニル〕-1,1,1,3,3,3-ヘキサフルオロプロパン、2,2-ビス〔4-(4-アミノフェノキシ)フェニル〕-1,1,1,3,3,3-ヘキサフルオロプロパンなどのベンゼン核4つのジアミン。
(i)ビフェニルテトラカルボン酸単位及びピロメリット酸単位からなる群から選ばれる少なくとも一種のテトラカルボン酸単位と、芳香族ジアミン単位とからなる芳香族ポリイミド、
(ii)テトラカルボン酸単位と、ベンゼンジアミン単位、ジアミノジフェニルエーテル単位及びビス(アミノフェノキシ)フェニル単位からなる群から選ばれる少なくとも一種の芳香族ジアミン単位とからなる芳香族ポリイミド、
及び/又は、
(iii)ビフェニルテトラカルボン酸単位及びピロメリット酸単位からなる群から選ばれる少なくとも一種のテトラカルボン酸単位と、ベンゼンジアミン単位、ジアミノジフェニルエーテル単位及びビス(アミノフェノキシ)フェニル単位からなる群から選ばれる少なくとも一種の芳香族ジアミン単位とからなる芳香族ポリイミド。
本発明で使用され得るPES多孔質膜は、ポリエーテルスルホンを含み、典型的には実質的にポリエーテルスルホンからなる。ポリエーテルスルホンは当業者に公知の方法で合成されたものであってよく、例えば、二価フェノール、アルカリ金属化合物及びジハロゲノジフェニル化合物を有機極性溶媒中で重縮合反応させる方法、二価フェノールのアルカリ金属二塩を予め合成しジハロゲノジフェニル化合物と有機極性溶媒中で重縮合反応させる方法等によって製造できる。
対数粘度0.5~1.0のポリエーテルスルホンの0.3質量%~60質量%と有機極性溶媒40質量%~99.7質量%とを含むポリエーテルスルホン溶液を、フィルム状に流延し、ポリエーテルスルホンの貧溶媒又は非溶媒を必須成分とする凝固溶媒に浸漬又は接触させて、空孔を有する凝固膜を作製する工程、及び
前記工程で得られた空孔を有する凝固膜を熱処理して前記空孔を粗大化させて、PES多孔質膜を得る工程
を含み、前記熱処理は、前記空孔を有する凝固膜を、前記ポリエーテルスルホンのガラス転移温度以上、もしくは240℃以上まで昇温させることを含む、方法で製造されてもよい。
前記マクロボイド層は、前記表面層A及びBに結合した隔壁と、当該隔壁並びに前記表面層A及びBに囲まれた、膜平面方向の平均孔径が10μm~500μmである複数のマクロボイドとを有し、
前記マクロボイド層の隔壁は、厚さが0.1μm~50μmであり、
前記表面層A及びBはそれぞれ、厚さが0.1μm~50μmであり、
前記表面層A及びBのうち、一方が平均孔径5μm超200μm以下の複数の細孔を有し、かつ他方が平均孔径0.01μm以上200μm未満の複数の細孔を有し、
表面層A及び表面層Bの、一方の表面開口率が15%以上であり、他方の表面層の表面開口率が10%以上であり、
前記表面層A及び前記表面層Bの前記細孔が前記マクロボイドに連通しており、
前記PES多孔質膜は、総膜厚が5μm~500μmであり、かつ空孔率が50%~95%である、
PES多孔質膜である。
本明細書において、「細胞培養モジュール」とは、細胞培養容器及び細胞培養装置に適用可能な細胞培養基材をいう。
本発明の細胞培養モジュールの一態様は、
頂部と、
底部と、
培養液流出入口を有する側部と、
前記頂部、底部及び側部によって形成される空間を仕切る、培養液流出入口を有する複数の仕切り部と、
頂部とそれに隣接する仕切り部との間の隙間空間、底部とそれに隣接する仕切り部との間の隙間空間、及び隣接する仕切り部同士の間の複数の隙間空間から選択される、2つ以上の隙間空間のそれぞれに固定される、ポリマー多孔質膜と、
を備える、細胞培養モジュールであって、
ここで、前記ポリマー多孔質膜は、複数の孔を有する表面層A及び表面層Bと、前記表面層A及び表面層Bの間に挟まれたマクロボイド層とを有する三層構造のポリマー多孔質膜であり、ここで前記表面層Aに存在する孔の平均孔径は、前記表面層Bに存在する孔の平均孔径よりも小さく、前記マクロボイド層は、前記表面層A及びBに結合した隔壁と、当該隔壁並びに前記表面層A及びBに囲まれた複数のマクロボイドとを有し、前記表面層A及びBにおける孔が前記マクロボイドに連通している、
細胞培養モジュールである。以下、「細胞培養モジュールA」とも呼ぶ。
培養液流出入口を有する頂部と、
培養液流出入口を有する底部と、
培養液流出入口を有する側部と、
前記頂部、底部及び側部によって形成される空間を仕切る、培養液流出入口を有する5つの仕切り部と、
頂部とそれに隣接する仕切り部との間の隙間空間、底部とそれに隣接する仕切り部との間の隙間空間、及び隣接する仕切り部同士の間の複数の隙間空間から選択される、4つの隙間空間のそれぞれに固定される、ポリマー多孔質膜と、
