Culture medium for stem cell suspension culture

Abstract

Stem cells can be cultured by suspension culture while improving functionalities of the stem cells by treating the stem cells with a water-soluble polymer (for example, culturing in the presence of the water-soluble polymer). Such a method is a more versatile culture method with low cost.

Description

Stem cell suspension culture medium

[0001] The present invention relates to cell culture technology, particularly to a suspension culture medium and culture method for stem cells.

[0002] With the industrialization of regenerative medicine, technological development (expansion and differentiation) of 3D pluripotent stem cells is progressing. Many media for suspension culture of stem cells, particularly pluripotent stem cells such as iPS cells, have already been reported for large-scale cell production, but issues remain. For example, some existing media can only support suspension culture of specific cell lines while maintaining the pluripotency of iPS cells. Other reported media (Non-Patent Document 1, Patent Document 1) enable suspension culture of many cell lines, presumably due to their high content of a multifunctional protein called albumin. In fact, Patent Document 1 states that "the inclusion of high concentrations of albumin together with CHIR99021 resulted in significant PSC expansion and maintenance of the pluripotency of expanded PSCs," suggesting that formulating a high concentration of albumin is advantageous for culture. However, for clinical use, the albumin species must be human, resulting in a very expensive medium. Furthermore, one report (Patent Document 2) successfully maintained the pluripotency of pluripotent stem cells by culturing them in an undifferentiated state by adding a differentiation-inhibiting reagent. However, differentiation-inhibiting reagents are also expensive, making their constant use an industrial challenge. Meanwhile, due to global population growth and improved dietary standards, a protein shortage (protein crisis) is predicted in the near future. Furthermore, livestock farming places a heavy burden on the environment, including the emission of large amounts of greenhouse gases, and ethical issues regarding raising animals in poor conditions have been raised. To address these issues, cultured meat, produced using cell culture techniques from livestock-derived stem cells, has attracted attention as a new protein source. Culture media containing serum, such as fetal bovine serum, are widely used in the culture of animal cells. However, from the perspectives of safety and economy, the development of serum-free culture media is desired. For example, serum-free media have been developed in which serum is replaced with alternative components such as growth factors. However, the large amount of albumin contained in serum-free media poses significant cost challenges.

[0003] U.S. Patent Application Publication No. 2021 / 0395698 International Patent Application Publication No. 2021 / 162090

[0004] Torizal FG et al., Commun Biol. 2021, 4(1):1316.

[0005] An object of the present invention is to provide a more versatile and low-cost culture method for suspension culture of stem cells, particularly pluripotent stem cells, and a culture medium therefor.

[0006] In view of the above problems, the present inventors focused on components that reduce the amount of expensive albumin and discovered that by using a water-soluble polymer, stem cell lines that were previously difficult to culture in suspension can be cultured at a high rate of maintaining undifferentiation. Furthermore, they discovered that using a water-soluble polymer in suspension culture allows the size of the resulting cell aggregates to be controlled to be small. Furthermore, they discovered that using a water-soluble polymer in animal-derived stem cells allows the number of cells obtained to be increased. That is, the present invention is as follows.

[0007] [1] A method for suspension culture of stem cells while improving their functionality, comprising treating the stem cells with a water-soluble polymer. [2] The method of [1], further comprising treating the stem cells with an albumin. [3] The method of [2], wherein the albumin is serum albumin. [4] The method of any of [1] to [3], wherein the water-soluble polymer is at least one selected from the group consisting of poloxamers and polysaccharides in which 80% or more of the glycosidic bonds are β-glycosidic bonds. [5] The method of [4], wherein the poloxamer is poloxamer-188. [6] The method of [4], wherein the polysaccharide in which 80% or more of the glycosidic bonds are β-glycosidic bonds is at least one selected from the group consisting of hyaluronic acids and celluloses. [7] The method of [6], wherein the hyaluronic acids are hyaluronic acid. [8] The method of [6], wherein the cellulose is cellulose ether. [9] The method of [8], wherein the cellulose ether is at least one selected from the group consisting of hydroxyalkyl cellulose, carboxymethyl cellulose, methyl cellulose, and hydroxyalkyl methyl cellulose.

[10] The method of [9], wherein the hydroxyalkyl cellulose is hydroxyethyl cellulose (HEC) and / or hydroxypropyl cellulose (HPC).

[11] The method of any one of [1] to

[10] , wherein the functionality of the stem cells is at least one selected from the group consisting of proliferation ability, the ability to maintain an undifferentiated state, and the ability to differentiate.

[12] The method of any one of [1] to

[10] , wherein the functionality of the stem cells is proliferation ability.

[13] The method of any one of [1] to

[10] , wherein the functionality of the stem cells is the ability to maintain an undifferentiated state.

[14] The method of any one of [1] to

[13] , wherein the stem cells are pluripotent stem cells, muscle stem cells, or adipose stem cells.

[15] The method of

[14] , wherein the stem cells are pluripotent stem cells.

[16] The method of

[14] , wherein the stem cells are muscle stem cells or adipose stem cells.

[17] A medium for suspension culture of stem cells while improving functionality, comprising a water-soluble polymer.

[18] The medium according to

[17] , further comprising an albumin.

[19] The medium according to

[18] , wherein the albumin is serum albumin.

[20] The medium according to any one of

[17] to

[19] , wherein the water-soluble polymer is at least one selected from the group consisting of poloxamers and polysaccharides in which 80% or more of the glycosidic bonds are β-glycosidic bonds.

