Additive for suspension culture of animal cells, culture medium for suspension culture, and suspension culture method

Abstract

To provide a culture method for efficiently proliferating animal cells for their application to research, substance production, medical treatment, and the like.SOLUTION: The present invention relates to a method for the suspension culture of a stem cell through stirring, shaking, circulation, or gas bubbling within an insulin-added medium for stem cell suspension culture, the method employing one or more selected from the group consisting of polyvinyl alcohols, polyoxyethylene polyoxypropylene block copolymers, and polyoxyethylene sorbitan mono-fatty acid esters in order to suppress insulin deposition within the medium caused by the stirring, shaking, circulation, or gas bubbling.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an additive for suspension culture of animal cells, a medium for suspension culture, and a suspension culture method.

Background Art

[0002] Many animal cells, including stem cells such as embryonic stem cells and induced pluripotent stem cells, have been maintained and proliferated by adherent culture using a scaffold material such as Matrigel or a human recombinant matrix such as vitronectin or laminin. However, in order to apply animal cells to research, substance production, medicine, etc., a culture method for efficiently growing them is required. As methods for culturing a large amount of animal cells, a method of suspension culture by stirring with a stirring blade, a method of culturing by refluxing the medium using a peristaltic pump, a method of culturing while performing gas aeration using a sparger from the bottom surface, etc. are widely used. In addition, in the culture of many animal cells, a serum-free medium that does not contain serum that may contain unknown factors, prions, viruses, etc., or a low albumin medium with a low albumin content is used. When suspension culture accompanied by stirring or the like is performed using such a medium, it has been reported that precipitation occurs in the medium, and it is considered that insulin added as a factor necessary for cell growth in the serum-free medium or low albumin medium precipitated due to physical stimuli such as stirring, reflux, and gas aeration (Non-Patent Document 1). When such precipitation of medium components occurs, the cell growth rate decreases. Therefore, in order to efficiently culture animal cells, it is desirable to suppress such precipitation.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

[0004] The present invention has been made under the above circumstances. The inventors confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) that the precipitate generated by physical stimulation in suspension culture using serum-free medium or low-albumin medium is insulin. Accordingly, 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 that can suppress precipitation of medium components such as insulin due to physical stimulation such as stirring, shaking, reflux, and gas aeration in suspension culture of animal cells, and improve the culture efficiency of animal cells and the quality of cultured cells. [Means for Solving the Problems]

[0005] As a result of intensive studies to solve the above problems, the inventors have found that by adding a water-soluble polymer to a medium for suspension culture of animal cells containing insulin or the like, precipitation of medium components such as insulin caused by physical stimulation can be well suppressed, and the present invention has been completed.

[0006] That is, the present invention relates to the following. [1] An additive for suspension culture of animal cells containing a water-soluble polymer. [2] The additive according to [1], wherein the water-soluble polymer is a nonionic water-soluble polymer having surface activity. [3] The additive according to [2], wherein 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. [4] The additive according to any one of [1] to [3], wherein the animal cell is a stem cell. [5] The additive according to [4], wherein the stem cell is one or more selected from the group consisting of adult stem cells, embryonic stem cells, and induced pluripotent stem cells. The additive according to any one of [1] to [5], which is an additive for suppressing precipitation of a medium component. The additive according to [6], wherein the medium component is insulin. A medium for suspension culture of animal cells, containing a water-soluble polymer. The medium according to [8], wherein the water-soluble polymer is a nonionic water-soluble polymer having surface activity. The medium according to [9], wherein 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 mono-fatty acid ester. The medium according to any one of [8] to

[10] , which is for suspension culture of stem cells. The medium according to

[11] , 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 medium according to any one of [8] to

[12] , wherein precipitation of the medium component is suppressed. The medium according to

[13] , wherein the medium component is insulin. A method for suspension culture of animal cells, including suspension-culturing animal cells in a medium containing a water-soluble polymer. The culture method according to

[15] , wherein the water-soluble polymer is a nonionic water-soluble polymer having surface activity. The culture method according to

[16] , wherein 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 mono-fatty acid ester. The culture method according to any one of

[15] to

[17] , wherein the animal cells are stem cells. 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.

[20] The culture method according to any one of

[15] to

[19] , wherein suspension culture is performed by stirring, shaking, refluxing, or gas aeration.

[21] The culture method according to any one of

[15] to

[20] , wherein animal cells are suspension-cultured in a medium in which precipitation of medium components is suppressed.

[22] The culture method according to

[21] , wherein the medium component is insulin.

[23] The culture method according to any one of

[15] to

[22] , wherein animal cells are suspension-cultured by forming cell aggregates.

Advantages of the Invention

[0007] According to the present invention, an additive capable of favorably suppressing precipitation of medium components such as insulin caused by physical stimuli such as stirring, shaking, refluxing, and gas aeration can be provided by adding it to a medium for suspension culture of animal cells containing insulin or the like. Further, according to the present invention, a medium for suspension culture of animal cells containing insulin or the like, in which precipitation of medium components such as insulin is favorably suppressed even when physical stimuli such as stirring, shaking, refluxing, and gas aeration are applied, can be provided, and suspension culture of animal cells can be performed while performing stirring, shaking, refluxing, gas aeration, etc. using the medium for suspension culture of animal cells containing insulin or the like. As a result, cell aggregates with controlled size can be efficiently formed, and the culture efficiency of animal cells and the quality of cultured cells can be improved. In particular, for 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 both the undifferentiated maintenance rate during maintenance culture and the differentiation rate during differentiation induction can be improved.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Figure 3

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Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0009] The present invention provides an additive for suspension culture of animal cells (hereinafter also referred to as "the additive of the present invention") added to a medium for suspension culture of animal cells containing insulin or the like. The additive of the present invention contains a water-soluble polymer.

[0010] In the present invention, 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 usually, a polymer showing water miscibility or water solubility with a weight average molecular weight of about 1,000 to 100,000 measured by size exclusion chromatography is used.