を備え、
ポリマー多孔質膜が固定されている隙間空間に隣接する隙間空間の少なくとも一方は、ポリマー多孔質膜を含まない、
立方体形状の細胞培養モジュール、とみなすことができる。したがって当該細胞培養モジュール複合体は、細胞培養モジュールAの一実施形態とみなすことができる。
複数の細胞培養サブモジュールと、
前記複数の細胞培養サブモジュールを積層して収容するための、培養液流出入口を有する細胞培養サブモジュール収容用ケーシングと
を備える、細胞培養モジュールであって、
ここで、前記細胞培養サブモジュールは、
ポリマー多孔質膜と、
培養液流出入口を有するポリマー多孔質膜収容用ケーシングであって、前記ポリマー多孔質膜が固定されて収容される、前記ポリマー多孔質膜収容用ケーシングと
を備え、
前記ポリマー多孔質膜は、複数の孔を有する表面層A及び表面層Bと、前記表面層A及び表面層Bの間に挟まれたマクロボイド層とを有する三層構造のポリマー多孔質膜であり、ここで前記表面層Aに存在する孔の平均孔径は、前記表面層Bに存在する孔の平均孔径よりも小さく、前記マクロボイド層は、前記表面層A及びBに結合した隔壁と、当該隔壁並びに前記表面層A及びBに囲まれた複数のマクロボイドとを有し、前記表面層A及びBにおける孔が前記マクロボイドに連通している、
細胞培養モジュールである。以下、「細胞培養モジュールB」とも呼ぶ。
細胞培養モジュール複合体10を用いた細胞培養
図5及び6に示される細胞培養モジュール複合体10を用いて、細胞培養を行った。図1~4に示される細胞培養モジュール1を2つ用意し、それぞれの頂部を対向させ、半田ごてによって熱融着して連結し、立方体形状の細胞培養モジュール複合体10を調製した。本実施例で使用されたポリイミド多孔質膜のサイズは、1.4cm×1.4cmである。細胞培養モジュール複合体10は、10枚で1セットのポリイミド多孔質膜積層体を4つ有し、その総面積は80cm2となる。細胞培養モジュール複合体10を、希釈したミルトン溶液(キョーリン製薬製)、超純水、70%エタノール含有水にて洗浄し、滅菌的に乾燥した後に使用した。
細胞培養モジュール20を用いた細胞培養
図7及び8に示される細胞培養モジュール20を用いて、細胞培養を行った。細胞培養モジュール20は、希釈したミルトン溶液(キョーリン製薬社製)、超純水、70%エタノール含有水にて洗浄し、滅菌的に乾燥した後に使用した。なお、本実施例で使用された細胞培養モジュール20で使用したポリイミド多孔質膜のサイズは1.0cm×1.0cmである。当該ポリイミド多孔質膜を6枚1セットとした積層体を3セット用意し、それぞれの間に中敷きを挟持した。本実施例で使用された細胞培養モジュール20中のポリイミド多孔質膜の総枚数は108枚であり、総面積は108cm2となる。細胞培養モジュール20を本実施例において5個使用したので、ポリイミド多孔質膜の総枚数は540枚であり、総面積は540cm2となる。使用された細胞培養モジュール20の細胞培養サブモジュール収容用ケーシング及びポリマー多孔質膜収容用ケーシングの材質は、いずれもポリオレフィン系樹脂である。また、使用された中敷きはNBCメッシュテック社製メッシュである。
図9及び10に示される細胞培養サブモジュール30を30個用意して、滅菌した。ポリイミド多孔質膜の総枚数は540枚であり、総面積は540cm2となる。当該サブモジュールを充填した150mLストレージボトル(コーニング社製)にCHO細胞単層培養用培地KBM270(コージンバイオ株式会社製)を26.7mL添加し、CO2インキュベータ内で10分間、プログラムシェーカー(ケニス社製)を用いて35rpmの回転条件で浸漬した。CHO DP-12浮遊細胞培養液(総細胞数6.16×106cells/ml、生細胞数5.41×106cells/ml、死細胞数7.51×105cells/ml、生細胞率88%)6.0mLと、浮遊細胞用培地(BalanCD(商標) CHO Growth A)7.3mLの混合液を添加し、約14時間、35rpmの回転条件で細胞を吸着させた(シート1枚当たりの細胞吸着数5.83×104cells)。回収培地から見出された細胞数から計算した細胞吸着率は97%であった。培地交換は毎日行い、培地中の一日当たりのグルコース消費量、乳酸産生量、乳酸脱水素酵素量および抗体の産生量を、Cedex Bio(ロシュ・ダイアグノスティックス社製)を用いて測定した(図11)。抗体産生量は、細胞培養モジュールを用いた場合(実施例2)の方が高かった。