[21] The medium according to

[20] , wherein the poloxamer is poloxamer-188.

[22] The medium according to

[20] , wherein the polysaccharide in which 80% or more of the glycosidic bonds are β-glycosidic bonds is at least one selected from the group consisting of hyaluronic acids and celluloses.

[23] The medium according to

[22] , wherein the hyaluronic acids are hyaluronic acid.

[24] The medium according to

[22] , wherein the celluloses are cellulose ethers.

[25] The medium according to

[24] , wherein the cellulose ether is at least one selected from the group consisting of hydroxyalkyl cellulose, carboxymethyl cellulose, methyl cellulose, and hydroxyalkyl methyl cellulose.

[26] The medium according to

[25] , wherein the hydroxyalkyl cellulose is hydroxyethyl cellulose (HEC) and / or hydroxypropyl cellulose (HPC).

[27] The medium according to any one of

[17] to

[26] , wherein the functionality of the stem cells is at least one selected from proliferation ability, the ability to maintain an undifferentiated state, and differentiation ability.

[28] The medium according to any one of

[17] to

[26] , wherein the functionality of the stem cells is proliferation ability.

[29] The medium according to any one of

[17] to

[26] , wherein the functionality of the stem cells is the ability to maintain an undifferentiated state.

[30] The medium according to any one of

[17] to

[29] , wherein the stem cells are pluripotent stem cells, muscle stem cells, or adipose stem cells.

[31] The medium according to

[30] , wherein the stem cells are pluripotent stem cells.

[32] The medium according to

[30] , wherein the stem cells are muscle stem cells or adipose stem cells.

[33] A method for producing stem cells with improved functionality, comprising a step of suspension culturing in the medium according to any one of

[17] to

[26] .

[34] The method according to

[33] , wherein the functionality is at least one selected from proliferation ability, the ability to maintain an undifferentiated state, and differentiation ability.

[35] The method according to

[33] , wherein the functionality is proliferation ability.

[36] The method of

[33] , wherein the functionality is the ability to maintain an undifferentiated state.

[37] The method of

[33] , wherein the stem cells are pluripotent stem cells, muscle stem cells, or adipose stem cells.

[38] The method of

[37] , wherein the stem cells are pluripotent stem cells.

[39] The method of

[37] , wherein the stem cells are muscle stem cells or adipose stem cells.

[40] A method for producing a medium for suspension culture of stem cells, comprising the step of adding a water-soluble polymer to a basal medium, wherein the functionality of the stem cells during suspension culture is improved.

[41] The method of

[40] , further comprising the step of adding albumins.

[42] The method of

[41] , wherein the albumins are serum albumin.

[43] The method of claim 40, wherein the water-soluble polymer is at least one selected from the group consisting of poloxamers and polysaccharides in which 80% or more of the glycosidic bonds are β-glycosidic bonds.

[44] The method of

[43] , wherein the poloxamers are poloxamer-188.

[45] The method of

[43] , wherein the polysaccharide in which 80% or more of the glycosidic bonds are β-glycosidic bonds is at least one selected from the group consisting of hyaluronic acids and celluloses.

[46] The method of

[45] , wherein the hyaluronic acids are hyaluronic acid.

[47] The method according to

[45] , wherein the cellulose is a cellulose ether.

[48] The method according to

[47] , wherein the cellulose ether is at least one selected from the group consisting of hydroxyalkyl cellulose, carboxymethyl cellulose, methyl cellulose, and hydroxyalkyl methyl cellulose.

[49] The method according to

[48] , wherein the hydroxyalkyl cellulose is hydroxyethyl cellulose (HEC) and / or hydroxypropyl cellulose (HPC).

[50] The method according to any one of

[40] to

[49] , wherein the functionality of the stem cells is at least one selected from the group consisting of proliferation ability, the ability to maintain an undifferentiated state, and differentiation ability.

[51] The method according to any one of

[40] to

[49] , wherein the functionality of the stem cells is proliferation ability.

[52] The method according to any one of

[40] to

[49] , wherein the functionality of the stem cells is the ability to maintain an undifferentiated state.

[53] The method according to any one of

[40] to

[42] , wherein the stem cells are pluripotent stem cells, muscle stem cells, or adipose stem cells.

[54] The method according to

[53] , wherein the stem cells are pluripotent stem cells.

[55] The method according to

[53] , wherein the stem cells are muscle stem cells or adipose stem cells.

[56] Use of a water-soluble polymer for improving the functionality of stem cells during suspension culture.

[57] The use according to

[56] , in combination with albumins.

[58] The use according to

[57] , wherein the albumin is serum albumin.

[59] The use according to any one of

[56] to

[58] , wherein the water-soluble polymer is at least one selected from the group consisting of poloxamers and polysaccharides in which 80% or more of the glycosidic bonds are β-glycosidic bonds.

[60] The use according to

[59] , wherein the poloxamer is poloxamer-188.

[61] The use according to

[59] , wherein the polysaccharide in which 80% or more of the glycosidic bonds are β-glycosidic bonds is at least one selected from the group consisting of hyaluronic acids and celluloses.

[62] The use according to

[61] , wherein the hyaluronic acids is hyaluronic acid.

[63] The use according to

[61] , wherein the cellulose is a cellulose ether.

[64] The use according to

[63] , wherein the cellulose ether is at least one selected from the group consisting of hydroxyalkyl cellulose, carboxymethyl cellulose, methyl cellulose, and hydroxyalkyl methyl cellulose.