[0011] Examples of such water-soluble polymers include carboxyvinyl polymer; polyvinyl alcohol; polyvinylpyrrolidone; polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene fatty acid ester, polyoxyethylene polyhydric alcohol fatty acid partial ester (such as polyoxyethylene glycerin fatty acid partial ester, polyoxyethylene sorbitol fatty acid partial ester, polyoxyethylene sorbitan fatty acid partial ester, etc.), polyoxyethylene hydrogenated castor oil, polyoxyethylene alkylamine and other polyoxyethylene type nonionic surfactants; polyoxyethylene polyoxypropylene random copolymer, polyoxyethylene polyoxypropylene block copolymer (poloxamer), polyoxyethylene polyoxypropylene alkyl ether and other polyoxyethylene polyoxypropylene type nonionic surfactants; polyglycerin fatty acid ester and other polyglycerin type nonionic surfactants.

[0012] For the purpose of the present invention, as the water-soluble polymer, a nonionic water-soluble polymer having surface activity is preferably used, and polyvinyl alcohol, polyoxyethylene type nonionic surfactant, polyoxyethylene polyoxypropylene type nonionic surfactant, and polyglycerin type nonionic surfactant are exemplified as preferable water-soluble polymers. Among them, polyvinyl alcohol, polyoxyethylene polyhydric alcohol fatty acid partial ester, and polyoxyethylene polyoxypropylene block copolymer (poloxamer) are more preferably used, and polyvinyl alcohol, polyoxyethylene sorbitan mono-fatty acid ester (polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, etc.), and polyoxyethylene polyoxypropylene block copolymer (poloxamer) are particularly preferably used.

[0013] For the additive of the present invention, one kind of water-soluble polymer may be selected and used alone, or two or more kinds may be selected and 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.

[0014] In the present invention, the above-mentioned water-soluble polymer may be used as the additive of the present invention as it is, or it may be dissolved or dispersed in a solvent such as water or polyhydric alcohol, in a liquid form such as an aqueous solution or a dispersion, or mixed with additives generally used in formulation such as excipients and binders, and sized, granulated, tabletted, etc. to be an additive in a solid form such as powder, granule, tablet, etc. Also, 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 it is easy to add to the medium for suspension culture of animal cells and easy to mix with the medium, the additive of the present invention is preferably provided in the form of liquid, powder, granule, tablet, etc.

[0015] The additive of the present invention is preferably prepared by sterilization treatment. The method of 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 sterilization, etc., and can be appropriately selected according to the form of the additive of the present invention.

[0016] 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 the preparation of the medium for suspension culture of animal cells, and can also be added to and used in the medium for suspension culture of animal cells described below.

[0017] By adding the additive of the present invention to a medium for suspension culture of animal cells containing insulin or the like, particularly when the medium for suspension culture of the animal cells is a serum-free medium or a low albumin medium, precipitation of medium components such as insulin caused by physical stimuli such as stirring, shaking, reflux, and gas aeration can be well suppressed, efficient formation of cell aggregates with controlled size is possible, and the culture efficiency of animal cells and the quality of cultured cells can be improved. By adding the additive of the present invention to a maintenance medium or a differentiation induction medium for undifferentiated cells such as stem cells, precipitation of medium components during maintenance culture and differentiation induction can be suppressed, and both the undifferentiated maintenance rate during maintenance culture and the differentiation rate during differentiation induction can be improved.

[0018] The present invention also provides a medium for suspension culture of animal cells (hereinafter also referred to as "the medium of the present invention" in this specification). Here, examples of animal cells include normal cells, stem cells, and progenitor cells derived from mammals.

[0019] Examples of normal cells derived from mammals include germ cells such as sperm and eggs, and somatic cells constituting the living body. Examples of somatic cells that make up a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, osteocytes, bone marrow cells, pericytes, dendritic cells, adipocytes, mesenchymal cells, epithelial cells, epidermal cells (e.g., keratinocytes, horny cells, etc.), endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nerve cells, glial cells, oligodendrocytes (oligodendroglia), microglia (microglia), astrocytes (astrocytes), heart cells, esophageal cells, muscle cells (e.g., smooth muscle cells, skeletal muscle cells), pancreatic beta cells, melanocytes, and mononuclear cells, etc. The somatic cells include cells collected from any tissue such as, 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, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eye, brain, nerve tissue, etc.

[0020] A stem cell refers to a cell that has the ability of self-renewal and the ability to differentiate into another type of cell and can proliferate without limit. For example, adult stem cells such as hematopoietic stem cells, satellite cells, neural stem cells, mesenchymal stem cells, mammary stem cells, olfactory mucosa 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, etc.; pluripotent stem cells such as embryonic stem cells (ES cells), embryonic carcinoma cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), etc.; cancer stem cells, etc.

[0021] A progenitor cell is a cell in an intermediate stage of differentiating from the stem cell into a specific somatic cell or germ cell, and examples include satellite cells, pancreatic progenitor cells, vascular progenitor cells, vascular endothelial progenitor cells, hematopoietic progenitor cells (CD34-positive cells derived from umbilical cord blood, etc.).

[0022] 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, and even more preferably provided as a medium for suspension culture of embryonic stem cells and induced pluripotent stem cells.

[0023] The medium of the present invention contains a water-soluble polymer together with the medium components usually used for suspension culture of the above-mentioned animal cells. Regarding the water-soluble polymer contained in the medium of the present invention, it is as described above in the additives of the present invention. In the medium of the present invention, the water-soluble polymer can be contained alone as one kind or in combination of two or more kinds. In the present invention, the water-soluble polymer may be contained together with the medium components in the state prepared as the additive of the present invention described above, or may be directly added to the medium components. The content of the water-soluble polymer in the medium of the present invention is usually 0.1 μg / mL to 10 mg / mL, preferably 1 μg / mL to 5 mg / mL, more preferably 10 μg / mL to 5 mg / mL, and still more preferably 10 μg / mL to 1 mg / mL as the final concentration during culture.

