WO2020022511A1 - 動物細胞の浮遊培養用の添加物、浮遊培養用培地および浮遊培養方法 - Google Patents
動物細胞の浮遊培養用の添加物、浮遊培養用培地および浮遊培養方法 Download PDFInfo
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- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0031—Serum-free culture media
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0043—Medium free of human- or animal-derived components
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2513/00—3D culture
Definitions
- the present invention relates to an additive for suspension culture of animal cells, a medium for suspension culture, and a suspension culture method.
- stem cells such as embryonic stem cells and induced pluripotent stem cells
- matricels such as vitronectin and laminin as scaffolds, and the like.
- Methods for culturing large quantities of animal cells include a method of floating culture with stirring with stirring blades, a method of culturing by refluxing the medium with a peristaltic pump, and a culture with gas sparging from the bottom using a sparger.
- a sparger are widely used.
- a serum-free medium containing no serum that may contain unknown factors, prions, viruses, etc., or a low albumin medium having a low albumin content is used. It has been reported that when used in suspension culture with stirring or the like as described above, precipitation occurs in the medium, and the precipitate is necessary for cell growth in a serum-free medium or a low albumin medium. It has been considered that insulin added as a factor may have been precipitated by physical stimulation such as stirring, reflux, gas aeration and the like (Non-Patent Document 1). When the precipitation of the medium component occurs, cell growth is reduced. Therefore, it is desirable to suppress such precipitation in order to efficiently culture animal cells.
- an object of the present invention is to suppress the precipitation of a medium component such as insulin due to physical stimulation such as stirring, shaking, reflux, and gas aeration in the suspension culture of animal cells, thereby improving the culture efficiency of animal cells and the culture cells.
- An object of the present invention is to provide an additive for suspension culture of animal cells, a medium for suspension culture, and a suspension culture method capable of improving the quality.
- the present inventors have found that by adding a water-soluble polymer to a suspension culture medium for animal cells containing insulin or the like, a medium component such as insulin generated by physical stimulation is added.
- a medium component such as insulin generated by physical stimulation is added.
- the present inventors have found that precipitation of can be favorably suppressed, and completed the present invention.
- the present invention relates to the following.
- An additive for suspension culture of animal cells containing a water-soluble polymer.
- the nonionic water-soluble polymer having surface activity is one or more selected from the group consisting of polyvinyl alcohol, polyoxyethylene polyoxypropylene block copolymer, and polyoxyethylene sorbitan monofatty acid ester. , [2].
- the additive according to [4], wherein the stem cells are one or more selected from the group consisting of adult stem cells, embryonic stem cells, and induced pluripotent stem cells.
- the additive according to [6], wherein the medium component is insulin.
- the water-soluble polymer is a nonionic water-soluble polymer having surface activity.
- the nonionic water-soluble polymer having surface activity is one or more selected from the group consisting of polyvinyl alcohol, polyoxyethylene polyoxypropylene block copolymer, and polyoxyethylene sorbitan monofatty acid ester. , [9].
- the stem cells are one or more selected from the group consisting of adult stem cells, embryonic stem cells, and induced pluripotent stem cells.
- the medium according to [13], wherein the medium component is insulin.
- a method for suspending and culturing animal cells which comprises suspending and culturing animal cells in a medium containing a water-soluble polymer.
- the nonionic water-soluble polymer having surface activity is one or more selected from the group consisting of polyvinyl alcohol, polyoxyethylene polyoxypropylene block copolymer, and polyoxyethylene sorbitan monofatty acid ester. , [16].
- the culture method according to any one of [15] to [17], wherein the animal cell is a stem cell.
- the culture method according to [18], wherein the stem cells are one or more selected from the group consisting of adult stem cells, embryonic stem cells, and induced pluripotent stem cells.
- the culture method according to any one of [15] to [20], wherein the animal cells are cultured in suspension in a medium in which the precipitation of medium components is suppressed.
- the culture method according to [21], wherein the medium component is insulin.
- the precipitation of medium components such as insulin caused by physical stimulation such as stirring, shaking, reflux, and gas aeration can be suppressed well.
- a medium for suspension culture of animal cells containing insulin or the like, wherein even if a physical stimulus such as stirring, shaking, reflux, or gas aeration is applied, precipitation of medium components such as insulin can be prevented. It is possible to provide a well-suppressed culture medium for suspension culture of animal cells, and use a medium for suspension culture of animal cells containing insulin and the like to perform stirring, shaking, reflux, gas ventilation, etc. Suspension culture of cells can be performed.
- a cell cluster with a controlled size can be efficiently formed, and the culture efficiency of animal cells and the quality of cultured cells can be improved.
- undifferentiated cells such as stem cells
- precipitation of medium components is suppressed both during maintenance culture in a maintenance medium and during differentiation induction in a differentiation induction medium, and the undifferentiated maintenance rate and differentiation induction during maintenance culture are suppressed. Both the differentiation rate at the time can be improved.
- FIG. 1 is a graph showing the effects of the respective concentrations of polyvinyl alcohol (PVA) and poloxamer (Kolliphor P188 BIO) on the effect of inhibiting the precipitation of insulin in Example 2.
- the bar in the figure indicates 500 ⁇ m.
- FIG. 2 is a diagram showing the effects of the addition concentration of poloxamer (Kolliphor P188 BIO) on the cell growth rate, cell viability, and undifferentiation maintenance rate of human iPS cells in Example 3.
- FIG. 3 is a diagram showing the effect of the concentration of poloxamer (Kolliphor P407) on the cell proliferation rate of human iPS cells in Example 4.
- FIG. 4 is a diagram showing the effect of the addition concentration of polyvinyl alcohol (PVA) on the cell growth rate of human iPS cells in Example 5.
- FIG. 5 is a diagram showing the effect of the concentration of polyoxyethylene sorbitan monolaurate (Kolliphor PS20) on the cell growth rate of human iPS cells in Example 6.
- FIG. 6 is a diagram showing the effect of poloxamer (Kolliphor P188 BIO) on insulin precipitation by stirring in Example 7. The bar in the figure indicates 100 ⁇ m.
- FIG. 7 is a diagram showing the effects of poloxamer (Kolliphor P188 BIO) and polyvinyl alcohol (PVA) on the precipitation of insulin by reflux in Example 8. The bar in the upper diagram indicates 500 ⁇ m.
- FIG. 8 is a diagram showing the effects of poloxamer (Kolliphor P188 BIO) and polyvinyl alcohol (PVA) on the precipitation of insulin by stirring in Example 9.
- the bar in the figure indicates 500 ⁇ m.
- FIG. 9 is a diagram showing the effect of the addition of poloxamer (Kolliphor P188 BIO) on the induction of human iPS cell differentiation in Example 10.
- the present invention provides an additive for suspension culture of animal cells (hereinafter, also referred to as “additive of the present invention” in the present specification), which is added to a medium for suspension culture of animal cells containing insulin or the like. I do.
- the additive of the present invention contains a water-soluble polymer.
- the “water-soluble polymer” refers to a polymer having a hydrophilic group in the molecule and being miscible or soluble in water. In the present invention, those having a solubility in water at 25 ° C. of 5% by weight or more are preferably used.
- the water-soluble polymer is not particularly limited, but a water-miscible or water-soluble polymer having a weight average molecular weight of about 1,000 to 100,000 as measured by size exclusion chromatography is usually used. Used.
- water-soluble polymers examples include carboxyvinyl polymer; polyvinyl alcohol; polyvinylpyrrolidone; polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene fatty acid ester, polyoxyethylene polyhydric alcohol fatty acid partial ester (polyoxyethylene Polyoxyethylene-type nonionic surfactants such as glycerin fatty acid partial ester, polyoxyethylene sorbitol fatty acid partial ester, polyoxyethylene sorbitan fatty acid partial ester), polyoxyethylene hydrogenated castor oil, polyoxyethylene alkylamine, etc .; Ethylene polyoxypropylene random copolymer, polyoxyethylene polyoxypropylene block copolymer (poloxamer) Polyoxyethylene polyoxypropylene-type nonionic surfactant polyoxyethylene polyoxypropylene alkyl ethers; polyglycerol type nonionic surfactants such as polyglycerol fatty acid ester and the like.
- a nonionic water-soluble polymer having surface activity is preferably used as the water-soluble polymer, and polyvinyl alcohol, a polyoxyethylene type nonionic surfactant, and a polyoxyethylene polyoxypropylene type non-ionic surfactant.
- Ionic surfactants and nonionic surfactants of the polyglycerin type are exemplified as preferred water-soluble polymers.
- polyvinyl alcohol, polyoxyethylene polyhydric alcohol fatty acid partial ester and polyoxyethylene polyoxypropylene block copolymer (poloxamer) are more preferably used, and polyvinyl alcohol, polyoxyethylene sorbitan monofatty acid ester (polyoxyethylene sorbitan monolau) are used.
- polyoxyethylene sorbitan monopalmitate polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, etc.
- polyoxyethylene polyoxypropylene block copolymer polyoxyethylene polyoxypropylene block copolymer
- one kind of water-soluble polymer may be selected and used alone, or two or more kinds may be used in combination.
- the content of the water-soluble polymer in the additive of the present invention is set so that the content of the water-soluble polymer in the medium composition when added to the medium is within the range of the content described below.
- the above-mentioned water-soluble polymer may be used as an additive of the present invention as it is, or may be dissolved or dispersed in a solvent such as water or a polyhydric alcohol, and may be in the form of a liquid such as an aqueous solution or a dispersion, or shaped.
- Agents, binders, etc. are mixed with additives generally used for formulation, sized, granulated, tableted, etc. to obtain solid-form additives such as powders, granules, tablets, etc. Is also good.