[65] The use according to

[64] , wherein the hydroxyalkyl cellulose is hydroxyethyl cellulose (HEC) and / or hydroxypropyl cellulose (HPC).

[66] The use according to any one of

[56] to

[65] , wherein the functionality of the stem cells is at least one selected from proliferation ability, ability to maintain undifferentiation, and ability to differentiate.

[67] The use according to any one of

[56] to

[65] , wherein the functionality of the stem cells is proliferation ability.

[68] The use according to any one of

[56] to

[65] , wherein the functionality of the stem cells is ability to maintain undifferentiation.

[69] The use according to any one of

[56] to

[68] , wherein the stem cells are pluripotent stem cells, muscle stem cells, or adipose stem cells.

[70] The use according to

[69] , wherein the stem cells are pluripotent stem cells.

[71] The use according to

[69] , wherein the stem cells are muscle stem cells or adipose stem cells.

[72] The agent for improving stem cell functionality during suspension culture, comprising a water-soluble polymer.

[73] The agent according to

[72] , wherein the agent is used in combination with albumins.

[74] The agent of

[73] , wherein the albumin is serum albumin.

[75] The agent of any of

[72] to [4], wherein the water-soluble polymer is at least one selected from the group consisting of poloxamers and polysaccharides in which 80% or more of the glycosidic bonds are β-glycosidic bonds.

[76] The agent of

[75] , wherein the poloxamer is poloxamer-188.

[77] The agent according to

[75] , wherein the polysaccharide in which 80% or more of the glycosidic bonds are β-glycosidic bonds is at least one selected from the group consisting of hyaluronic acids and celluloses.

[78] The agent according to

[77] , wherein the hyaluronic acids are hyaluronic acid.

[79] The agent according to

[77] , wherein the celluloses are cellulose ethers.

[80] The agent according to

[79] , wherein the cellulose ether is at least one selected from the group consisting of hydroxyalkyl cellulose, carboxymethyl cellulose, methyl cellulose, and hydroxyalkyl methyl cellulose.

[81] The agent according to

[80] , wherein the hydroxyalkyl cellulose is hydroxyethyl cellulose (HEC) and / or hydroxypropyl cellulose (HPC).

[82] The agent according to any one of

[72] to

[81] , wherein the functionality of the stem cells is at least one selected from the group consisting of proliferation ability, the ability to maintain undifferentiation, and the ability to differentiate.

[83] The agent according to any one of

[72] to

[81] , wherein the functionality of the stem cells is proliferation ability.

[84] The agent according to any one of

[72] to

[81] , wherein the functionality of the stem cells is the ability to maintain undifferentiated state.

[85] The agent according to any one of

[72] to

[84] , wherein the stem cells are pluripotent stem cells, muscle stem cells, or adipose stem cells.

[86] The agent according to

[84] , wherein the stem cells are pluripotent stem cells.

[87] The agent according to

[84] , wherein the stem cells are muscle stem cells or adipose stem cells.

[0008] According to the present invention, it is possible to perform versatile (cell line-independent) suspension culture of stem cells, particularly pluripotent stem cells, and the functionality of stem cells, particularly the proliferation ability and / or ability to maintain an undifferentiated state of pluripotent stem cells, can be improved.