[0024] Examples of medium components that can be included in the medium of the present invention include medium components commonly used for culturing animal cells. For example, sugars such as glucose, fructose, sucrose, and maltose; amino acids such as asparagine, aspartic acid, glutamine, and glutamic acid; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soy peptide; serum; vitamins such as vitamin A, vitamin B group (thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, nicotinamide, etc.), vitamin C, and vitamin E; fatty acids such as oleic acid, arachidonic acid, and linoleic acid; lipids such as cholesterol; inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate; trace elements such as zinc, copper, and selenium; buffers such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; cell adhesion factors and extracellular matrix components such as Type I collagen, Type II collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and activin A; hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin, etc. Appropriate components can be selected and used according to the type of animal cells to be cultured.

[0025] When animal cells are undifferentiated cells such as stem cells, components that suppress the differentiation of the stem cells or the like can be added to the maintenance medium for maintaining the stem cells or the like in an undifferentiated state. In addition, components that induce or promote the differentiation of stem cells or the like can be added to the differentiation-inducing medium for inducing the differentiation of stem cells or the like. Examples of components that suppress the differentiation of stem cells or the like include leukemia inhibitory factor (LIF), which is a differentiation inhibitor of embryonic stem cells, fibroblast growth factor (FGF), transforming growth factor (TGF)-β, bone morphogenetic protein (BMP) that suppresses the differentiation of neural stem cells, Notch protein, and polycomb complex that suppresses the differentiation of embryonic stem cells and iPS cells. Examples of components that induce or promote the differentiation of stem cells or the like include activin A, which induces embryonic stem cells into endodermal cells, retinoic acid, bone morphogenetic protein (BMP) inhibitor (such as noggin) that induces the differentiation of iPS cells into neuroectoderm, transforming growth factor (TGF)-β, extracellular secreted glycoprotein (WNT) that induces the differentiation of iPS cells into mesoderm, activin that induces the differentiation of iPS cells into mesoderm and endoderm, glycogen synthase kinase 3 (GSK3) inhibitor, and the like. After initial differentiation into ectodermal cells, mesodermal cells, and endodermal cells, when differentiating into cells of each organ or tissue, necessary growth factors, nutritional factors, etc. can be added according to the organ or tissue to be induced for differentiation. For example, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), etc. are used.

[0026] Since serum may contain unknown factors, prions, viruses, etc., it is preferable that the medium of the present invention does not contain serum as a medium component. In addition, when the medium of the present invention is prepared as a medium for culturing human cells, it is preferable that the medium does not contain components derived from animals other than humans.

[0027] In the present invention, the water-soluble polymer may be contained in a medium commonly used for suspension culture of animal cells such as the above-described normal cells, stem cells, and progenitor cells derived from mammals, and used as the medium of the present invention. Examples of media used for culturing normal cells derived from mammalian cells include Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s Nutrient Mixture F12, DMEM / F12 medium, McCoy’s 5A medium, Minimum Essential Medium (MEM), Eagle’s Minimum Essential Medium (EMEM), alpha Modified Eagle’s Minimum Essential Medium (αMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove’s Modified Dulbecco’s Medium (IMDM), MCDB131 medium, William’s Medium E, Fischer’s Medium, and the like.

[0028] Examples of media used for culturing stem cells include STEMPRO (registered trademark) hESC SFM medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), TeSR-E8 medium (STEMCELL Technologies), Essential 8 medium (Life Technologies), HEScGRO (trademark) Serum-Free Medium for hES cells (Millipore), PluriSTEM (trademark) Human ES / iPS Medium (EMD Millipore), NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), NutriStem (trademark) XF / FF Culture Medium (Stemgent), AF NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), S-medium (DS Pharma Biomedical), StemFit (registered trademark) AK03N medium (Ajinomoto), hESF9 medium, hESF-FX medium, CDM medium, DEF-CS 500 Xeno-Free 3D Spheroid Culture Medium (Cellartis), StemFlex medium (Thermo Fisher Scientific), and the like.

[0029] Examples of media used for culturing progenitor cells include HPGM (trademark) (Chemicon), QBSF-60 (Quality Biological), and the like.

[0030] In the present invention, a water-soluble polymer may also be added to a differentiation induction medium for stem cells or the like. Examples of the differentiation induction medium for stem cells or the like include TeSR-E6 medium (STEMCELL Technologies), TeSR-E7 medium (STEMCELL Technologies), Essential 6 (Thermo Fisher Scientific), and the like.

[0031] For the purpose of the present invention, it is preferable to use a feeder-free medium for culturing animal cells, more preferably a serum-free medium or a low-albumin medium. Further, for use as a medium for culturing human cells, a medium that does not contain components derived from animals other than humans (xeno-free medium) is preferable. In addition, since the effects of the present invention can be obtained more remarkably, the medium of the present invention is preferably a serum-free medium or a low-albumin medium containing insulin.

[0032] Furthermore, from the viewpoint of use in suspension culture of animal cells, the medium of the present invention is preferably in a liquid form such as a solution or a dispersion.

[0033] The medium of the present invention can be prepared by appropriately selecting components from the above-described medium components according to a known composition, adding them together with a water-soluble polymer to a solvent such as water, and dissolving or dispersing them. In addition, the medium of the present invention can be prepared by adding a water-soluble polymer to the above-described animal cell culture medium provided by each company or institution and dissolving or dispersing it. Furthermore, the medium of the present invention can be prepared in a concentrated state higher than the concentration at the time of use or in a lyophilized powder state, and diluted with a solvent such as water or dissolved in a solvent such as water at the time of use. The medium of the present invention is preferably prepared by performing the sterilization treatment as described above.

[0034] When suspension-culturing animal cells using the medium of the present invention, precipitation of medium components such as insulin caused by physical stimuli such as stirring, shaking, reflux, and gas aeration can be favorably suppressed, efficient formation of cell aggregates with controlled sizes is possible, and the culture efficiency of animal cells and the quality of cultured cells can be improved. The medium of the present invention can be suitably used as a medium for maintenance culture of undifferentiated cells such as stem cells and as a medium for induction of differentiation. When maintaining or inducing differentiation of undifferentiated cells such as stem cells, precipitation of medium components can be suppressed, and both the undifferentiated cell maintenance rate during maintenance culture and the differentiation rate during induction of differentiation can be improved.