- the above-mentioned water-soluble polymer may be mixed with a part of the medium components described below such as carbohydrates and inorganic salts to prepare the additive of the present invention. From the viewpoint that the addition to the medium for suspension culture of animal cells is simple and that the mixture with the medium is easy, the additive of the present invention is preferably in the form of a liquid, powder, granule, tablet or the like. Provided.
- the additive of the present invention is preferably prepared by sterilizing.
- the method of the sterilization treatment is not particularly limited, and examples thereof include autoclave sterilization at 121 ° C. for 20 minutes, radiation sterilization, ethylene oxide gas sterilization, filter filtration sterilization, and the like, which are appropriately selected depending on the form of the additive of the present invention. be able to.
- the additive of the present invention can be added to the components of the medium for suspension culture of animal cells described below, and used for preparing the medium for suspension culture of animal cells. It can be added and used.
- the additive of the present invention By adding the additive of the present invention to a suspension culture medium for animal cells containing insulin or the like, especially when the suspension culture medium for animal cells is a serum-free medium or a low albumin medium, stirring is performed. Precipitation of medium components such as insulin caused by physical stimulation such as shaking, reflux, gas aeration and the like can be suppressed well, efficient formation of cell mass of controlled size is possible, and animal cell Culture efficiency and quality of cultured cells can be improved.
- the additive of the present invention can suppress the precipitation of medium components during maintenance culture and differentiation induction by being added to the maintenance medium or differentiation induction medium for undifferentiated cells such as stem cells, Both the maintenance rate and the differentiation rate during differentiation induction can be improved.
- the present invention also provides a medium for suspension culture of animal cells (hereinafter, also referred to as “medium of the present invention” in the present specification).
- animal cells include normal cells, stem cells and progenitor cells derived from mammals.
- Examples of normal cells derived from mammals include germ cells such as sperm and ovum, and somatic cells constituting living bodies.
- Examples of somatic cells constituting a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, bone Cells, bone marrow cells, pericytes, dendritic cells, adipocytes, mesenchymal cells, epithelial cells, epidermal cells (eg, keratinocytes (keratinocytes), keratinocytes, etc.), endothelial cells, vascular endothelial cells, hepatocytes , Chondrocytes, cumulus cells, neurons, glial cells, oligodendrocytes (oligodendrocytes), microglia (microglia), astrocytes (astroglia), heart cells, esophageal cells, muscle
- somatic cells include, for example, skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, It includes cells obtained from any tissue such as vascular tissue, blood (including cord blood), bone marrow, heart, eye, brain, and nerve tissue.
- Stem cells refer to cells that have the ability to self-renew and differentiate into different types of cells and can proliferate without limit.
- adult stem cells such as hematopoietic stem cells, satellite cells, neural stem cells, mesenchymal stem cells, mammary gland stem cells, olfactory mucosal stem cells, neural crest stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, hair follicle stem cells, and the like
- Pluripotent stem cells such as embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ cells, induced pluripotent stem cells (iPS cells), and cancer stem cells.
- Progenitor cells are cells that are in the process of differentiating from the stem cells to specific somatic cells or germ cells, and are satellite cells, pancreatic progenitor cells, vascular progenitor cells, vascular endothelial progenitor cells, hematopoietic progenitor cells (derived from cord blood CD34 positive cells).
- the medium of the present invention is preferably provided as a medium for suspension culture of stem cells, more preferably provided as a medium for suspension culture of adult stem cells, embryonic stem cells and induced pluripotent stem cells, still more preferably embryonic stem cells and artificial stem cells. It is provided as a medium for suspension culture of pluripotent stem cells.
- the medium of the present invention contains a water-soluble polymer together with the above-mentioned medium components usually used for suspension culture of animal cells.
- the water-soluble polymer contained in the medium of the present invention is as described above in the additive of the present invention.
- the water-soluble polymer may be used alone or in combination of two or more. It can be contained.
- the water-soluble polymer may be contained together with the above-mentioned medium component in a state prepared as the above-mentioned additive of the present invention, or may be added directly to the medium component.
- the content of the water-soluble polymer in the medium of the present invention is usually 0.1 ⁇ g / mL to 10 mg / mL, preferably 1 ⁇ g / mL to 5 mg / mL, more preferably 10 ⁇ g / mL, as the final concentration during culture. It is from 5 to 5 mg / mL, more preferably from 10 ⁇ g / mL to 1 mg / mL.
- Examples of the medium components that can be contained in the medium of the present invention include those commonly used for culturing animal cells, and include, for example, sugars such as glucose, fructose, sucrose, and maltose; asparagine, aspartic acid, glutamine, and glutamic acid.
- Proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soy peptide; serum; vitamin A and vitamin B group (thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, nicotinamide, etc.) Vitamins such as vitamin C and vitamin E; fatty acids such as oleic acid, arachidonic acid and linoleic acid; lipids such as cholesterol; sodium chloride, potassium chloride, calcium chloride, magnesium sulfate and sodium dihydrogen phosphate Inorganic elements; trace elements such as zinc, copper, and selenium; N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid) (BES )), 4- (2-hydroxyethyl) -1-
- a component for suppressing the differentiation of the stem cell or the like can be added to a maintenance medium for maintaining the stem cell or the like in an undifferentiated state.
- a component for inducing or promoting differentiation of stem cells or the like can be added to the differentiation-inducing medium for inducing differentiation of stem cells or the like.
- Components that suppress differentiation of stem cells and the like include, for example, leukemia inhibitory factor (LIF), fibroblast growth factor (FGF), and transforming growth factor (TGF), which are differentiation inhibitors of embryonic stem cells.
- BMP bone morphogenetic protein
- Notch protein that suppress differentiation of neural stem cells
- polycomb complex that suppresses differentiation of embryonic stem cells and iPS cells.
- Components that induce or promote differentiation of stem cells and the like include, for example, activin A that induces embryonic stem cells into endodermal cells, retinoic acid, and bone morphogenetic factor (BMP) that induces differentiation of iPS cells into neuroectoderm.
- Inhibitors such as Noggin
- TGF transforming growth factor
- WNT extracellular glycoprotein
- iPS cells mesoderm
- Activin glycogen synthase kinase 3 (GSK3) inhibitor and the like.
- BDNF cranial nerve-derived neurotrophic factor
- GDNF glial cell-derived neurotrophic factor
- FGF fibroblast growth factor
- BMP bone morphogenetic factor
- HGF hepatocyte Growth factors
- the medium of the present invention preferably does not contain serum as a medium component.
- the medium of the present invention is prepared as a culture medium for human cells, it is preferable that the medium does not contain any animal-derived components other than humans.
- the water-soluble polymer may be contained in a medium commonly used for suspension culture of animal cells such as normal cells derived from mammals, stem cells and progenitor cells described above, and may be used as the medium of the present invention.
- a medium commonly used for suspension culture of animal cells such as normal cells derived from mammals, stem cells and progenitor cells described above, and may be used as the medium of the present invention.
- Examples of the medium used for culturing normal cells derived from mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, and DMEM / F12's medium.
- McCoy's 5A medium Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), ⁇ -modified Eagle's Minimum Essential Medium (alpha) Eagle's Minimum Essential Medium ( ⁇ MEM), Roswell Park Memorial (Roswell Park Memor) al Institute) (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E (William's Medium E), Fisher's Medium (Fischer's Medium, etc.) Is mentioned.
- MEM Minimum Essential Medium
- EMEM Eagle's Minimum Essential Medium
- alpha Eagle's Minimum Essential Medium
- RPMI Roswell Park Memorial (Roswell Park Memor) al Institute
- STEMPRO registered trademark
- hESC @ SFM medium Life Technologies
- mTeSR1 medium ST
- TeSR2 medium ST
- TeSR-E8 medium SEmark
- HESCGRO HESCGRO
- TM Serum-Free Medium Medium
- hES cells Millipore ST
- EM Millipore
- EMD Millipore EMD Millipore
- NutriStem registered trademark
- hESC XF medium Bio Industries Israel Beit-Haemek Co., Ltd.
- Examples of a medium used for culturing progenitor cells include HPGM TM (Kembrex), QBSF-60 (Quality Biologicals) and the like.
- a water-soluble polymer may be added to a differentiation-inducing medium such as a stem cell.
- a differentiation-inducing medium such as a stem cell.
- examples of the medium for inducing differentiation of stem cells and the like include TeSR-E6 medium (STEMCELL Technologies), TeSR-E7 medium (STEMCELL Technologies), and Essential 6 (Thermo Fisher Scientific). Company).
- a feeder-free culture medium for animal cells more preferably to use a serum-free medium or a low albumin medium, and to use a culture medium for human cells, Those containing no components derived from animals other than humans (Xeno-free medium) are preferred.
- the medium of the present invention is preferably a serum-free medium or a low albumin medium containing insulin, since the effects of the present invention can be more remarkably obtained.
- the medium of the present invention is preferably in a liquid form such as a solution or a dispersion.
- the medium of the present invention can be prepared by adding a component appropriately selected from the above-mentioned medium components to a solvent such as water, together with a water-soluble polymer, and dissolving or dispersing the medium according to a known composition.
- the medium of the present invention can be prepared by adding or dissolving or dispersing a water-soluble polymer to the above-described animal cell culture medium provided by each company or institution.
- the medium of the present invention is prepared in a state more concentrated than the concentration at the time of use or in a lyophilized powder state, and diluted with a solvent such as water at the time of use, or dissolved in a solvent such as water. It can also be used.
- the medium of the present invention is preferably prepared by performing the sterilization treatment as described above.
- the medium of the present invention is used as a medium for maintaining and culturing undifferentiated cells such as stem cells, and can be suitably used as a medium for inducing differentiation. Precipitation of medium components can be suppressed, and both the undifferentiation maintenance rate during maintenance culture and the differentiation rate during differentiation induction can be improved.