[0009] FIG. 1 shows the results of flow cytometry of the CD30 positivity rate in cells of the 1210B2 strain after two passages of suspension culture using StemFit (registered trademark) AK03N. The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows relative fluorescence intensity. FIG. 2 shows the results of flow cytometry of the CD30 positivity rate in cells of the 1383D2 strain after two passages of suspension culture using StemFit (registered trademark) AK03N. The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows relative fluorescence intensity. FIG. 3 shows the results of flow cytometry of the CD30 positivity rate in cells of the 201B7 strain after two passages of suspension culture using StemFit (registered trademark) AK03N. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. FIG. 4 shows the results of flow cytometry analysis of the CD30 positivity rate in cells of the 253G4 strain after two passages of suspension culture using StemFit® AK03N. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. FIG. 5 shows the results of an investigation into the effect of increasing the amount of albumin on improving cell quality. The CD30 positivity rate in cells of the 201B7 strain after two passages of suspension culture using StemFit® AK03N containing rHA(i) at various concentrations (2.5 g / L, 10 g / L) was examined by flow cytometry. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. Figure 6 shows the results of investigating the effect of increasing the amount of albumin on improving cell quality. The CD30 positivity rate of cells after two passages of suspension culture of the 201B7 strain using StemFit (registered trademark) AK03N containing BSA at various concentrations (2.5 g / L, 10 g / L) was examined by flow cytometry. The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows the relative fluorescence intensity. Figure 7 shows the results of investigating the effect of improving cell quality when a water-soluble polymer is used instead of albumin.The CD30 positivity rate of the 201B7 strain after one passage in suspension culture using StemFit® AK03N containing albumin (rHA(i)) or various concentrations of water-soluble polymers (PVA, KP188, Hya, PGA, HEC(i)) was examined by flow cytometry. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. Figure 8 shows the results of examining the effect of co-addition of albumin and water-soluble polymers on improving cell quality. The CD30 positivity rate of the 201B7 strain after one passage in suspension culture using StemFit® AK03N containing various concentrations of albumin (rHA(i)) and / or predetermined concentrations of water-soluble polymers (KP188, HEC(i)) was examined by flow cytometry. The vertical axis represents the number of cells exhibiting fluorescence (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. FIG. 9 shows the results of examining the amount of albumin and water-soluble polymer added. The 201B7 strain was cultured in suspension for two passages using StemFit® AK03N containing albumin (rHA(i)) and / or a water-soluble polymer (HEC(i)) at a predetermined concentration, and the CD30 positivity rate in the cells after the two passages was examined by flow cytometry. The vertical axis represents the number of cells exhibiting fluorescence (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. FIG. 10 is a graph showing the results of examining the amount of albumin and water-soluble polymer added. The increase in cell number after two passages of the 201B7 strain was cultured in suspension for two passages using StemFit® AK03N containing albumin (rHA(i)) and / or a water-soluble polymer (HEC(i)) at a predetermined concentration was examined. The vertical axis shows the cumulative cell growth fold, and the horizontal axis shows the concentrations of rHA(i) and HEC(i) in the medium. Figure 11 shows the results of examining the amounts of albumin and water-soluble polymer added. The CD30 positivity rate in cells after two passages of suspension culture of the 201B7 strain using StemFit (registered trademark) AK03N containing albumin (rHA(ii)) and / or water-soluble polymer (HEC(i)) at a predetermined concentration was examined by flow cytometry. The vertical axis shows the number of cells showing fluorescence (i.e., the number of CD30-positive cells), and the horizontal axis shows the relative fluorescence intensity. Figure 12 shows the results of examining the amounts of albumin and water-soluble polymer added.The CD30 positivity rate of the 201B7 strain after one passage in suspension culture using StemFit® AK03N containing albumin (rHA(iii) or BSA) and / or water-soluble polymer (HEC(i)) at a predetermined concentration was examined by flow cytometry. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. FIG. 13 shows the results of examining the amount and type of albumin and water-soluble polymer added. The CD30 positivity rate of the 201B7 strain after one passage in suspension culture using StemFit® AK03N containing albumin (rHA(i)) and / or water-soluble polymer (HEC(i) to HEC(v)) at a predetermined concentration was examined by flow cytometry. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. Figure 14 shows the results of examining the amount of albumin and water-soluble polymer added. The CD30 positivity rate of cells after one passage of suspension culture of the 253G4 strain using StemFit® AK03N containing a predetermined concentration of albumin (rHA(i)) and / or water-soluble polymer (HEC(i)) was examined by flow cytometry. The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows the relative fluorescence intensity. Figure 15 shows the results of examining the amount and type of albumin and water-soluble polymer added. The CD30 positivity rate of cells after two passages of suspension culture of the 201B7 strain using StemFit® AK03N containing a predetermined concentration of albumin (rHA(i)) and / or water-soluble polymer (any of HPC(i) to HPC(iii)) was examined by flow cytometry. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. Fig. 16 is a bright-field image showing aggregates in a medium containing a water-soluble polymer (Hya). Fig. 17 shows the results of flow cytometry analysis of the CD30 positivity rate in cells after one passage of suspension culture of the 201B7 strain using StemFit (registered trademark) AK03N supplemented with HEC(i). The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity.Figure 18 shows the results of flow cytometry of the CD30 positivity rate in cells of the 201B7 strain after one passage of agitation culture using StemFit (registered trademark) AK03N supplemented with HEC(i). The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows relative fluorescence intensity. Figure 19 shows the results of flow cytometry of the CD30 positivity rate in cells of the 201B7 strain after one passage of suspension culture using StemFit (registered trademark) AK03N supplemented with MC. The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows relative fluorescence intensity. Figure 20 shows the results of flow cytometry of the CD30 positivity rate in cells of the 201B7 strain after one passage of suspension culture using StemFit (registered trademark) AK03N supplemented with CMC. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. FIG. 21 shows the results of flow cytometry analysis of the CD30 positivity rate in cells of the 201B7 strain after one passage in suspension culture using StemFit (registered trademark) AK03N supplemented with HPMC. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. FIG. 22 shows the results of flow cytometry analysis of the CD30 positivity rate in cells of the 201B7 strain after one passage in suspension culture using StemFit (registered trademark) AK03N supplemented with γ-CD. The vertical axis represents the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis represents the relative fluorescence intensity. Figure 23 shows the results of flow cytometry analysis of the CD30 positivity rate in cells after one passage of suspension culture of the 201B7 strain using StemFit (registered trademark) AK03N supplemented with GG. The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows relative fluorescence intensity. Figure 24 shows the results of flow cytometry analysis of the CD30 positivity rate in cells after one passage of suspension culture of the 201B7 strain using medium supplemented with rHA(i) or rHA(i) and Dex(i) or Dex(ii) for 03NΔAlb. The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows relative fluorescence intensity.Figure 25 shows the results of flow cytometry analysis of the CD30 positivity rate in cells of the 201B7 strain after one passage in suspension culture using a medium containing 03NΔAlb supplemented with rHA(i) or rHA(i) and DSS. The vertical axis shows the number of fluorescent cells (i.e., the number of CD30-positive cells), and the horizontal axis shows the relative fluorescence intensity. Figure 26 shows the results of flow cytometry analysis of the CD30 positivity rate in cells of the 201B7 strain after one passage in suspension culture using a medium containing 03NΔAlb supplemented with rHA(i) or rHA(i) and DSS. ＴＭ 1 is a graph showing the results of investigating the proliferation fold when animal-derived stem cells were suspension cultured using media containing various water-soluble polymers (medium). The vertical axis shows the proliferation fold relative to the control, and the horizontal axis shows the type of each polymer added to the medium.