[0035] Furthermore, the present invention provides a method for suspension-culturing animal cells (hereinafter, also referred to as "the culturing method of the present invention" in this specification). The culturing method of the present invention includes suspension-culturing animal cells in a medium for suspension-culturing animal cells containing a water-soluble polymer.

[0036] The "medium for suspension-culturing animal cells containing a water-soluble polymer" is as described above. In the present invention, the water-soluble polymer contained in the medium for suspension-culturing animal cells may be prepared and added as the above-described additive of the present invention, or the water-soluble polymer itself may be directly added. Also, in the present invention, the water-soluble polymer is added to the medium so as to have a concentration of usually 0.1 μg / mL to 10 mg / mL as the final concentration during culture, preferably added to the medium so as to have a concentration of 1 μg / mL to 5 mg / mL, more preferably added to the medium so as to have a concentration of 10 μg / mL to 5 mg / mL, and even more preferably added to the medium so as to have a concentration of 10 μg / mL to 1 mg / mL.

[0037] In the culture method of the present invention, the suspension culture of animal cells can be carried out according to the method of ordinary suspension culture. That is, according to the culture scale, appropriate culture utensils or culture devices such as cell culture plates, cell culture flasks, and bioreactors are used, and animal cells are seeded in the suspension culture medium for animal cells added with the medium of the present invention or the additive of the present invention described above, and usually at 25°C to 39°C, preferably 33°C to 39°C, usually in the presence of 4% by volume to 10% by volume, preferably 4% by volume to 6% by volume of carbon dioxide, and usually in the presence of 1% by volume to 25% by volume, preferably 4% by volume to 20% by volume of oxygen, and cultured for usually 1 day to 30 days, preferably 2 days to 14 days. In addition, the medium is changed every 2 to 3 days. For medium change, after separating animal cells and the medium by centrifugation or filtration, a new medium may be added to the animal cells. Alternatively, after appropriately concentrating the animal cells by centrifugation or filtration, a new medium may be added to this concentrated cell solution. The gravitational acceleration (G) during the above centrifugation is usually 50G to 1,000G, preferably 100G to 500G, and the pore size of the filter used for filtration is usually 10 μm to 200 μm.

[0038] The culture method of the present invention can be carried out by performing stirring, shaking, reflux, gas aeration, etc. Stirring can be carried out using a bioreactor, a culture tank with stirring blades, etc. Stirring is usually carried out at a stirring speed of 10 rpm to 2,000 rpm, preferably 40 rpm to 1,000 rpm. Shaking can be carried out using a shaker or a shaking incubator. Shaking is usually carried out at a shaking speed of 10 rpm to 500 rpm, preferably 50 rpm to 250 rpm. Reflux can be carried out using a peristaltic pump, a tubing pump, etc. As the reflux tube, a peristaltic pump tube made of silicone, neoprene (chloroprene rubber), marprene (polypropylene·ethylene propylene rubber), a tubing pump tube, etc. are used. The reflux is usually carried out at a flow rate of 10 μL / min to 1000 mL / min, preferably 1 mL / min to 100 mL / min. Gas aeration can be carried out using various spargers such as a micro sparger and a filter sparger. Gas aeration can usually be carried out at an aeration rate of 1 mL / min to 1000 mL / min, preferably 50 mL / min to 200 mL / min. In order to efficiently obtain cell aggregates with controlled size, suspension culture of animal cells is preferably carried out with stirring or shaking.

[0039] The cultured animal cells can be recovered by centrifugation or filtration using a filter. Centrifugation is carried out at 50 G to 1,000 G, preferably 100 G to 500 G for about 1 minute to 10 minutes. Also, filtration can be carried out using a filter having pores of about 10 μm to 200 μm.

[0040] 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.).

[0041] By the culture method of the present invention, when suspension culture of animal cells is carried out with stirring, shaking, reflux, gas aeration, etc., precipitation of medium components such as insulin caused by physical stimulation can be favorably suppressed, and cell aggregates with controlled size can be formed for suspension culture. 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 both maintenance culture and differentiation induction of undifferentiated cells such as stem cells. When performing maintenance culture or differentiation induction of undifferentiated cells such as stem cells, precipitation of medium components can be suppressed, and both the undifferentiated maintenance rate during maintenance culture and the differentiation rate during differentiation induction can be improved.

Example

[0042] Hereinafter, the present invention will be described in more detail with reference to examples.

[0043] In the following examples, using the following culture medium for stem cells and the following water-soluble polymers, undifferentiated human iPS cells (hiPSC) were used as stem cells, and suspension culture by stirring was performed as follows.

[0044] (1) As the culture medium for stem cells, Essential 8 medium (Thermo Fisher Scientific, A1517001), StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.), and the differentiation induction medium described later were used.

[0045] (2) As the water-soluble polymers, polyvinyl alcohol (PVA) (Nippon Synthetic Chemical Industry Co., Ltd., EG-03P), poloxamer (polyoxyethylene (160) polyoxypropylene (27) block copolymer) (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) were used.

[0046] (3) Evaluation of precipitation of insulin in the medium by stirring As a stirring culture device, a single-use bioreactor with a volume of 5 mL (ABLE Corporation, S-1467) was used. 5 mL of medium was added to the culture device, and stirring was carried out at 120 rpm for 24 hours under the conditions of 37 °C, 5% by volume of carbon dioxide, and 20% by volume of oxygen. Then, the medium was transferred to a 6-well cell culture plate and observed with an inverted microscope ("CKX41", Olympus Corporation, magnification = 40 times), and photography was performed.