- the present invention provides a method for suspension culture of animal cells (hereinafter, also referred to as “the culture method of the present invention” in the present specification).
- the culturing method of the present invention includes culturing animal cells in suspension in a medium for animal cell suspension culture containing a water-soluble polymer.
- the “culture medium for suspension culture of animal cells containing a water-soluble polymer” is as described above, and in the present invention, the water-soluble polymer contained in the culture medium for suspension culture of animal cells is the additive of the present invention described above. And the water-soluble polymer itself may be directly added. Further, in the present invention, the water-soluble polymer is added to the medium so as to have a final concentration of 0.1 ⁇ g / mL to 10 mg / mL as a final concentration at the time of culturing, and is added to a concentration of 1 ⁇ g / mL to 5 mg / mL.
- the suspension culture of animal cells can be performed according to a usual suspension culture method. That is, using a culture instrument or culture apparatus such as a cell culture plate, a cell culture flask, a bioreactor or the like as appropriate according to the culture scale, for the suspension culture of animal cells to which the above-described medium of the present invention or the additive of the present invention is added
- the animal cells are seeded on a medium, usually at 25 ° C. to 39 ° C., preferably 33 ° C. to 39 ° C., usually in the presence of 4% to 10% by volume, preferably 4% to 6% by volume of carbon dioxide.
- cultivation is carried out in the presence of 1 to 25% by volume, preferably 4 to 20% by volume of oxygen, usually for 1 to 30 days, preferably for 2 to 14 days.
- the medium is changed every two to three days.
- the medium may be exchanged by separating the animal cells from the medium by centrifugation or filtration, and then adding a new medium to the animal cells.
- animal cells may be appropriately concentrated by centrifugation or filtration, and then a new medium may be added to the concentrated cell solution.
- the gravitational acceleration (G) during the centrifugation is generally 50 G to 1,000 G, preferably 100 G to 500 G, and the size of the pores of the filter used for filtration is usually 10 ⁇ m to 200 ⁇ m.
- the culturing method of the present invention can be carried out by performing stirring, shaking, reflux, gas aeration, and the like.
- Agitation can be performed using a bioreactor, a culture tank with stirring blades, or the like.
- the stirring is performed at a stirring speed of usually 10 rpm to 2,000 rpm, preferably 40 rpm to 1,000 rpm.
- Shaking can be performed using a shaking machine or a shaking culture machine. Shaking is usually performed at a shaking speed of 10 rpm to 500 rpm, preferably 50 rpm to 250 rpm.
- the reflux can be performed using a peristaltic pump, a tubing pump, or the like.
- a tube for a peristaltic pump made of silicone, neoprene (chloroprene rubber), marprene (polypropylene / ethylene propylene rubber), a tube for a tubing pump, or the like is used.
- the reflux is usually performed at a flow rate of 10 ⁇ L / min to 1000 mL / min, preferably 1 mL / min to 100 mL / min.
- Gas ventilation can be performed using various spargers such as a micro sparger and a filter sparger.
- the gas can be ventilated at a flow rate of usually 1 mL / min to 1000 mL / min, preferably 50 mL / min to 200 mL / min.
- the suspension culture of animal cells is preferably performed with stirring or shaking.
- the cultured animal cells can be collected by centrifugation or filtration using a filter. Centrifugation is performed at 50 G to 1,000 G, preferably 100 G to 500 G, for about 1 minute to 10 minutes. The filtration can be performed using a filter having pores of about 10 ⁇ m to 200 ⁇ m.
- the cultured animal cells are preferably stored in liquid nitrogen using a freezing medium containing a cryoprotectant such as STEM-CELLBANKER (Nippon Zenyaku Kogyo Co., Ltd.).
- a cryoprotectant such as STEM-CELLBANKER (Nippon Zenyaku Kogyo Co., Ltd.).
- the culture method of the present invention when performing suspension culture of animal cells by performing stirring, shaking, reflux, gas aeration, and the like, it is possible to satisfactorily suppress precipitation of a medium component such as insulin caused by physical stimulation.
- the suspension culture can be performed by forming a cell mass of a controlled size. As a result, the culture efficiency of animal cells and the quality of cultured cells can be improved.
- the culture method of the present invention can be suitably used for any of maintenance culture of undifferentiated cells such as stem cells, and induction of differentiation. Precipitation can be suppressed, and both the undifferentiated maintenance rate during maintenance culture and the differentiation rate during differentiation induction can be improved.
- suspension culture was performed by stirring as follows using undifferentiated human iPS cells (hiPSC) as stem cells using the following stem cell culture medium and the following water-soluble polymer.
- hiPSC undifferentiated human iPS cells
- polyvinyl alcohol (Nippon Synthetic Chemical Industry Co., Ltd., EG-03P), poloxamer (polyoxyethylene (160) polyoxypropylene (27) block copolymer) (Kolliphor (Kolliphor) (Registered trademark) P188 @ BIO) (BASF), poloxamer (polyoxyethylene (202) polyoxypropylene (56) block copolymer) (Kolliphor (registered trademark) P407) (BASF) ), Poloxamer (polyoxyethylene (20) polyoxypropylene (20) block copolymer) (Kollisolv P124) (BASF), polyoxyethylene (20) sorbitan monolaurate (Kolliphor) PS 20 (BASF) Polyoxyethylene (20) sorbitan monopalmitate (Kolliphor PS # 40) (BASF) and polyoxyethylene (20) sorbitan monooleate (Kolliphor PS # 80) (BASF ) Company was used.
- stirring culture was performed at 80 rpm. After the second day, the medium was replaced. The medium was replaced by extracting the amount of medium supernatant described in each example, centrifuging at 200 G for 5 minutes, removing the supernatant, adding the same amount of new medium, suspending the pellet, and adding the resulting mixture to the bioreactor. I went.
- the measurement of cell mass and length in cultured stem cells the measurement of cell number and viability, the undifferentiation maintenance rate of cells and the measurement of differentiation rate to definitive endoderm cells are as follows: I went as follows.
- the pellet was crushed by performing tapping twice, and cells were resuspended by pipetting after adding a medium containing 1 mL of Rho-linked kinase inhibitor (Y-27632). After passing through a strainer (BD Falcon (Corning), 2-1919-02), further inhibit 1 mL of Rho-bound kinase The cell strainer was co-washed with a medium containing (Y-27632), and the recovered cell suspension was analyzed using a viable-cell auto analyzer Vi-CELL XR (Beckman Coulter, Inc.) to determine the cell number and viability. Was measured. The viability of the cells was determined by dividing the number of living cells in the collected cells by the total number of collected cells, and the cell growth rate (times) was obtained by dividing the number of living cells by the number of seeded cells.
- a viable-cell auto analyzer Vi-CELL XR Vi-CELL XR
- BD Perm / Wash buffer (trademark), divided into a sample for double staining, a sample for single staining, a sample for isotype control, and a sample for non-staining, and dispensed into centrifuge tubes. Centrifugation was performed at 2,000 rpm for 2 minutes, and the supernatant was removed. Double and single stains are Alexa Fluor® 488 mouse anti-oct3 / 4 (Becton Dickinson, 560253) and 1:10 (10 ⁇ ) dilutions at 1: 5 (5 ⁇ ) dilution.
- Alexa Fluor® 647 mouse anti-SSEA-4 (Becton Dickinson, Inc., 560796) of BD Perm / Wash buffer (trademark) was added at room temperature. Incubation was performed for 20 minutes under light shielding. Alexa Fluor® 488 Mouse IgG1 ⁇ Isotype Control (Becton Dickinson, 557721) or 1:20 (20 ⁇ a) dilution of Alexa Fluor® 488 Mouse IgG1 ⁇ Isotype Control for Isotype Control samples.
- BD Perm / Wash buffer (trademark) supplemented with Fluor (registered trademark) 647 Mouse IgG3 and ⁇ Isotype Control (Becton Dickinson, Inc., 560803) were added thereto, followed by incubation at room temperature under light shielding for 20 minutes. After each of the above reactions, 500 ⁇ L of BD Perm / Wash buffer (trademark) was added, centrifuged at 5,000 rpm for 2 minutes, and the supernatant was removed.
- Thermo Fisher Scientific 1 mL of Focusing fluid (Thermo Fisher Scientific, 4848621) was added to each sample, and the mixture was centrifuged again at 5,000 rpm for 2 minutes, and then 200 ⁇ L of Focusing fluid (Thermo Fisher Scientific) was added. Fisher Scientific), 4848621).
- the prepared sample was analyzed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). Alexa Fluor (registered trademark) 488 was detected with BL1, and Alexa Fluor (registered trademark) 647 was detected with RL1.
- the undifferentiated maintenance rate of the cells can be represented by the Oct3 / 4 / SSEA4 positive rate in the cultured cells.
- the stained cells were washed once with 1000 ⁇ L of FACS buffer, then centrifuged again to form a pellet, and a cell immobilization / cell permeation solution (BD Cytofix / Cytoperm TM Kit (BD Biosciences), 554714)). Specifically, cells were fixed by adding 200 ⁇ L of Cytofix / Cytoperm and allowing the mixture to stand on ice for 20 minutes. Next, 1 mL of BD Perm / Wash buffer was added, and the mixture was centrifuged at 5,000 rpm for 2 minutes to remove the supernatant, and suspended in 200 ⁇ L of BD Perm / Wash buffer (trademark).
- BD Cytofix / Cytoperm TM Kit BD Biosciences
- BD Pharmingen TM PE Mouse anti-Human Sox17 (BD Biosciences, 561591) or BD Pharmingen TM PE Mouse IgG1, ⁇ Isotype Control, Bios Biosciences, Inc. 400139) was added thereto, and incubated at room temperature under light shielding for 20 minutes to react. After the above reaction, 500 ⁇ L of BD Perm / Wash buffer (trademark) was added, and the mixture was centrifuged at 5,000 rpm for 2 minutes to remove the supernatant.