[0010] The present invention is described below. Terms used in this specification have the meanings commonly used in the art unless otherwise specified. 1. Medium for stem cell suspension culture (medium of the present invention) The present invention provides a medium for suspension culture of stem cells while improving stem cell functionality, which medium is characterized by containing a water-soluble polymer (hereinafter, this medium will also be referred to as the "medium of the present invention").

[0011] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation potential. Multipotent stem cells refer to stem cells that have the ability to differentiate into multiple types of tissues and cells, although not all types. Unipotent stem cells refer to stem cells that have the ability to differentiate into specific tissues and cells. Known examples of multipotent or unipotent stem cells include adult stem cells (also called somatic stem cells or tissue stem cells). Specific examples include neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, adipose stem cells, germline stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Pluripotent stem cells are stem cells that can be cultured in vitro and have the ability to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm). In the present invention, the stem cells are preferably pluripotent stem cells, muscle stem cells, and adipose stem cells. Known pluripotent stem cells include embryonic stem cells (ES cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells), nuclear transfer ES cells, somatic cell-derived ES cells (ntES cells), and induced pluripotent stem cells (iPS cells). Examples of pluripotent stem cells include pluripotent stem cells induced and selected by stress or cell stimulation. Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei can also be used as pluripotent stem cells (Nature, 385, 810 (1997); Science, 280, 1256 (1998); Nature Biotechnology, 17, 456 (1999); Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); Nature Genetics, 24, 109 (2000)). In the present invention, iPS cells are preferred as pluripotent stem cells.iPS cells include 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 836B3, FF-I14s03, FF-I01s04, MH09s01, Ff-XT18s02, Ff-WIs03, Ff-WJs513, Ff-CLs14, Ff-KVs09, QHJI14s03, QHJI01s04, RWMH09s01, DRXT18s02, RJWIs03, YZWJs513, ILCLs14, GLKVs09, Ff-XT28s05-ABo_To, Ff-I01s04-ABII-KO, Ff-I14s04-ABII-KO (all from iPS Academia Japan, RIKEN BioResource Center, or Kyoto University iPS Research Foundation), Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeaky (JCRB1329 strain), Toe (JCRB1338 strain), and Lollipop (JCRB1336 strain) (all from the National Center for Child Health and Development, Department of Rare and Intractable Diseases and Disease Resources Research, National Institute of Biomedical Innovation, JCRB Cell Bank), UTA-1 and UTA-1-SF-2-2 strains (all from the University of Tokyo), 21526, 21528, 21530, 21531, 31536, 3 Strains such as 1538 (all Fujifilm Cellular Dynamics), ATCC-DYP0730, ATCC-DYP0250, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-DYS0100, ATCC-HYS0103, ATCC-CYS0105, KYOU-DXR0109B, ATCC-BYS0110, ATCC-BYS0111, ATCC-BYS0112, ATCC-BYS0113, ATCC-BXS0114, ATCC-BXS0115, ATCC-BXS0116, and ATCC-BXS0117 (all American Type Culture Collection, a non-profit organization) can be used. The identity of iPS cells can be confirmed using undifferentiation markers, which are attributable to the undifferentiated nature of iPS cells, such as alkaline phosphatase, Oct3 / 4, Sox2, Nanog, ERas, and Esgl.Furthermore, CD30 can be used as an undifferentiation marker according to a previous report (MA Lagarkova et al., Cell Cycle, 7, 3610-3612 (2008)). Methods for detecting these undifferentiation markers include mRNA detection methods (using primers or probes) and immunological detection methods (using antibodies or labels).

[0012] Muscle stem cells (also known as myogenic stem cells) are specialized to differentiate into muscle fiber cells (myoblasts and myocytes) that make up skeletal muscle and do not normally differentiate into other cell types; therefore, they are considered unipotent stem cells. They are primarily known as satellite cells. These cells are located between the basement membrane and sarcolemma of muscle fibers and are inactive in their resting state. However, they are activated in response to muscle damage or stimulation. After activation, they proliferate and differentiate into myoblasts, forming new muscle cells and muscle fibers. These cells are useful in research into the treatment of muscle diseases and muscle weakness due to aging. Furthermore, because of their self-renewal ability, muscle stem cells are also useful in the production of cultured meat. By proliferating and differentiating muscle stem cells under appropriate conditions, it is expected that edible muscle tissue can be produced without slaughtering animals. Muscle stem cells can be obtained by directly isolating them from living organisms, by purchasing commercially available cells (suppliers: ATCC, Lonza, PromoCell, etc.), or by inducing differentiation from stem cells such as pluripotent stem cells or multipotent stem cells. Identification of muscle stem cells can be confirmed using the expression of markers specific to muscle stem cells. Examples of such markers include cell surface markers such as Pax7 and CD56 (NCAM), and genes associated with muscle differentiation such as Myf5, MyoD, and Myogenin. Methods for detecting these markers include mRNA detection (using primers and probes) and immunological detection (using antibodies and labels).

[0013] Adipose-derived stem cells (ADSCs) are stem cells derived from adipose tissue. They can be induced to differentiate into adipocytes, bone cells, chondrocytes, muscle cells, and nerve cells (under certain conditions), making them multipotent stem cells. Adipose stem cells are useful in regenerative medicine, tissue engineering, immunomodulatory therapy, and even cosmetic medicine. Adipose stem cells can be obtained by directly isolating them from living organisms, by purchasing commercially available cells (supplied by ATCC, Lonza, etc.), or by inducing differentiation from stem cells such as pluripotent stem cells or multipotent stem cells. Identification of adipose stem cells can be confirmed by the expression of markers specific to adipose stem cells. Examples of such markers include CD29, CD44, CD73, CD90, CD105, and CD117. Methods for detecting these markers include mRNA detection (using primers and probes) and immunological detection (using antibodies and labels).