[0047] (4) Suspension cell culture of undifferentiated human iPS cells (hiPSC) by stirring As the undifferentiated human iPS cells (hiPSC), hiPS cells of the 1210B2 strain (see Nakagawa, M. et al., Sci. Rep. 4, 3594, 2014) were used. Suspension cell culture by stirring was performed using a single-use bioreactor with a volume of 5 mL (ABLE Corporation, S-1467) as the culture device. 5 mL of a medium containing 10 μM of Rho-associated coiled-coil kinase inhibitor (Y-27632) (FUJIFILM Wako Pure Chemical Corporation, 034-24024) was added to the above bioreactor, single-cell hiPSC was added, and stirring culture was carried out at 80 rpm under the conditions of 37 °C, 5% by volume of carbon dioxide, and 20% by volume of oxygen. Medium exchange was performed after the second day. The medium exchange was carried out by extracting the amount of medium supernatant described in each example, centrifuging at 200G for 5 minutes, removing the supernatant, adding the same amount of new medium, suspending the pellet, and then adding it to the bioreactor.

[0048] Also, in each of the following examples, the measurement of the number of cell aggregates and the major axis, the measurement of the number of cells and the survival rate, the undifferentiated maintenance rate of the cells, and the differentiation rate into embryonic endoderm cells of the cultured stem cells were carried out as follows.

[0049] (1) Measurement of the number of cell aggregates and the major axis 500 μL of the culture supernatant containing cell aggregates was aliquoted into a 24-well plate. After shaking the cell aggregates to disperse them, the entire well was photographed using a BZ-X fluorescence microscope (Keyence Corporation). By performing a macro cell count on the photographed image, the number of cell aggregates and the average major axis length were determined.

[0050] (2) Measurement of cell number and viability The entire volume of the culture supernatant containing cell aggregates was collected and centrifuged at 500 G for 5 min. After removing the supernatant, tapping was performed 10 times, 1 mL of cell separation / dispersion solution (Accumax (Millipore, SCR006)) was added, and the cell aggregate pellet was suspended. After incubating at room temperature for 5 min, the cell aggregates were resuspended by pipetting. After incubating at room temperature for 5 min again, the cell aggregates were made into single cells by pipetting. 4 mL of medium was added and centrifuged at 500 G for 5 min. After removing the supernatant, the pellet was disrupted by tapping 10 times, and the cells were resuspended by adding 1 mL of medium containing Rho-associated coiled-coil kinase inhibitor (Y-27632) and pipetting. The suspension was passed through a 40-μm cell strainer (BD Falcon (Corning), 2-1919-02), and the cell strainer was co-washed with 1 mL of medium containing Rho-associated coiled-coil kinase inhibitor (Y-27632). The collected cell suspension was analyzed using a viable cell autoanalyzer Vi-CELL XR (Beckman Coulter, Inc.) to measure the cell number and viability. The viability of the cells was determined by dividing the number of live cells in the collected cells by the total number of collected cells, and the growth rate (fold) of the cells was determined by dividing the number of live cells by the number of seeded cells.

[0051] (3) Measurement of undifferentiated maintenance rate of cells The cells that had been single-celled after culturing were fixed with a cell immobilization / cell permeabilization solution (BD Cytofix / Cytoperm™ Kit (BD Biosciences, 554714)). Specifically, 200 μL of Cytofix / Cytoperm was added, and the hiPSCs were fixed by allowing them to stand on ice for 20 minutes. Next, 1 mL of BD Perm / Wash buffer™ (BD Biosciences, 554723) was added, and centrifugation was performed at 5,000 rpm for 2 minutes to remove the supernatant. Next, it was suspended in an appropriate amount of BD Perm / Wash buffer™, divided into samples for double staining, single staining, isotype control, and non-staining, and each was dispensed into a centrifuge tube, followed by centrifugation at 5,000 rpm for 2 minutes to remove the supernatant. Double staining and single staining were performed by adding 100 μL of a solution in which both, or one of, Alexa Fluor® 488 mouse anti-oct3 / 4 (Becton Dickinson, 560253) diluted 1:5 (5-fold) and Alexa Fluor® 647 mouse anti-SSEA-4 (Becton Dickinson, 560796) diluted 1:10 (10-fold) was added to BD Perm / Wash buffer™, and incubating at room temperature in the dark for 20 minutes. For the isotype control sample, 100 μL of BD Perm / Wash buffer™ supplemented with Alexa Fluor® 488 Mouse IgG1 κ Isotype Control (Becton Dickinson, 557721) diluted 1:20 (20-fold) or Alexa Fluor® 647 Mouse IgG3, κ Isotype Control (Becton Dickinson, 560803) diluted 1:20 (20-fold) was added, and incubation was carried out in the dark at room temperature for 20 min in the same manner. After each of the above reactions, 500 μL of BD Perm / Wash buffer™ was added, and centrifugation was performed at 5,000 rpm for 2 min to remove the supernatant. 1 mL of Focusing fluid (Thermo Fisher Scientific, 4488621) was added to each sample, centrifugation was performed again at 5,000 rpm for 2 min, and then the sample was suspended in 200 μL of Focusing fluid (Thermo Fisher Scientific, 4488621). The prepared samples were analyzed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). Alexa Fluor® 488 was detected using BL1, and Alexa Fluor® 647 was detected using RL1. The undifferentiated maintenance rate of cells can be represented by the positive rate of Oct3 / 4 / SSEA4 in the cultured cells.