- BD Perm / Wash buffer (trademark) was added, and the mixture was centrifuged at 5,000 rpm for 2 minutes to remove the supernatant.
- Thermo Fisher Scientific 1 mL of Focusing fluid (Thermo Fisher Scientific, 4848621) was added to each sample, and the mixture was centrifuged again at 5,000 rpm for 2 minutes, and then 200 ⁇ L of Focusing fluid (Thermo Fisher Scientific) was added. Fisher Scientific), 4848621). The prepared sample was analyzed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). PE was detected by BL1 and APC was detected by RL1. The differentiation rate of the cells can be represented by the CXCR4 or SOX17 positive rate in the cultured cells.
- Example 1 Examination of the effect of various water-soluble polymers on insulin precipitation To each of Essential 8 medium containing insulin, 1 mg / mL of each of the above-mentioned water-soluble polymers was added, and as described above, at 120 rpm in a stirring culture device. Stirring was performed for 24 hours. Thereafter, the state of the medium was observed with an inverted microscope, and the inhibitory effect of the water-soluble polymer on the precipitation of insulin was evaluated based on the degree of insulin precipitation in the medium according to the following evaluation criteria. For comparison, the same treatment was carried out without adding a water-soluble polymer, and the degree of precipitation of insulin in the medium was evaluated. The results are shown in Table 1. ⁇ Evaluation criteria> Very good (precipitation of insulin is completely suppressed); ++ Good (insulin precipitation is almost suppressed); + No inhibitory effect (precipitation of insulin is observed);
- Example 1 As shown in Table 1, when polyvinyl alcohol, various poloxamers, and various polyoxyethylene sorbitan monofatty acid esters were added, it was recognized that the precipitation of insulin due to stirring was favorably suppressed. From the above results of Example 1, it was clarified that by adding a water-soluble polymer such as polyvinyl alcohol to the insulin-containing medium, the precipitation of insulin generated when the medium was stirred could be suppressed.
- a water-soluble polymer such as polyvinyl alcohol
- Example 2 Investigation of the effect of each concentration of polyvinyl alcohol and poloxamer on the inhibitory effect of insulin precipitation on 500 mg / mL to 10 mg / mL polyvinyl alcohol (PVA) or 100 ng / mL in Essential 8 medium containing insulin. Poloxamer (Kolliphor P188 BIO) at 11 mg / mL was added and agitated at 120 rpm for 24 hours in a 5 mL bioreactor as described above. After stirring for 24 hours, the medium was observed with an inverted microscope. For comparison, the same treatment was carried out without adding polyvinyl alcohol and poloxamer, and a photograph was taken under an inverted microscope. A photograph taken under a microscope is shown in FIG.
- Example 3 Examination of Effect of Poloxamer (Kolliphor P188 BIO) on HiPSC Proliferation 0.1 ⁇ g / mL to 1 mg / mL poloxamer (Kolliphor P188 BIO) was added to Essential 8 medium containing insulin, and hiPSC was added. The suspension was stirred and suspended for 4 days to evaluate the effect of the poloxamer at each concentration. 1 ⁇ 10 6 cells of the 1210B2 strain hiPSC were inoculated into a 5 mL bioreactor, and cultivation with stirring was performed at a stirring speed of 80 rpm as described above. The medium was replaced with 3.5 mL on the third day, and the cell mass was disrupted on the fourth day, and the cell growth rate, viability, and undifferentiation maintenance rate were determined. The results are shown in FIG.
- Example 4 Investigation of Effect of Poloxamer (Kolliphor P407) on HiPSC Proliferation 1 ⁇ g / mL to 1 mg / mL poloxamer (Kolliphor P407) was added to Essential 8 medium containing insulin, and the hiPSC was stirred and floated for 5 days. After culturing, the effect of each concentration of poloxamer was evaluated. 1 ⁇ 10 6 cells of the 1210B2 strain hiPSC were inoculated into a 5 mL bioreactor, and cultivation with stirring was performed at a stirring speed of 80 rpm as described above. The medium was replaced with 3.5 mL on the second and third days, and the cell mass was disrupted on the fifth day to determine the cell growth rate. The results are shown in FIG.
- Example 6 Examination of the effect of polyoxyethylene sorbitan monolaurate (Kolliphor PS 20) on the growth of hiPSC In Essential 8 medium containing insulin, 100 ng / mL to 10 ⁇ g / mL of polyoxyethylene sorbitan monolaurate (Kolliphor) was used. PS20) was added, and the hiPSCs were cultured with stirring for 4 days to evaluate the effects of polyoxyethylene sorbitan monolaurate at each concentration. 1 ⁇ 10 6 cells of the 1210B2 strain of hiPSC were seeded in a 5 mL bioreactor, and agitation suspension culture was performed at an agitation speed of 80 rpm as described above. On the second day, 3.5 mL of the medium was replaced, and on the third day, the cell mass was crushed, and the cell growth rate was determined. The results are shown in FIG.
- Example 7 Examination of the Effect of Poloxamer on Precipitation of Insulin by Stirring Using a Stirring Impeller
- a stirring cultivation device a 2 L animal cell culture tank (lower magnet stirring type) (ABLE Corporation) and Using a stainless steel (SUS316L) stirring blade, the effect of the poloxamer on the precipitation of insulin by stirring was evaluated.
- 500 mL of Essential 8 medium containing insulin and 1 mg / mL of poloxamer (Kolliphor P188 BIO) were added, and the mixture was stirred at 37 ° C. and 150 rpm for 8 hours.
- Example 8 Investigation of Effect of Water-Soluble Polymer on Precipitation of Insulin by Reflux Using a peristaltic pump (“Perista Biominipump AC-2120” (ATTO Corporation)) as a medium for medium reflux.
- a peristaltic pump (“Perista Biominipump AC-2120” (ATTO Corporation)) as a medium for medium reflux.
- the effect of the water-soluble polymer on the precipitation of insulin by reflux was evaluated, and a silicone tube having an inner diameter of 3 mm and an outer diameter of 5 mm was used as the reflux tube.
- the precipitation of insulin was caused by refluxing the medium, but the precipitation of insulin was suppressed by adding poloxamer (Kolliphor P188 BIO) or polyvinyl alcohol (PVA) to the medium.
- poloxamer polyoxyethylene (160) polyoxypropylene (27) block copolymer
- polyvinyl alcohol polyvinyl alcohol
- Example 9 Investigation of Effect of Water-Soluble Polymer on Precipitation of Insulin in Stem Cell Differentiation Induction Medium Supplement in insulin-containing differentiation induction medium (4 (w / v)% TeSR-E6 medium (STEMCELL Technologies 1) Polyvinyl alcohol (PVA) at 1 mg / mL and poloxamer (Kolliphor P188 BIO) at 1 mg / mL were added to the RPMI1640 medium supplemented with) Co., Ltd.), and 48 hours at 80 rpm in a 5 mL bioreactor as described above. Stirring was performed. Thereafter, the medium was observed with an inverted microscope CKX41. For comparison, the same treatment was carried out without adding polyvinyl alcohol and poloxamer, and observed under an inverted microscope. A photograph taken under a microscope is shown in FIG.
- Example 10 Examination of effect of poloxamer (Kolliphor P188 BIO) on induction of differentiation of hiPSCs into definitive endoderm cells To a 30 mL bioreactor, 30 mL of StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.) was added. 6 ⁇ 10 6 cells of the 1210B2 strain of hiPSC were seeded and subjected to stirring suspension culture at 120 rpm for 6 days to form a cell mass of hiPSC.
- StemFit registered trademark
- AK03N medium Ajinomoto Co., Inc.
- a 5 mL cell mass was transferred to a 5 mL bioreactor and supplemented with a differentiation-inducing medium (4 (w / v)% TeSR-E6 medium supplemented with 0.1 mg / mL poloxamer (Kolliphor P188 BIO) (stem cell).
- Embryos were stirred and suspended at 80 rpm for 5 days in 2 ⁇ M glycogen-generating kinase 3 inhibitor (CHIR99021) and RPMI1640 medium supplemented with 100 ng / mL of activin A (Activin A). Differentiation into endoderm cells was induced. For comparison, agitation suspension culture was similarly performed without adding poloxamer to induce differentiation into definitive endoderm cells.
- a medium component such as insulin is added to a medium for suspension culture of animal cells containing insulin or the like by physical stimulation such as stirring, shaking, reflux, or gas aeration.
- a medium for suspension culture of animal cells containing insulin or the like wherein even if a physical stimulus such as stirring, shaking, reflux, or gas aeration is applied, precipitation of medium components such as insulin can be prevented. It is possible to provide a well-suppressed culture medium for suspension culture of animal cells, and use a medium for suspension culture of animal cells containing insulin and the like to perform stirring, shaking, reflux, gas ventilation, etc. Suspension culture of cells can be performed.
- suspension culture of animal cells can be performed by forming a cell mass having a controlled size, and the culture efficiency of animal cells and the quality of cultured cells can be improved.
- undifferentiated cells such as stem cells
- precipitation of medium components is suppressed both during maintenance culture in a maintenance medium and during differentiation induction in a differentiation induction medium, and the undifferentiated maintenance rate and differentiation induction during maintenance culture are suppressed. Both the differentiation rate at the time can be improved.