[0014] In the present invention, "suspension culture" refers to a cell culture method performed without cells adhering to the culture vessel. In the present invention, suspension culture may or may not involve external pressure or vibration applied to the liquid medium, or shaking or rotation within the liquid medium. Suspension culture with shaking is also referred to as "shaking suspension culture." Examples include methods using vessels with non-cell-adhesive surfaces, the hanging drop method, the rotation culture method, the agitation culture method, the rotary culture method, the three-dimensional scaffold method, the centrifugation method, and methods using aggregation by an electric field or a magnetic field. Another example is a method of culturing cells in spherical or fibrous carriers made of alginate gel, etc. Both methods can be performed in accordance with methods commonly practiced in the art.

[0015] Culturing stem cells in the medium of the present invention allows for improved stem cell functionality during cultivation. Here, "stem cell functionality" refers to any property that stem cells can or should possess, but is not limited to, such as proliferation, maintenance of undifferentiated state, and differentiation. The "proliferation ability" of stem cells refers to the ability of stem cells to self-replicate and generate new cells. This is the process by which stem cells multiply before generating (differentiating into) specific types of cells (differentiated cells). Stem cells typically exist in an undifferentiated state or in a state that is not differentiated to a certain extent. Therefore, the "ability to maintain undifferentiated state" of stem cells refers to the ability of stem cells to maintain themselves, self-replicate (synonymous with the proliferation ability described above), and continue to exist without differentiation. Stem cells are cells that can transform (differentiate) into different cells in response to stimuli such as differentiation-inducing factors. Therefore, the "differentiation ability" of stem cells refers to the ability of stem cells to transform into different cell types. For example, stem cells can transform from an undifferentiated state into specific cell types (e.g., muscle cells, nerve cells, and adipocytes). The differentiation potential of stem cells varies depending on the degree to which the stem cells are multipotent, unipotent, or pluripotent, but in the present invention, all of these are referred to as "differentiation potential."

[0016] When stem cells are cultured using the medium of the present invention, the functionality of the stem cells (described above) can be significantly improved compared to when the medium is not used. Here, "improvement" varies depending on the type of functionality of the target stem cells, but for example, improvement of "proliferative potential" or "undifferentiated potential" means that stem cells in an undifferentiated state increase more rapidly and / or in greater numbers. Furthermore, extending the time it takes for stem cells to change into a different cell type is also an improvement in proliferative potential or undifferentiated potential. Improvement of "differentiation potential" means that the change from stem cells to a desired different cell type, i.e., induction of differentiation, can be carried out more accurately and efficiently.

[0017] The medium of the present invention is characterized by containing a water-soluble polymer. The term "water-soluble polymer" refers to a polymer that has a hydrophilic group in the molecule and is water-miscible or water-soluble. The water-soluble polymer is not particularly limited, but typically has a weight-average molecular weight of approximately 1,000 to 1,500,000, preferably 10,000 to 1,000,000, and more preferably 100,000 to 1,000,000, as measured by size exclusion chromatography. The viscosity of the water-soluble polymer is not particularly limited as long as it does not adversely affect the overall ease of handling of the medium when added to the medium. This viscosity is determined appropriately depending on the type of water-soluble polymer used and the amount added to the medium. Generally, however, water-soluble polymers with a "viscosity of a 2 wt% aqueous solution at 20°C" of approximately 100-500 mPa·s are preferred. In the present invention, "water-miscible or water-soluble" refers to a polymer that disperses or dissolves uniformly in water; for example, a polymer with a solubility in water of 0.5 wt% or more at 25°C is preferably used.

[0018] The water-soluble polymer may be natural, semi-synthetic or synthetic polymer compound, and may be nonionic, anionic, cationic or zwitterionic.Specifically, natural or semi-synthetic polymers include hyaluronic acids (e.g., hyaluronic acid, sodium hyaluronate), sodium chondroitin sulfate, alginic acid, sodium alginate, ammonium alginate, carboxymethyl amylose, agar, funori, casein, glue, pullulan, gum tragacanth, gum arabic, nonionic modified guar gum, locust bean gum, tamarind gum, celluloses (e.g., methylcellulose, carboxymethylcellulose, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC)), curdlan, paramylon, glucomannan, chitin, etc. Examples of synthetic surfactants include carboxyvinyl polymers; polyvinyl alcohol; polyvinylpyrrolidone; polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene fatty acid esters, polyoxyethylene polyhydric alcohol fatty acid partial esters (polyoxyethylene glycerin fatty acid partial esters, polyoxyethylene sorbitol fatty acid partial esters, polyoxyethylene sorbitan fatty acid partial esters, etc.), polyoxyethylene hydrogenated castor oil, polyoxyethylene alkylamines and other polyoxyethylene polyoxypropylene type nonionic surfactants; polyoxyethylene polyoxypropylene random copolymers, polyoxyethylene polyoxypropylene block copolymers (poloxamers), polyoxyethylene polyoxypropylene alkyl ethers and other polyglycerin type nonionic surfactants.

[0019] A preferred embodiment of the water-soluble polymer to be contained in the medium of the present invention is poloxamers.