[0052] (4) Measurement of the differentiation rate of hiPSCs into definitive endoderm cells The cells that were single-celled after culturing were centrifuged at 5,000 rpm for 2 minutes. The cell pellet was suspended in 100 μL of BD Perm / Wash buffer (trademark) (BD Biosciences, 554723), and 1 μL of BD Pharmingen (trademark) APC Mouse Anti-Human CD184 (BD Biosciences, 560936) or 1 μL of BD Pharmingen (trademark) APC Mouse IgG2a, κ Isotype Control (BD Biosciences, 555576) was added, and staining was performed by incubating at room temperature in the dark for 20 minutes. The stained cells were washed once with 1000 μL of FACS buffer, centrifuged again to obtain a pellet, and fixed with a cell fixation / cell permeabilization solution (BD Cytofix / Cytoperm (trademark) Kit, BD Biosciences, 554714). Specifically, 200 μL of Cytofix / Cytoperm was added, and the cells were fixed by allowing them to stand on ice for 20 minutes. Next, 1 mL of BD Perm / Wash buffer was added, centrifuged at 5,000 rpm for 2 minutes to remove the supernatant, and suspended in 200 μL of BD Perm / Wash buffer (trademark). 1 μL of BD Pharmingen (trademark) PE Mouse anti-Human Sox17 (BD Biosciences, 561591) or BD Pharmingen (trademark) PE Mouse IgG1,κ Isotype Control (BD Biosciences, 400139) was added, and the reaction was allowed to proceed by incubating at room temperature in the dark for 20 minutes. After the above reaction, 500 μL of BD Perm / Wash buffer (trademark) was added, and centrifugation was performed at 5,000 rpm for 2 minutes. The supernatant was removed. 1 mL of Focusing fluid (Thermo Fisher Scientific, 4488621) was added to each sample, and after centrifugation at 5,000 rpm for 2 minutes again, the sample was suspended in 200 μL of Focusing fluid (Thermo Fisher Scientific, 4488621). The prepared samples were analyzed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). Detection of PE was performed using BL1, and detection of APC was performed using RL1. The differentiation rate of cells can be represented by the positive rate of CXCR4 or SOX17 in the cultured cells.

[0053] [Example 1] Examination of the effects of various water-soluble polymers on insulin precipitation To the Essential 8 medium containing insulin, 1 mg / mL of each of the above water-soluble polymers was added, and stirring was performed at 120 rpm for 24 hours in a stirring culture apparatus as described above. Then, the state of the medium was observed with an inverted microscope, and the insulin precipitation inhibitory effect of the water-soluble polymer was evaluated according to the following evaluation criteria based on the degree of insulin precipitation in the medium. For comparison, the same treatment was performed without adding the water-soluble polymer, and the degree of insulin precipitation in the medium was evaluated. The results are shown in Table 1. <Evaluation criteria> Very good (insulin precipitation is completely suppressed); ++ Good (insulin precipitation is almost suppressed); + No inhibitory effect (insulin precipitation is observed); -

[0054]

Table 1

[0055] As shown in Table 1, when polyvinyl alcohol, various poloxamers, and various polyoxyethylene sorbitan monofatty acid esters were added, it was found that the precipitation of insulin by stirring was well suppressed. From the above results of Example 1, it became clear that by adding a water-soluble polymer such as polyvinyl alcohol to the insulin-containing medium, the precipitation of insulin that occurs when the medium is stirred can be suppressed.

[0056] [Example 2] Examination of the influence of the addition concentrations of polyvinyl alcohol and poloxamer on the insulin precipitation inhibitory effect To the Essential 8 medium containing insulin, polyvinyl alcohol (PVA) at 500 ng / mL to 10 mg / mL or poloxamer (Kolliphor P188 BIO) at 100 ng / mL to 1 mg / mL was added, and as described above, stirring was carried out at 120 rpm for 24 hours in a 5 mL bioreactor. 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 photography was performed under an inverted microscope. The photographs taken under the microscope are shown in Figure 1.

[0057] As shown in Figure 1, when the Essential 8 medium containing insulin was stirred in the bioreactor, precipitation of insulin was observed. In contrast, when each concentration of polyvinyl alcohol (PVA) and poloxamer (Kolliphor P188 BIO) was added, it was confirmed that the precipitation of insulin was suppressed at any concentration. From the above results of Example 2, it became clear that by adding polyvinyl alcohol at 500 ng / mL or more and poloxamer (polyoxyethylene (160) polyoxypropylene (27) block copolymer) at 100 ng / mL or more to the insulin-containing medium, the precipitation of insulin caused by stirring the medium can be suppressed.

[0058] [Example 3] Examination of the effect of poloxamer (Kolliphor P188 BIO) on the proliferation of hiPSCs Poloxamer (Kolliphor P188 BIO) at a concentration of 0.1 μg / mL to 1 mg / mL was added to Essential 8 medium containing insulin, and hiPSCs were cultured by stirring and suspension for 4 days to evaluate the effect of each concentration of poloxamer. hiPSCs of the 1210B2 strain were seeded at 1x10 6 cells in a 5 mL bioreactor, and stirred culture was performed at a stirring speed of 80 rpm as described above. On the 3rd day, 3.5 mL of the medium was exchanged, and on the 4th day, the cell mass was disrupted to determine the cell growth rate, survival rate, and undifferentiated maintenance rate. The results are shown in Figure 2.

[0059] As shown in Figure 2, when hiPSCs were cultured by stirring and suspension in a medium supplemented with poloxamer (Kolliphor P188 BIO) at each concentration, the cell growth rate, survival rate, and undifferentiated maintenance rate were improved. From the above results of Example 3, by adding poloxamer (polyoxyethylene (160) polyoxypropylene (27) block copolymer) (Kolliphor P188 BIO) to the medium at a concentration of 0.1 μg / mL to 1 mg / mL and performing stirring and suspension culture of hiPSCs, precipitation of insulin in the medium was suppressed, growth of cell masses was promoted, and it was revealed that it became possible to efficiently proliferate hiPSCs in good condition.

[0060] [Example 4] Examination of the effect of poloxamer (Kolliphor P407) on the proliferation of hiPSCs Poloxamer (Kolliphor P407) at a concentration of 1 μg / mL to 1 mg / mL was added to Essential 8 medium containing insulin, and hiPSCs were cultured by stirring and suspension for 5 days to evaluate the effect of each concentration of poloxamer. hiPSCs of the 1210B2 strain were seeded at 1x10 6Cells were seeded and cultured with stirring at a stirring speed of 80 rpm as described above. On the 2nd and 3rd days, 3.5 mL of the medium was exchanged. On the 5th day, the cell mass was disrupted and the cell growth rate was determined. The results are shown in Figure 3.