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Abstract
Description
しかし、動物細胞を研究、物質生産、医療などに応用するために、効率よく増殖させる培養方法が求められている。動物細胞を大量に培養する方法としては、撹拌羽根で撹拌して浮遊培養する手法、ペリスタルティックポンプを用いて培地を還流させて培養する手法、底面よりスパージャーを用いて気体通気を行いながら培養する手法などが広く用いられている。
また、多くの動物細胞の培養において、未知の因子やプリオン、ウイルス等が含まれる可能性のある血清を含有しない無血清培地や、アルブミン含有量の少ない低アルブミン培地が用いられるが、かかる培地を用いて、上記のような撹拌等を伴う浮遊培養を行うと、培地中に析出を生じることが報告されており、該析出物は、無血清培地や低アルブミン培地に、細胞の成長に必要な因子として添加されたインスリンが、撹拌、還流、気体通気などによる物理的な刺激により析出したのではないかと考察されている(非特許文献1)。
かかる培地成分の析出が生じると細胞の増殖が低下するため、効率的な動物細胞の培養を行うためには、そのような析出を抑制することが望ましい。
従って、本発明の目的は、動物細胞の浮遊培養において、撹拌、振とう、還流、気体通気等の物理的刺激によるインスリン等の培地成分の析出を抑制し、動物細胞の培養効率および培養細胞の品質を向上させ得る動物細胞の浮遊培養用の添加物、浮遊培養用培地、および浮遊培養方法を提供することである。
[1]水溶性ポリマーを含有する、動物細胞の浮遊培養用添加物。
[2]水溶性ポリマーが、界面活性を有する非イオン性の水溶性ポリマーである、[1]に記載の添加物。
[3]界面活性を有する非イオン性の水溶性ポリマーが、ポリビニルアルコール、ポリオキシエチレンポリオキシプロピレンブロックコポリマーおよびポリオキシエチレンソルビタンモノ脂肪酸エステルからなる群より選択される1種または2種以上である、[2]に記載の添加物。
[4]動物細胞が幹細胞である、[1]~[3]のいずれかに記載の添加物。
[5]幹細胞が、成体幹細胞、胚性幹細胞および人工多能性幹細胞からなる群より選択される1種または2種以上である、[4]に記載の添加物。
[6]培地成分の析出を抑制するための添加物である、[1]~[5]のいずれかに記載の添加物。
[7]培地成分がインスリンである、[6]に記載の添加物。
[8]水溶性ポリマーを含有する、動物細胞の浮遊培養用培地。
[9]水溶性ポリマーが、界面活性を有する非イオン性の水溶性ポリマーである、[8]に記載の培地。
[10]界面活性を有する非イオン性の水溶性ポリマーが、ポリビニルアルコール、ポリオキシエチレンポリオキシプロピレンブロックコポリマーおよびポリオキシエチレンソルビタンモノ脂肪酸エステルからなる群より選択される1種または2種以上である、[9]に記載の培地。
[11]幹細胞の浮遊培養用である、[8]~[10]のいずれかに記載の培地。
[12]幹細胞が、成体幹細胞、胚性幹細胞および人工多能性幹細胞からなる群より選択される1種または2種以上である、[11]に記載の培地。
[13]培地成分の析出が抑制されている、[8]~[12]のいずれかに記載の培地。
[14]培地成分がインスリンである、[13]に記載の培地。
[15]水溶性ポリマーを含有する培地にて動物細胞を浮遊培養することを含む、動物細胞の浮遊培養方法。
[16]水溶性ポリマーが、界面活性を有する非イオン性の水溶性ポリマーである、[15]に記載の培養方法。
[17]界面活性を有する非イオン性の水溶性ポリマーが、ポリビニルアルコール、ポリオキシエチレンポリオキシプロピレンブロックコポリマーおよびポリオキシエチレンソルビタンモノ脂肪酸エステルからなる群より選択される1種または2種以上である、[16]に記載の培養方法。
[18]動物細胞が幹細胞である、[15]~[17]のいずれかに記載の培養方法。
[19]幹細胞が、成体幹細胞、胚性幹細胞および人工多能性幹細胞からなる群より選択される1種または2種以上である、[18]に記載の培養方法。
[20]撹拌、振とう、還流または気体通気を行って浮遊培養する、[15]~[19]のいずれかに記載の培養方法。
[21]培地成分の析出が抑制された培地にて動物細胞を浮遊培養する、[15]~[20]のいずれかに記載の培養方法。
[22]培地成分がインスリンである、[21]に記載の培養方法。
[23]細胞塊を形成させて動物細胞を浮遊培養する、[15]~[22]のいずれかに記載の培養方法。
また、本発明により、インスリン等を含有する動物細胞の浮遊培養用培地であって、撹拌、振とう、還流、気体通気等の物理的刺激が付加されても、インスリン等の培地成分の析出が良好に抑制された動物細胞の浮遊培養用培地を提供することができ、インスリン等を含有する動物細胞の浮遊培養用培地を用いて、撹拌、振とう、還流、気体通気等を行いながら、動物細胞の浮遊培養を行うことができる。
その結果、大きさの制御された細胞塊を効率よく形成させることができ、動物細胞の培養効率および培養細胞の品質を向上させることができる。
特に、幹細胞等の未分化細胞について、維持培地での維持培養時、および分化誘導培地での分化誘導時のいずれにおいても培地成分の析出が抑制され、維持培養時の未分化維持率、分化誘導時の分化率の双方を向上させることができる。
本発明の添加物は、水溶性ポリマーを含有する。
また、水溶性ポリマーとしては、特に限定されるものではないが、通常、サイズ排除クロマトグラフィーにより測定される重量平均分子量が1,000~100,000程度の水混和性または水溶性を示すポリマーが用いられる。
本発明の添加物における水溶性ポリマーの含有量は、培地に添加した際の培地組成物中における水溶性ポリマーの含有量が、後述する含有量の範囲内となるように設定される。
また、上記した水溶性ポリマーを、炭水化物、無機塩等の以下に述べる培地成分の一部と混合して、本発明の添加物として調製してもよい。
動物細胞の浮遊培養用培地への添加が簡便で、培地との混和も容易であるとの観点から、本発明の添加物は、好ましくは液状、粉末状、顆粒状、錠剤状等の形態で提供される。
本発明の添加物は、幹細胞等の未分化細胞の維持培地や分化誘導培地に添加することにより、維持培養および分化誘導時の培地成分の析出を抑制することができ、維持培養時の未分化維持率、分化誘導時の分化率の双方を向上させることができる。
ここで、動物細胞としては、哺乳動物由来の正常細胞、幹細胞および前駆細胞が挙げられる。
生体を構成する体細胞の例としては、以下に限定されるものではないが、線維芽細胞、骨髄細胞、Bリンパ球、Tリンパ球、好中球、赤血球、血小板、マクロファージ、単球、骨細胞、骨髄細胞、周皮細胞、樹状細胞、脂肪細胞、間葉細胞、上皮細胞、表皮細胞(たとえば、角化細胞(ケラチノサイト)、角質細胞等)、内皮細胞、血管内皮細胞、肝実質細胞、軟骨細胞、卵丘細胞、神経細胞、グリア細胞、オリゴデンドロサイト(希突起膠細胞)、マイクログリア(小膠細胞)、アストロサイト(星状膠細胞)、心臓細胞、食道細胞、筋肉細胞(たとえば、平滑筋細胞、骨格筋細胞)、膵臓ベータ細胞、メラニン細胞および単核細胞等が挙げられる。
当該体細胞には、たとえば皮膚、腎臓、脾臓、副腎、肝臓、肺、卵巣、膵臓、子宮、胃、結腸、小腸、大腸、膀胱、前立腺、精巣、胸腺、筋肉、結合組織、骨、軟骨、血管組織、血液(臍帯血を含む)、骨髄、心臓、眼、脳、神経組織などの任意の組織から採取される細胞が含まれる。
たとえば、造血幹細胞、衛星細胞、神経幹細胞、間葉系幹細胞、乳腺幹細胞、嗅粘膜幹細胞、神経冠幹細胞、肝幹細胞、膵幹細胞、筋幹細胞、生殖幹細胞、腸管幹細胞、毛包幹細胞等の成体幹細胞;胚性幹細胞(ES細胞)、胚性腫瘍細胞、胚性生殖幹細胞、人工多能性幹細胞(iPS細胞)等の多能性幹細胞;癌幹細胞等が挙げられる。
本発明の培地に含有される水溶性ポリマーについては、本発明の添加物において上記した通りであり、本発明の培地には、水溶性ポリマーは1種を単独で、または2種以上を組み合わせて含有させることができる。
本発明においては、水溶性ポリマーは、上記した本発明の添加物として調製された状態で、前記培地成分とともに含有されてもよく、または培地成分に対して直接添加されてもよい。
本発明の培地における水溶性ポリマーの含有量は、培養時における終濃度として、通常0.1μg/mL~10mg/mLであり、好ましくは1μg/mL~5mg/mLであり、より好ましくは10μg/mL~5mg/mLであり、さらに好ましくは10μg/mL~1mg/mLである。
また、幹細胞等の分化を誘導する分化誘導培地には、幹細胞等の分化を誘導もしくは促進する成分を添加することができる。
幹細胞等の分化を抑制する成分としては、たとえば、胚性幹細胞の分化抑制因子である白血病阻害因子(leukemia inhibitory factor,LIF)、線維芽細胞増殖因子(FGF)、トランスフォーミング増殖因子(TGF)-β、神経幹細胞の分化を抑制する骨形成因子(bone morphogenetic protein; BMP)やNotchタンパク質、胚性幹細胞やiPS細胞の分化を抑制するポリコーム複合体等が挙げられる。
幹細胞等の分化を誘導もしくは促進する成分としては、たとえば、胚性幹細胞を内胚葉系細胞に誘導するアクチビンA、レチノイン酸、iPS細胞の神経外胚葉への分化を誘導する骨形成因子(BMP)阻害剤(ノギン等)、トランスフォーミング増殖因子(TGF)-β、iPS細胞の中胚葉への分化を誘導する細胞外分泌糖タンパク質(WNT)、iPS細胞の中胚葉や内胚葉への分化を誘導するアクチビン、グリコーゲン合成酵素キナーゼ3(GSK3)阻害剤等が挙げられる。
外胚葉系細胞、中胚葉系細胞、内胚葉系細胞に初期分化させた後、各臓器や組織の細胞に分化させる際には、分化誘導しようとする臓器や組織に応じて、必要な増殖因子や栄養因子等を添加することができ、たとえば、脳神経由来神経栄養因子(BDNF)、グリア細胞由来神経栄養因子(GDNF)、線維芽細胞増殖因子(FGF)、骨形成因子(BMP)、肝細胞増殖因子(HGF)等が用いられる。