[0020] Another preferred embodiment of the water-soluble polymer to be contained in the medium of the present invention is a polysaccharide containing β-glycosidic bonds as glycosidic bonds. Preferably, the polysaccharide has 80% or more of its glycosidic bonds. Polysaccharides are polymeric compounds formed by linking multiple monosaccharide molecules via glycosidic bonds, and are known to take on a variety of forms depending on the type of constituent monosaccharides, the bonding pattern, and whether or not they are branched. The water-soluble polymer used in the present invention preferably has 80% or more, more preferably 85% or more, and particularly preferably 90% or more of its glycosidic bonds. Examples of such β-glycosidic bonds include β-1,4 glycosidic bonds, β-1,3 glycosidic bonds, β-1,6 glycosidic bonds, and β-1,2 glycosidic bonds. The polysaccharide may be composed of one type of β-glycosidic bond or two or more types of β-glycosidic bonds. For example, in celluloses, β-D-glucose units are linked via β-1,4 bonds, while in hyaluronic acids, β-1,4 and β-1,3 bonds are alternately linked. The β-glycosidic bonds are preferably β-1,4 glycosidic bonds. A preferred embodiment of polysaccharides in which 80% or more of the glycosidic bonds are β-glycosidic bonds and which can be included as a water-soluble polymer in the medium of the present invention are hyaluronic acids and celluloses in which 80% or more of the glycosidic bonds are β-glycosidic bonds.

[0021] The water-soluble polymers contained in the culture medium of the present invention are preferably poloxamers (e.g., poloxamer-P188), hyaluronic acids (e.g., hyaluronic acid), and celluloses (e.g., cellulose ether). Cellulose ethers are derivatives in which the hydroxyl groups (—OH) in the cellulose molecule are replaced with ether groups (—OR). Cellulose has a structure in which β-D-glucose units are linked together via 1,4-glycosidic bonds, and cellulose ethers refer to a group of compounds that retain this basic structure but have been chemically modified to impart specific properties. Examples of cellulose ethers include methyl cellulose, ethyl cellulose, ethyl methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, hydroxyalkyl celluloses (e.g., hydroxyethyl cellulose, hydroxypropyl cellulose), hydroxyalkyl methyl celluloses (e.g., hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose), benzyl cellulose, trityl cellulose, cyanoethyl cellulose, and aminoethyl cellulose. Preferred are methyl cellulose, carboxymethyl cellulose, hydroxyalkyl celluloses (e.g., hydroxyethyl cellulose, hydroxypropyl cellulose), and hydroxyalkyl methyl celluloses (e.g., hydroxypropyl methyl cellulose). The cellulose ether may be further modified.

[0022] In the medium of the present invention, one water-soluble polymer may be selected and used alone, or two or more water-soluble polymers may be selected and used in combination.

[0023] In the present invention, the water-soluble polymers described above can be used as they are, or they can be dissolved or dispersed in a solvent such as water or a polyhydric alcohol, and used in a liquid form such as an aqueous solution or dispersion, or mixed with additives commonly used in formulations such as excipients and binders, and then sized, granulated, tableted, etc. to be used as an additive in a solid form such as a powder, granules, tablet, etc. Alternatively, the water-soluble polymers described above can be mixed with some of the medium components described below, such as carbohydrates and inorganic salts, and then the mixture can be added to the basal medium.

[0024] The water-soluble polymer or an additive containing the same is preferably prepared by sterilization. The sterilization method is not particularly limited, and examples thereof include autoclave sterilization at 121°C for 20 minutes, radiation sterilization, ethylene oxide gas sterilization, and filter sterilization, and can be appropriately selected depending on the form of the water-soluble polymer or the additive containing the same.

[0025] The content of the water-soluble polymer in the medium of the present invention is adjusted appropriately depending on the type of water-soluble polymer used, but the final concentration during culture is usually 0.1 μg / mL to 10 mg / mL, preferably 1 μg / mL to 8 mg / mL, more preferably 10 μg / mL to 7 mg / mL, even more preferably 100 μg / mL to 6 mg / mL, and even more preferably 200 μg / mL to 5 mg / mL.

[0026] Examples of medium components other than the water-soluble polymer that can be contained in the medium of the present invention include medium components that are commonly used for suspension culture of animal cells, such as 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 soybean peptide; serum; vitamins such as vitamin A, B vitamins (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 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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; Type I collagen, Type II collagen, and the like; Examples of such components include cell adhesion factors and extracellular matrix components such as 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; and hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the type of animal cells to be cultured.

[0027] Among the medium components other than water-soluble polymers that can be contained in the medium of the present invention, albumin is preferred. Albumin may be commercially available or home-prepared. In one embodiment, the albumin is a full-length albumin polypeptide. In another embodiment, the albumin may be a partial peptide or a mutant peptide (collectively referred to as albumins) as long as it has the desired function, such as functioning as an additional component in the medium. Preferably, the albumin is a full-length albumin polypeptide. Albumin can be from any suitable species, and in one embodiment, the albumin is human-derived. In another embodiment, the albumin is bovine-derived. In another embodiment, the albumin is porcine-derived. Albumin may be derived from any source, for example, from blood (e.g., whole blood, plasma, serum). In one embodiment, the albumin is recombinant albumin. Recombinant albumin may be produced in any host cell, including, but not limited to, bacteria, yeast, fungi, plants, mammalian cells, and insect cells. In the present invention, albumin is preferably human-derived albumin, more preferably human serum albumin, and even more preferably recombinant human serum albumin.