[0061] As shown in Figure 3, when cultured in a medium supplemented with 1 μg / mL to 1 mg / mL of poloxamer (Kolliphor P407), the cell growth rate was improved. From the above results of Example 4, by adding poloxamer (polyoxyethylene (202) polyoxypropylene (56) block copolymer) (Kolliphor P407) to the medium at a concentration of 1 μg / mL to 1 mg / mL and performing stirring suspension culture of hiPSCs, precipitation of insulin in the medium was suppressed, growth of cell masses was promoted, and it was revealed that it became possible to efficiently proliferate hiPSCs in good condition.

[0062] [Example 5] Examination of the effect of polyvinyl alcohol (PVA) on the proliferation of hiPSCs 50 μg / mL to 5 mg / mL of polyvinyl alcohol (PVA) was added to Essential 8 medium containing insulin, and hiPSCs were cultured with stirring for 4 days to evaluate the effect of each concentration of polyvinyl alcohol. hiPSCs of the 1210B2 strain were seeded into a 5 mL bioreactor at 1x10 6 Cells were seeded and cultured with stirring suspension at a stirring speed of 80 rpm as described above. On the 2nd day of culture, 3.5 mL of the medium was exchanged. On the 4th day, the cell mass was disrupted and the cell growth rate was determined. The results are shown in Figure 4.

[0063] As shown in Figure 4, when cultured in a medium supplemented with 50 μg / mL to 5 mg / mL of polyvinyl alcohol (PVA), the cell growth rate was improved. From the above results of Example 5, by adding polyvinyl alcohol (PVA) to the medium at a concentration of 50 μg / mL to 5 mg / mL and performing stirring suspension culture of hiPSCs, precipitation of insulin in the medium was suppressed, growth of cell masses was promoted, and it was revealed that it became possible to efficiently proliferate hiPSCs in good condition.

[0064] [Example 6] Examination of the effect of polyoxyethylene sorbitan monolaurate (Kolliphor PS 20) on the growth of hiPSCs Polyoxyethylene sorbitan monolaurate (Kolliphor PS 20) at a concentration of 100 ng / mL to 10 μg / mL was added to Essential 8 medium containing insulin, and hiPSCs were cultured with stirring for 4 days to evaluate the effect of polyoxyethylene sorbitan monolaurate at each concentration. hiPSCs of the 1210B2 strain were seeded at 1x10 6 cells in a 5 mL bioreactor, and suspension culture with stirring was performed at a stirring speed of 80 rpm as described above. On the second day, 3.5 mL of the medium was exchanged, and on the third day, the cell clumps were disrupted to determine the cell growth rate. The results are shown in Fig. 5.

[0065] As shown in Fig. 5, when cultured in a medium supplemented with polyoxyethylene sorbitan monolaurate (Kolliphor PS 20) at a concentration of 100 ng / mL to 10 μg / mL, the cell growth rate was improved. From the above results of Example 6, by adding polyoxyethylene sorbitan monolaurate (Kolliphor PS 20) to the medium at a concentration of 100 ng / mL to 10 μg / mL and performing stirring culture of hiPSCs, precipitation of insulin in the medium was suppressed, growth of cell clumps was promoted, and it was clarified that it became possible to efficiently grow hiPSCs in good condition.

[0066] [Example 7] Examination of the effect of poloxamer on insulin precipitation by stirring using a stirring blade As a stirring culture device, an animal cell culture tank with a liquid volume of 2 L (lower magnetic stirring type) (ABLE Corporation) and a stirring blade made of stainless steel (SUS316L) were used to evaluate the effect of poloxamer on insulin precipitation by stirring. 500 mL of Essential 8 medium containing insulin and 1 mg / mL of poloxamer (Kolliphor P188 BIO) were added to the above animal cell culture tank, and stirring was carried out at 37 °C and 150 rpm for 8 hours. Then, the medium was transferred to a 6-well cell culture plate and observed with an inverted microscope ("CKX53", Olympus Corporation) (magnification = 100 times), and photography was performed. For comparison, the same treatment was carried out without adding poloxamer, and photography was performed under an inverted microscope. The photographs taken under the microscope are shown in Fig. 6.

[0067] As shown in Fig. 6, it was confirmed that insulin precipitated due to the stirring of the medium, but the addition of poloxamer (Kolliphor P188 BIO) suppressed the precipitation of insulin. From the above results of Example 7, it was clarified that by adding a poloxamer (polyoxyethylene (160) polyoxypropylene (27) block copolymer) at a concentration of 1 mg / mL to a medium containing insulin, the precipitation of insulin caused thereby can be suppressed even when the medium is stirred using a stirring blade.

[0068] [Example 8] Examination of the effect of water-soluble polymers on the precipitation of insulin by reflux As equipment for medium reflux, a peristaltic pump ("Perista·Bio Mini Pump AC-2120" (ATTO Corporation)) was used to evaluate the effect of water-soluble polymers on the precipitation of insulin by reflux. As the reflux tube, a silicone tube with an inner diameter of 3 mm and an outer diameter of 5 mm was used. In a 50 mL plastic tube, 45 mL of Essential 8 medium containing insulin, and 1 mg / mL each of poloxamer (Kolliphor P188 BIO) and polyvinyl alcohol (PVA) were added, and reflux was carried out at room temperature for 24 hours at a flow rate of 5 mL / min using the peristaltic pump. Then, the medium was transferred to a 6-well cell culture plate and observed with an inverted microscope ("CKX41", Olympus Corporation) (magnification = 40 times and 100 times), and photographs were taken. For comparison, the same treatment was carried out without adding poloxamer and polyvinyl alcohol, and photographs were taken under an inverted microscope. The photographs taken under the microscope are shown in Fig. 7.

[0069] As shown in Fig. 7, precipitation of insulin occurred by refluxing the medium, but precipitation of insulin was suppressed by adding poloxamer (Kolliphor P188 BIO) or polyvinyl alcohol (PVA) to the medium. From the above results of Example 8, it was revealed that by adding poloxamer (polyoxyethylene (160) polyoxypropylene (27) block copolymer) and polyvinyl alcohol at a concentration of 1 mg / mL each to a medium containing insulin, precipitation of insulin occurring during reflux of the medium can be suppressed.