哺乳動物細胞由来の正常細胞の培養に用いられる培地としては、ダルベッコ改変イーグル培地(Dulbecco’s Modified Eagle’s Medium)(DMEM)、ハムF12培地(Ham’s Nutrient Mixture F12)、DMEM/F12培地、マッコイ5A培地(McCoy’s 5A medium)、最小必須培地(Minimum Essential Medium)(MEM)、イーグル最小必須培地(Eagle’s Minimum Essential Medium)(EMEM)、α改変型イーグル最小必須培地(alpha Modified Eagle’s Minimum Essential Medium)(αMEM)、ロズウェルパーク記念研究所(Roswell Park Memorial Institute)(RPMI)1640培地、イスコフ改変ダルベッコ培地(Iscove’s Modified Dulbecco’s Medium)(IMDM)、MCDB131培地、ウィリアム培地E(William’s Medium E)、フィッシャー培地(Fischer’s Medium)等が挙げられる。
幹細胞等の分化誘導培地としては、TeSR-E6培地(ステムセルテクノロジーズ(STEMCELL Technologies)社)、TeSR-E7培地(ステムセルテクノロジーズ(STEMCELL Technologies)社)、Essential 6(サーモフィッシャー サイエンティフィック(Thermo Fisher Scientific)社)などが挙げられる。
また、本発明の効果がより顕著に得られることから、本発明の培地は、無血清培地または低アルブミン培地であって、インスリンを含有するものであることが好ましい。
また、本発明の培地は、各社・各機関から提供されている上記した動物細胞培養用培地に、水溶性ポリマーを添加して溶解または分散して調製することができる。
さらに、本発明の培地は、使用時の濃度よりも濃縮した状態や、凍結乾燥された粉末状の状態で調製し、使用時に水等の溶媒で希釈し、または水等の溶媒に溶解して用いる態様とすることもできる。
本発明の培地は、上記したような滅菌処理を施して調製されることが好ましい。
本発明の培地は、幹細胞等の未分化細胞の維持培養用の培地として、また、分化誘導用培地として好適に用いることができ、幹細胞等の未分化細胞の維持培養または分化誘導の際に、培地成分の析出を抑制することができ、維持培養時の未分化維持率、分化誘導時の分化率の双方を向上させることができる。
本発明の培養方法は、水溶性ポリマーを含有する動物細胞の浮遊培養用培地にて、動物細胞を浮遊培養することを含む。
また、本発明においては、水溶性ポリマーは、培養時における終濃度として、通常0.1μg/mL~10mg/mLの濃度となるように培地に添加され、1μg/mL~5mg/mLの濃度になるように培地に添加されることが好ましく、10μg/mL~5mg/mLの濃度となるように培地に添加されることがより好ましく、10μg/mL~1mg/mLの濃度となるように培地に添加されることがさらに好ましい。
培地交換は、遠心分離やろ過により動物細胞と培地とを分離した後、新しい培地を動物細胞に添加すればよい。あるいは、遠心分離やろ過を行うことにより動物細胞を適宜濃縮した後、新しい培地をこの濃縮細胞液に添加すればよい。
上記遠心分離の際の重力加速度(G)は、通常50G~1,000G、好ましくは、100G~500Gであり、ろ過に用いるフィルターの細孔の大きさは、通常10μm~200μmである。
撹拌は、バイオリアクター、撹拌羽根付き培養槽等を用いて行うことができる。
撹拌は、通常10rpm~2,000rpm、好ましくは40rpm~1,000rpmの撹拌速度で行う。
振とうは、振とう機や振とう培養機を用いて行うことができる。
振とうは、通常10rpm~500rpm、好ましくは50rpm~250rpmの振とう速度で行う。
還流は、ペリスタルティックポンプ、チュービングポンプ等を用いて行うことができる。還流用チューブとしては、シリコーン、ネオプレン(クロロプレンゴム)、マープレン(ポリプロピレン・エチレンプロピレンゴム)製等のペリスタルティックポンプ用チューブ、チュービングポンプ用チューブ等を用いる。
還流は、通常10μL/分~1000mL/分、好ましくは1mL/分~100mL/分の流速で行う。
気体通気は、マイクロスパージャー、フィルタースパージャー等、各種スパージャーを用いて行うことができる。
気体通気は、通常1mL/分~1000mL/分、好ましくは50mL/分~200mL/分の通気量で行うことができる。
大きさの制御された細胞塊を効率よく得るためには、動物細胞の浮遊培養は、撹拌または振とうを行って実施することが好ましい。
遠心分離は、50G~1,000G、好ましくは100G~500Gで1分間~10分間程度行う。
また、ろ過は、10μm~200μm程度の細孔を有するフィルターを用いて行うことができる。
本発明の培養方法は、幹細胞等の未分化細胞の維持培養、および分化誘導のいずれにも好適に用いることができ、幹細胞等の未分化細胞の維持培養または分化誘導の際に、培地成分の析出を抑制することができ、維持培養時の未分化維持率、分化誘導時の分化率の双方を向上させることができる。
撹拌培養器材として、シングルユースバイオリアクター5mL容量(エイブル株式会社(ABLE Corporation)、S-1467)を用いた。前記培養器材に培地を5mL添加し、37℃、5体積%二酸化炭素および20体積%酸素の条件下で、120rpmにて24時間撹拌を行った。その後、培地を6ウェル細胞培養プレートに移し、倒立顕微鏡(「CKX41」、オリンパス株式会社(Olympus Corporation)、倍率=40倍)で観察し、写真撮影を行った。
未分化ヒトiPS細胞(hiPSC)としては、1210B2株のhiPS細胞(Nakagawa, M.et al., Sci. Rep. 4, 3594, 2014参照)を用いた。
撹拌による浮遊細胞培養は、培養器材として、シングルユースバイオリアクター5mL容量(エイブル株式会社(ABLE Corporation)、S-1467)を用いて行った。
上記バイオリアクターに10μMのRho結合キナーゼ阻害剤(Y-27632)(富士フィルム和光純薬株式会社、034-24024)を含有する培地5mLを添加し、シングルセル化したhiPSCを添加して、37℃、5体積%二酸化炭素および20体積%酸素の条件下で、80rpmにて撹拌培養を行った。
2日目以降には培地交換を行った。培地交換は、各実施例に記載の量の培地上清を抜き取り、200G、5min遠心分離し、上清を除去後、同量の新しい培地を添加し、ペレットを懸濁後バイオリアクターに添加して行った。
細胞塊を含む培地上清を24ウェルプレートに500μL分取した。細胞塊を振とうして分散させたのち、BZ-X蛍光顕微鏡(株式会社キーエンス(Keyence))でウェル全体を撮影した。撮影した画像に対してマクロセルカウントを行うことで、細胞塊の個数と長径の平均を求めた。
細胞塊を含む培地上清を全量回収し、500G、5min遠心分離した。上清を除去後、10回タッピングを行い、1mLの細胞分離/分散溶液(Accumax(ミリポア(Millipore)社、SCR006)を添加し、細胞塊ペレットを懸濁した。室温で5minインキュベートした後、ピペッティングにより細胞塊を再懸濁した。再度室温で5minインキュベートした後、ピペッティングにより細胞塊をシングルセル化した。4mLの培地を添加し、500G、5min遠心分離した。上清を除去後、10回タッピングを行うことでペレットを破砕し、1mLのRho結合キナーゼ阻害剤(Y-27632)を含有する培地を添加してピペッティングすることにより細胞を再懸濁した。懸濁液を40μmのセルストレーナー(BD Falcon(コーニング社)、2-1919-02)に通し、さらに1mLのRho結合キナーゼ阻害剤(Y-27632)を含有する培地でセルストレーナーを共洗いした。回収した細胞懸濁液を生死細胞オートアナライザー Vi-CELL XR(ベックマン・コールター株式会社)で分析することで、細胞数と生存率の測定を行った。
回収した細胞中の生細胞数を回収した全細胞数で除して、細胞の生存率を求め、生細胞数を播種した細胞数で除して細胞の増殖率(倍)を求めた。
培養後シングルセル化した細胞を、細胞固定化/細胞透過化溶液(BD Cytofix/Cytoperm(商標) Kit(ビー ディー バイオサイエンス(BD Biosciences)社、554714))により固定化した。具体的には、200μLのCytofix/Cytopermを添加し、氷上で20分静置することでhiPSCの固定を行った。
次いで、1mLのBD Perm/Wash buffer(商標)(ビー ディー バイオサイエンス(BD Biosciences)社、554723)を添加し、5,000rpm、2min遠心分離を行い、上清を除いた。次に、適当量のBD Perm/Wash buffer(商標)に懸濁し、二重染色用サンプル、単染色用サンプル、isotype control用サンプル、非染色用サンプルに分けてそれぞれ遠沈管に分注し、5,000rpm、2min遠心分離を行い、上清を除去した。
二重染色および単染色は1:5(5倍)希釈のAlexa Fluor(登録商標) 488 mouse anti-oct3/4(ベクトン・ディッキンソン(Becton Dickinson)社、560253)および1:10(10倍)希釈のAlexa Fluor(登録商標) 647 mouse anti―SSEA-4(ベクトン・ディッキンソン(Becton Dickinson)社、560796)の両方、もしくは一方をBD Perm/Wash buffer(商標)に添加した溶液を100μL添加し、室温、遮光下で20minインキュベーションすることで行った。