抗ヒトIL-8抗体産生CHO-DP12細胞(ATCC CRL-12445)を馴化・浮遊化した細胞を、培地(BalanCD(商標) CHO GROWTH A)を用いて浮遊培養し、1mlあたりの細胞数が、2.0×106になるまで培養を継続した。
2 頂部
3 底部
4 側部
5 第一仕切り部
6 第二仕切り部
7a~7e ポリマー多孔質膜積層体
8a~8h 培養液流出入口
9 載置台
10 細胞培養モジュール複合体
20 細胞培養モジュール
21 細胞培養サブモジュール収容用ケーシング
22 隙間空間
30 細胞培養サブモジュール
31 ポリマー多孔質膜収容用ケーシング
32a、32b 中敷き
Claims (15)
- 頂部と、
底部と、
培養液流出入口を有する側部と、
前記頂部、底部及び側部によって形成される空間を仕切る、培養液流出入口を有する複数の仕切り部と、
頂部とそれに隣接する仕切り部との間の隙間空間、底部とそれに隣接する仕切り部との間の隙間空間、及び隣接する仕切り部同士の間の複数の隙間空間から選択される、2つ以上の隙間空間のそれぞれに固定される、ポリマー多孔質膜と、
を備える、細胞培養モジュールであって、
ここで、前記ポリマー多孔質膜は、複数の孔を有する表面層A及び表面層Bと、前記表面層A及び表面層Bの間に挟まれたマクロボイド層とを有する三層構造のポリマー多孔質膜であり、ここで前記表面層Aに存在する孔の平均孔径は、前記表面層Bに存在する孔の平均孔径よりも小さく、前記マクロボイド層は、前記表面層A及びBに結合した隔壁と、当該隔壁並びに前記表面層A及びBに囲まれた複数のマクロボイドとを有し、前記表面層A及びBにおける孔が前記マクロボイドに連通している、
細胞培養モジュール。 - ポリマー多孔質膜が固定されている隙間空間に隣接する隙間空間の少なくとも一方は、ポリマー多孔質膜を含まない、請求項1に記載の細胞培養モジュール。
- 頂部とそれに隣接する仕切り部との間の隙間空間、底部とそれに隣接する仕切り部との間の隙間空間、及び隣接する仕切り部同士の間の複数の隙間空間から選択される、2以上の隙間空間のそれぞれに、3~100枚のポリマー多孔質膜が積層されて配置される、請求項1又は2に記載の細胞培養モジュール。
- 前記ポリマー多孔質膜がポリイミド多孔質膜である、請求項1~3のいずれか1項に記載の細胞培養モジュール。
- 前記ポリマー多孔質膜がポリエーテルスルホン多孔質膜である、請求項1~3のいずれか1項に記載の細胞培養モジュール。
- 前記頂部及び底部は培養液流出入口を有する、請求項1~5のいずれか1項に記載の細胞培養モジュール。
- 直方体形状を有する、請求項1~6のいずれか1項に記載の細胞培養モジュール。
- 立方体形状を有する、請求項1~6のいずれか1項に記載の細胞培養モジュール。
- 卵形状を有する、請求項1~6のいずれか1項に細胞培養モジュール。
- 複数の請求項7又は8に記載の細胞培養モジュールが連結されてなる、細胞培養モジュール複合体。
- 複数の細胞培養サブモジュールと、
前記複数の細胞培養サブモジュールを積層して収容するための、培養液流出入口を有する細胞培養サブモジュール収容用ケーシングと
を備える、細胞培養モジュールであって、
ここで、前記細胞培養サブモジュールは、
ポリマー多孔質膜と、
培養液流出入口を有するポリマー多孔質膜収容用ケーシングであって、前記ポリマー多孔質膜が固定されて収容される、前記ポリマー多孔質膜収容用ケーシングと
を備え、
前記ポリマー多孔質膜は、複数の孔を有する表面層A及び表面層Bと、前記表面層A及び表面層Bの間に挟まれたマクロボイド層とを有する三層構造のポリマー多孔質膜であり、ここで前記表面層Aに存在する孔の平均孔径は、前記表面層Bに存在する孔の平均孔径よりも小さく、前記マクロボイド層は、前記表面層A及びBに結合した隔壁と、当該隔壁並びに前記表面層A及びBに囲まれた複数のマクロボイドとを有し、前記表面層A及びBにおける孔が前記マクロボイドに連通している、
細胞培養モジュール。 - 積層された前記複数の細胞培養サブモジュールと、細胞培養サブモジュール収容用ケーシングとの間に隙間空間を有する、請求項11に記載の細胞培養モジュール。
- 前記ポリマー多孔質膜収容用ケーシングに、3~100枚のポリマー多孔質膜が積層されて収容される、請求項11又は12に記載の細胞培養モジュール。
- 前記ポリマー多孔質膜がポリイミド多孔質膜である、請求項11~13のいずれか1項に記載の細胞培養モジュール。
- 前記ポリマー多孔質膜がポリエーテルスルホン多孔質膜である、請求項11~13のいずれか1項に記載の細胞培養モジュール。
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