[0070] [Example 9] Examination of the effect of water-soluble polymers on insulin precipitation in a stem cell differentiation induction medium To a differentiation induction medium containing insulin (RPMI1640 medium supplemented with 4 (w / v)% TeSR-E6 medium (STEMCELL Technologies)), 1 mg / mL of polyvinyl alcohol (PVA) and 1 mg / mL of poloxamer (Kolliphor P188 BIO) were added, and stirring was carried out at 80 rpm for 48 hours in a 5 mL bioreactor as described above. Then, the medium was observed with an inverted microscope CKX41. For comparison, the same treatment was carried out without adding polyvinyl alcohol and poloxamer, and the samples were observed under an inverted microscope. The photograph taken under the microscope was shown in Fig. 8 above.

[0071] As shown in Fig. 8, when the differentiation induction medium containing insulin was stirred in the bioreactor, precipitation of insulin was observed. In contrast, when 1 mg / mL each of polyvinyl alcohol (PVA) and poloxamer (Kolliphor P188 BIO) was added, precipitation of insulin was suppressed in both cases. From the above results of Example 9, it was confirmed that by adding poloxamer (polyoxyethylene (160) polyoxypropylene (27) block copolymer) and polyvinyl alcohol at a concentration of 1 mg / mL each to the differentiation induction medium containing insulin, precipitation of insulin caused by stirring the medium can be suppressed.

[0072] [Example 10] Examination of the effect of poloxamer (Kolliphor P188 BIO) on the induction of differentiation of hiPSCs into definitive endoderm cells 30 mL of StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.) was added to a 30 mL capacity bioreactor, and 6×10 6 hiPSCs of the 1210B2 strain were seeded, and stirred suspension culture was performed at 120 rpm for 6 days to form cell aggregates of hiPSCs. A 5 mL portion of the cell aggregates was transferred to a 5 mL capacity bioreactor, and differentiation induction culture was performed by stirring suspension at 80 rpm for 5 days in a differentiation induction medium (supplement in 4 (w / v)% TeSR-E6 medium (STEMCELL Technologies), 2 μM glycogen synthase kinase 3 inhibitor (CHIR99021), RPMI1640 medium supplemented with 100 ng / mL activin A) to which 0.1 mg / mL of poloxamer (Kolliphor P188 BIO) was added to induce differentiation into definitive endoderm cells. For comparison, stirred suspension culture was carried out in the same manner without adding poloxamer to induce differentiation into definitive endoderm cells. After induction of differentiation, the cell mass was disrupted, and the viable cell count and cell viability were measured as described above. Also, by the flow cytometry analysis described above, the percentage of positive cells was quantified for each of CXCR4 and SOX17, which are markers for definitive endoderm cells. The results are shown in Fig. 9.

[0073] As shown in Fig. 9, when hiPSCs were cultured with stirring and suspension in a differentiation induction medium supplemented with poloxamer (Kolliphor P188 BIO) to induce differentiation, an improvement in viable cell count and cell viability was observed. Also, for each of CXCR4 and SOX17, which are definitive endoderm cell markers, the percentage of positive cells increased. From the above results of Example 10, it was confirmed that by culturing hiPSCs with stirring and suspension in a differentiation induction medium containing poloxamer (polyoxyethylene (160) polyoxypropylene (27) block copolymer), the cell growth rate and viability were improved, and differentiation into definitive endoderm cells was also promoted.

Industrial Applicability

[0074] As described in detail above, according to the present invention, an additive that can favorably suppress precipitation of medium components such as insulin due to physical stimuli such as stirring, shaking, reflux, and gas aeration can be provided by adding it to a medium for suspension culture of animal cells containing insulin or the like. Also, according to the present invention, a medium for suspension culture of animal cells containing insulin or the like, in which precipitation of medium components such as insulin is favorably suppressed even when physical stimuli such as stirring, shaking, reflux, and gas aeration are applied, can be provided, and suspension culture of animal cells can be performed while performing stirring, shaking, reflux, gas aeration, etc. using the medium for suspension culture of animal cells containing insulin or the like. As a result, according to the present invention, suspension culture of animal cells can be performed by forming cell masses with controlled size, and the culture efficiency of animal cells and the quality of cultured cells can be improved. In particular, for 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 both the undifferentiated maintenance rate during maintenance culture and the differentiation rate during differentiation induction can be improved.

[0075] This application is based on Japanese Patent Application No. 2018-141909 filed in Japan, the content of which is incorporated herein in its entirety.

Claims

1. A method for the suspension culture of stem cells, which comprises culturing stem cells in suspension in a medium for suspension culture of stem cells containing insulin by stirring, shaking, circulating or aerating the stem cells, using one or more members selected from the group consisting of polyvinyl alcohol, polyoxyethylene polyoxypropylene block copolymers and polyoxyethylene sorbitan mono fatty acid esters to inhibit precipitation of insulin in the medium due to stirring, shaking, circulating or aerating the medium.

2. The method according to claim 1, 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.

3. The method according to claim 1 or 2, wherein the medium for suspension culture of stem cells is a maintenance medium for maintaining stem cells in an undifferentiated state.

4. The method according to claim 1 or 2, wherein the medium for suspension culture of stem cells is a differentiation-inducing medium that induces differentiation of stem cells.

5. The method according to any one of claims 1 to 4, wherein the medium for suspension culture of stem cells is a feeder-free medium.

6. The method according to any one of claims 1 to 4, wherein the medium for suspension culture of stem cells is a serum-free medium or a low-albumin medium.

7. The method according to any one of claims 1 to 4, wherein the medium for suspension culture of stem cells is a xeno-free medium.

8. The method according to any one of claims 1 to 7, wherein one or more selected from the group consisting of polyvinyl alcohol, polyoxyethylene polyoxypropylene block copolymer, and polyoxyethylene sorbitan mono fatty acid ester are added to give a final concentration during culture of 0.1 µg / mL to 10 mg / mL.

9. The method according to any one of claims 1 to 8, wherein the stem cells are cultured in suspension to form cell clusters.

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