Isotype control用サンプルには1:20(20倍)希釈のAlexa Fluor(登録商標) 488 Mouse IgG1 κ Isotype Control(ベクトン・ディッキンソン(Becton Dickinson)社、557721)または1:20(20倍)希釈のAlexa Fluor(登録商標) 647 Mouse IgG3、κ Isotype Control(ベクトン・ディッキンソン(Becton Dickinson)社、560803)を添加したBD Perm/Wash buffer(商標)を100μL加え、同様に室温、遮光下で20minインキュベーションした。
上記の各反応後、500μLのBD Perm/Wash buffer(商標)を添加して5,000rpm、2min遠心分離し、上清を除去した。各サンプルにFocusing fluid(サーモフィッシャー サイエンティフィック(Thermo Fisher Scientific)社、4488621)を1mL添加し、再度5,000rpm、2min遠心分離を行った上で200μLのFocusing fluid(サーモフィッシャー サイエンティフィック(Thermo Fisher Scientific)社、4488621)に懸濁した。調製したサンプルは、Attune NxT Flow Cytometer(サーモフィッシャー サイエンティフィック(Thermo Fisher Scientific)社)で解析を実施した。Alexa Fluor(登録商標) 488はBL1、Alexa Fluor(登録商標) 647はRL1で検出を行った。
細胞の未分化維持率は、培養された細胞におけるOct3/4/SSEA4陽性率により表すことができる。
培養後シングルセル化した細胞を、5,000rpm、2min遠心分離した。細胞ペレットを100μLのBD Perm/Wash buffer(商標)(ビー ディー バイオサイエンス(BD Biosciences)社、554723)に懸濁し、1μLのBD Pharmingen(商標) APC Mouse Anti-Human CD184(ビー ディー バイオサイエンス(BD Biosciences)社、560936)または1 μLのBD Pharmingen(商標) APC Mouse IgG2a, κ Isotype Control(ビー ディー バイオサイエンス(BD Biosciences)社、555576)を添加し、室温、遮光下で20minインキュベーションすることで染色を行った。染色した細胞は1000μLのFACS bufferで一度洗浄した後、再度遠心分離してペレットとし、細胞固定化/細胞透過化溶液(BD Cytofix/Cytoperm(商標) Kit(ビー ディー バイオサイエンス(BD Biosciences)社、554714))により固定化した。具体的には、200μLのCytofix/Cytopermを添加し、氷上で20分静置することで細胞の固定を行った。
次いで、1mLのBD Perm/Wash bufferを添加し、5,000rpm、2min遠心分離を行って上清を除き、200μLのBD Perm/Wash buffer(商標)に懸濁した。1μLのBD Pharmingen(商標)PE Mouse anti-Human Sox17 (ビー ディー バイオサイエンス(BD Biosciences)社、561591)またはBD Pharmingen(商標)PE Mouse IgG1,κ Isotype Control( ビー ディー バイオサイエンス(BD Biosciences)社、400139)を添加し、室温、遮光下で20minインキュベーションして反応させた。
上記の反応後、500μLのBD Perm/Wash buffer(商標)を添加して5,000rpm、2min遠心分離し、上清を除去した。各サンプルにFocusing fluid(サーモフィッシャー サイエンティフィック(Thermo Fisher Scientific)社、4488621)を1mL添加し、再度5,000rpm、2min遠心分離を行った上で200μLのFocusing fluid(サーモフィッシャー サイエンティフィック(Thermo Fisher Scientific)社、4488621)に懸濁した。調製したサンプルは、Attune NxT Flow Cytometer(サーモフィッシャー サイエンティフィック(Thermo Fisher Scientific)社)で解析を実施した。PEはBL1、APCはRL1で検出を行った。
細胞の分化率は、培養された細胞におけるCXCR4またはSOX17陽性率により表すことができる。
インスリンを含有するEssential 8培地に、上記した水溶性ポリマー各1mg/mLを添加し、上記した通り、撹拌培養器材中で120rpmにて24時間撹拌を行った。その後、培地の状態を倒立型顕微鏡で観察し、培地中のインスリンの析出の程度により、水溶性ポリマーのインスリン析出抑制効果を下記評価基準により評価した。
なお、比較のため、水溶性ポリマーを添加せずに同様に処理して、培地中のインスリンの析出の程度を評価した。
結果を表1に示した。
<評価基準>
非常に良好である(インスリンの析出が完全に抑制されている);++
良好である(インスリンの析出がほぼ抑制されている);+
抑制効果なし(インスリンの析出が認められる);-
実施例1の上記結果から、ポリビニルアルコール等の水溶性ポリマーをインスリン含有培地に添加することで、培地を撹拌した際に生ずるインスリンの析出を抑制できることが明らかになった。
インスリンを含有するEssential 8培地に、500ng/mL~10mg/mLのポリビニルアルコール(PVA)、または100ng/mL~1mg/mLのポロキサマー(Kolliphor P188 BIO)を添加し、上記の通り、5mLのバイオリアクター中で120rpmにて24時間撹拌を行った。24時間撹拌後に培地を倒立型顕微鏡で観察した。
なお、比較のため、ポリビニルアルコールおよびポロキサマーを添加せずに同様に処理し、倒立顕微鏡下にて写真撮影を行った。
顕微鏡下に撮影した写真を図1に示した。
実施例2の上記結果より、ポリビニルアルコールを500ng/mL以上、またポロキサマー(ポリオキシエチレン(160)ポリオキシプロピレン(27)ブロックコポリマー)を100ng/mL以上インスリン含有培地に添加することで、培地を撹拌することにより生じるインスリンの析出を抑制できることが明らかになった。
インスリンを含有するEssential 8培地に、0.1μg/mL~1mg/mLのポロキサマー(Kolliphor P188 BIO)を添加し、hiPSCを4日間撹拌浮遊培養して、各濃度のポロキサマーの効果を評価した。
1210B2株のhiPSCを5mLのバイオリアクターに1x106細胞播種し、上記の通り、80rpmの撹拌速度で撹拌培養を行った。3日目に3.5mLの培地交換を行い、4日目に細胞塊を破砕し、細胞の増殖率、生存率および未分化維持率を求めた。結果を図2に示した。
実施例3の上記結果より、ポロキサマー(ポリオキシエチレン(160)ポリオキシプロピレン(27)ブロックコポリマー)(Kolliphor P188 BIO)を、0.1μg/mL~1mg/mLの濃度で培地に添加してhiPSCの撹拌浮遊培養を行うことにより、培地中のインスリンの析出が抑制されるとともに、細胞塊の成長を促し、状態の良いhiPSCを効率よく増殖させることが可能となることが明らかになった。
インスリンを含有するEssential 8培地に、1μg/mL~1mg/mLのポロキサマー(Kolliphor P407)を添加し、hiPSCを5日間撹拌浮遊培養して、各濃度のポロキサマーの効果を評価した。
1210B2株のhiPSCを5mLのバイオリアクターに1x106細胞播種し、上記の通り、80rpmの撹拌速度で撹拌培養を行った。2、3日目に3.5mLの培地交換を行い、5日目に細胞塊を破砕し、細胞の増殖率を求めた。結果を図3に示した。
実施例4の上記結果より、ポロキサマー(ポリオキシエチレン(202)ポリオキシプロピレン(56)ブロックコポリマー)(Kolliphor P407)を1μg/mL~1mg/mLの濃度で培地に添加してhiPSCの撹拌浮遊培養を行うことにより、培地中のインスリンの析出が抑制されるとともに、細胞塊の成長を促し、状態の良いhiPSCを効率よく増殖させることが可能となることが明らかになった。
インスリンを含有するEssential 8培地に50μg/mL~5mg/mLのポリビニルアルコール(PVA)を添加し、hiPSCを4日間撹拌培養して、各濃度のポリビニルアルコールの効果を評価した。
1210B2株のhiPSCを5mLのバイオリアクターに1x106細胞播種し、上記の通り、80rpmの撹拌速度で撹拌浮遊培養を行った。培養2日目に3.5mLの培地交換を行い、4日目に細胞塊を破砕し、細胞の増殖率を求めた。結果を図4に示した。
実施例5の上記結果より、ポリビニルアルコール(PVA)を50μg/mL~5mg/mLの濃度で培地に添加してhiPSCの撹拌浮遊培養を行うことにより、培地中のインスリンの析出が抑制されるとともに、細胞塊の成長を促し、状態の良いhiPSCを効率よく増殖させることが可能となることが明らかになった。
インスリンを含有するEssential 8培地に100ng/mL~10μg/mLのポリオキシエチレンソルビタンモノラウレート(Kolliphor PS 20)を添加し、hiPSCを4日間撹拌培養して、各濃度のポリオキシエチレンソルビタンモノラウレートの効果を評価した。
1210B2株のhiPSCを5mLのバイオリアクターに1x106細胞播種し、上記の通り、80rpmの撹拌速度で撹拌浮遊培養を行った。2日目に3.5mLの培地交換を行い、3日目に細胞塊を破砕し、細胞の増殖率を求めた。結果を図5に示した。
実施例6の上記結果より、ポリオキシエチレンソルビタンモノラウレート(Kolliphor PS 20)を100ng/mL~10μg/mLの濃度で培地に添加してhiPSCの撹拌培養を行うことにより、培地中のインスリンの析出が抑制されるとともに、細胞塊の成長を促し、状態の良いhiPSCを効率よく増殖させることが可能となることが明らかになった。
撹拌培養器材として、液量2Lの動物細胞培養槽(下部マグネット撹拌型)(エイブル株式会社(ABLE Corporation))およびステンレス鋼(SUS316L)製撹拌羽根を用いて、撹拌によるインスリンの析出に対するポロキサマーの効果を評価した。
上記動物細胞培養槽に、インスリンを含有するEssential 8培地500mL、および1mg/mLのポロキサマー(Kolliphor P188 BIO)を添加して、37℃で150rpmにて8時間撹拌を行った。その後、培地を6ウェル細胞培養プレートに移し、倒立顕微鏡(「CKX53」、オリンパス株式会社(Olympus Corporation))で観察し(倍率=100倍)、写真撮影を行った。
なお、比較のため、ポロキサマーを添加せずに同様に処理し、倒立顕微鏡下にて写真撮影を行った。
顕微鏡下に撮影した写真を図6に示した。
実施例7の上記結果より、ポロキサマー(ポリオキシエチレン(160)ポリオキシプロピレン(27)ブロックコポリマー)を1mg/mLの濃度で、インスリンを含有する培地に添加することにより、撹拌羽根を用いて培地を撹拌する場合にも、それにより生じるインスリンの析出を抑制できることが明らかになった。
培地還流用器材として、ペリスタルティックポンプ(「ぺリスタ・バイオミニポンプ AC-2120」(アトー株式会社(ATTO Corporation))を用いて、還流によるインスリンの析出に対する水溶性ポリマーの影響を評価した。還流用チューブとしては、内径=3mm、外径=5mmのシリコーンチューブを用いた。
50mL容量のプラスティックチューブに、インスリンを含有するEssential 8培地45mL、およびポロキサマー(Kolliphor P188 BIO)およびポリビニルアルコール(PVA)各1mg/mLをそれぞれ添加し、上記ペリスタルティックポンプにより、室温で5mL/分の流速にて、24時間還流を行った。その後、培地を6ウェル細胞培養プレートに移し、倒立顕微鏡(「CKX41」、オリンパス株式会社(Olympus Corporation))で観察し(倍率=40倍および100倍)、写真撮影を行った。
なお、比較のため、ポロキサマーおよびポリビニルアルコールを添加せずに同様に処理し、倒立顕微鏡下にて写真撮影を行った。
顕微鏡下に撮影した写真を図7に示した。
実施例8の上記結果より、ポロキサマー(ポリオキシエチレン(160)ポリオキシプロピレン(27)ブロックコポリマー)およびポリビニルアルコールをそれぞれ1mg/mLの濃度で、インスリンを含有する培地に添加することにより、培地を還流する際に生じるインスリンの析出を抑制できることが明らかになった。
インスリンを含有する分化誘導培地(4(w/v)%のTeSR-E6培地中のサプリメント(ステムセルテクノロジーズ(STEMCELL Technologies)社)を添加したRPMI1640培地)に、1mg/mLのポリビニルアルコール(PVA)、1mg/mLのポロキサマー(Kolliphor P188 BIO)を添加し、上記の通り、5mLのバイオリアクター中で80rpmにて48時間撹拌を行った。その後、培地を倒立型顕微鏡CKX41で観察した。
なお、比較のため、ポリビニルアルコールおよびポロキサマーを添加せずに同様に処理し、倒立顕微鏡下にて観察した。
上記にて、顕微鏡下に撮影した写真を図8に示した。
実施例9の上記結果より、ポロキサマー(ポリオキシエチレン(160)ポリオキシプロピレン(27)ブロックコポリマー)およびポリビニルアルコールをそれぞれ1mg/mLの濃度で、インスリンを含有する分化誘導培地に添加することにより、培地を撹拌することにより生じるインスリンの析出を抑制できることが確認された。
30mL容量のバイオリアクターに、StemFit(登録商標)AK03N培地(味の素株式会社)を30mL添加し、1210B2株のhiPSCを6×106細胞播種し、120rpmにて6日間撹拌浮遊培養を行い、hiPSCの細胞塊を形成させた。5mL分の細胞塊を5mL容量のバイオリアクターに移し、0.1mg/mLのポロキサマー(Kolliphor P188 BIO)を添加した分化誘導培地(4(w/v)%のTeSR-E6培地中のサプリメント(ステムセルテクノロジーズ(STEMCELL Technologies)社)、2μMのグリコーゲン生成キナーゼ3阻害剤(CHIR99021)、100ng/mLのアクチビンA(Activin A)を添加したRPMI1640培地)中、80rpmにて5日間撹拌浮遊培養を行い、胚体内胚葉細胞への分化を誘導した。
なお、比較のため、ポロキサマーを添加せずに、同様に撹拌浮遊培養を行い、胚体内胚葉細胞への分化を誘導した。
分化誘導後、細胞塊を破砕し、上記の通り、生細胞数および細胞生存率を測定した。また、上記したフローサイトメトリー解析により、胚体内胚葉細胞のマーカーであるCXCR4およびSOX17のそれぞれについて、陽性細胞の割合を定量した。
結果を図9に示した。
また、胚体内胚葉細胞マーカーであるCXCR4およびSOX17のそれぞれについて、陽性である細胞の割合が増加した。
実施例10の上記結果より、ポロキサマー(ポリオキシエチレン(160)ポリオキシプロピレン(27)ブロックコポリマー)を含有する分化誘導培地にてhiPSCを撹拌浮遊培養することにより、細胞の増殖率および生存率が向上し、胚体内胚葉細胞への分化も促進されることが確認された。
また、本発明により、インスリン等を含有する動物細胞の浮遊培養用培地であって、撹拌、振とう、還流、気体通気等の物理的刺激が付加されても、インスリン等の培地成分の析出が良好に抑制された動物細胞の浮遊培養用培地を提供することができ、インスリン等を含有する動物細胞の浮遊培養用培地を用いて、撹拌、振とう、還流、気体通気等を行いながら、動物細胞の浮遊培養を行うことができる。
その結果、本発明により、大きさの制御された細胞塊を形成させて動物細胞の浮遊培養を行うことができ、動物細胞の培養効率および培養細胞の品質を向上させることができる。
特に、幹細胞等の未分化細胞について、維持培地での維持培養時、および分化誘導培地での分化誘導時のいずれにおいても培地成分の析出が抑制され、維持培養時の未分化維持率、分化誘導時の分化率の双方を向上させることができる。
Claims (23)
- 水溶性ポリマーを含有する、動物細胞の浮遊培養用添加物。
- 水溶性ポリマーが、界面活性を有する非イオン性の水溶性ポリマーである、請求項1に記載の添加物。
- 界面活性を有する非イオン性の水溶性ポリマーが、ポリビニルアルコール、ポリオキシエチレンポリオキシプロピレンブロックコポリマーおよびポリオキシエチレンソルビタンモノ脂肪酸エステルからなる群より選択される1種または2種以上である、請求項2に記載の添加物。
- 動物細胞が幹細胞である、請求項1~3のいずれか1項に記載の添加物。
- 幹細胞が、成体幹細胞、胚性幹細胞および人工多能性幹細胞からなる群より選択される1種または2種以上である、請求項4に記載の添加物。
- 培地成分の析出を抑制するための添加物である、請求項1~5のいずれか1項に記載の添加物。
- 培地成分がインスリンである、請求項6に記載の添加物。
- 水溶性ポリマーを含有する、動物細胞の浮遊培養用培地。
- 水溶性ポリマーが、界面活性を有する非イオン性の水溶性ポリマーである、請求項8に記載の培地。
- 界面活性を有する非イオン性の水溶性ポリマーが、ポリビニルアルコール、ポリオキシエチレンポリオキシプロピレンブロックコポリマーおよびポリオキシエチレンソルビタンモノ脂肪酸エステルからなる群より選択される1種または2種以上である、請求項9に記載の培地。
- 幹細胞の浮遊培養用である、請求項8~10のいずれか1項に記載の培地。
- 幹細胞が、成体幹細胞、胚性幹細胞および人工多能性幹細胞からなる群より選択される1種または2種以上である、請求項11に記載の培地。
- 培地成分の析出が抑制されている、請求項8~12のいずれか1項に記載の培地。
- 培地成分がインスリンである、請求項13に記載の培地。
- 水溶性ポリマーを含有する培地にて動物細胞を浮遊培養することを含む、動物細胞の浮遊培養方法。
- 水溶性ポリマーが、界面活性を有する非イオン性の水溶性ポリマーである、請求項15に記載の培養方法。
- 界面活性を有する非イオン性の水溶性ポリマーが、ポリビニルアルコール、ポリオキシエチレンポリオキシプロピレンブロックコポリマーおよびポリオキシエチレンソルビタンモノ脂肪酸エステルからなる群より選択される1種または2種以上である、請求項16に記載の培養方法。
- 動物細胞が幹細胞である、請求項15~17のいずれか1項に記載の培養方法。
- 幹細胞が、成体幹細胞、胚性幹細胞および人工多能性幹細胞からなる群より選択される1種または2種以上である、請求項18に記載の培養方法。
- 撹拌、振とう、還流または気体通気を行って浮遊培養する、請求項15~19のいずれか1項に記載の培養方法。
- 培地成分の析出が抑制された培地にて動物細胞を浮遊培養する、請求項15~20のいずれか1項に記載の培養方法。
- 培地成分がインスリンである、請求項21に記載の培養方法。
- 細胞塊を形成させて動物細胞を浮遊培養する、請求項15~22のいずれか1項に記載の培養方法。
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CN201980049708.7A CN112469814A (zh) | 2018-07-27 | 2019-07-26 | 动物细胞的悬浮培养用添加物、悬浮培养用培养基和悬浮培养方法 |
KR1020217005944A KR20210041003A (ko) | 2018-07-27 | 2019-07-26 | 동물 세포의 부유 배양용 첨가물, 부유 배양용 배지 및 부유 배양 방법 |
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EP3831932A1 (en) | 2021-06-09 |
JPWO2020022511A1 (ja) | 2021-08-02 |
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