[0028] Albumin may be present in the medium of the present invention at any suitable concentration to achieve the desired effect. The albumin concentration (final concentration) in the medium may range, for example, from 0.5 to 30 g / L, from 0.5 to 20 g / L, or from 0.5 to 15 g / L. In one embodiment, the albumin concentration may range from 1.0 to 15 g / L or from 2.5 g / L to 10 g / L. In another embodiment, the albumin concentration in the medium is in the range of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 g / L or more, and 15, 14, 13, 12, 11, 10, 9.0, 8.0, 7.0, 6.0, 5.0, 4.0 g / L or less. The albumin concentration in the medium can be measured using a method commonly used in the art or a method based thereon.

[0029] When the medium of the present invention contains albumins, the ratio of the albumins to the water-soluble polymer is not particularly limited as long as the desired effect is obtained, and is adjusted appropriately depending on the types of albumins and water-soluble polymers used.

[0030] When culturing (maintenance culture) undifferentiated stem cells, a component that suppresses the differentiation of stem cells, etc. can be added. Furthermore, a component that induces or promotes the differentiation of stem cells, etc. can be added to a differentiation-inducing medium that induces the differentiation of stem cells, etc. Examples of components that suppress the differentiation of stem cells, etc. include leukemia inhibitory factor (LIF), fibroblast growth factor (FGF), and transforming growth factor (TGF)-β, which are differentiation-inhibitory factors for embryonic stem cells, bone morphogenetic protein (BMP) and Notch protein, which suppress the differentiation of neural stem cells, and Polycomb complex, which suppresses the differentiation of embryonic stem cells and iPS cells. Examples of components that induce or promote differentiation of stem cells and the like include activin A, which induces embryonic stem cells into endodermal cells, retinoic acid, bone morphogenetic protein (BMP) inhibitors (such as noggin) that induce differentiation of iPS cells into neuroectoderm, transforming growth factor (TGF)-β, extracellular glycoprotein (WNT) that induces differentiation of iPS cells into mesoderm, activin, which induces differentiation of iPS cells into mesoderm or endoderm, glycogen synthase kinase 3 (GSK3) inhibitors, etc. When initially differentiating into ectodermal cells, mesodermal cells, or endodermal cells, and then differentiating them into cells of various organs or tissues, necessary growth factors, nutrient factors, etc. can be added depending on the organ or tissue to be differentiated, and examples of such factors include brain nerve-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and hepatocyte growth factor (HGF).

[0031] It is preferable that the medium of the present invention does not contain serum as a medium component, because serum may contain unknown factors, prions, viruses, etc. Furthermore, 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 any components derived from animals other than humans.

[0032] Furthermore, in the present invention, the water-soluble polymer may be contained in a so-called basal medium, which is commonly used for suspension culture of animal cells, particularly mammalian stem cells, to produce the medium of the present invention. As used herein, "basal medium" refers to a medium containing carbon sources, nitrogen sources, inorganic salts, and the like, which are essential for cell culture. The basal medium that can be used as a component of the medium of the present invention is not particularly limited and may be selected appropriately depending on the type of cell to be cultured. The basal medium may be prepared by a method known per se, or a commercially available product may be used. The medium of the present invention may contain additional medium components as necessary.

[0033] Examples of basal media include media used for culturing mammalian cells, media used for maintaining and culturing stem cells, and media used for inducing differentiation of stem cells. Examples of media used for culturing 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, and Fischer's Medium.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 (trademark) hESC XF medium (Sartorius). NutriStem™ XF / FF Culture Medium (Sartorius Stedim), AF NutriStem™ hESC XF Medium (Sartorius Stedim), S-medium (DS Pharma Biomedical Co., Ltd.), StemFit™ AK03N Medium (Ajinomoto Co., Inc.), StemFit™ Basic03 Medium (Ajinomoto Co., Inc.), StemFit™ Basic04CT Medium (Ajinomoto Co., Inc.), hESF9 Medium, hESF-FX Medium, CDM Medium, DEF-CS 500 Xeno-Free 3D Spheroid Examples of suitable medium include Cellartis Culture Medium and StemFlex Medium (Thermo Fisher Scientific).

[0034] For the purposes of the present invention, it is preferable to use a feeder-free basal medium, and it is even more preferable to use a serum-free medium. Furthermore, for use as a culture medium for human stem cells, it is preferable to use a medium that does not contain components derived from animals other than humans (xeno-free medium).

[0035] Furthermore, from the viewpoint of use in suspension culture of stem cells, the medium of the present invention is preferably in the form of a liquid such as a solution or dispersion when used.

[0036] 2. Method for Suspension Culturing of Stem Cells (Culture Method of the Present Invention) The present invention further provides a method for suspension culturing of stem cells (hereinafter also referred to as the "culture method of the present invention"). The culture method of the present invention is a method for suspension culturing stem cells while improving stem cell functionality, which comprises treating the stem cells with a water-soluble polymer. The culture method of the present invention may further comprise treating the stem cells with albumins, if desired. The improvement in stem cell functionality specifically refers to an improvement in the proliferation ability and / or ability of the stem cells to maintain an undifferentiated state. Therefore, the present invention provides a method for suspension culturing stem cells while improving the proliferation ability and / or ability of the stem cells to maintain an undifferentiated state. Such a method can also be said to be a method for maintaining and proliferating stem cells or a method for maintaining an undifferentiated state of stem cells. Furthermore, the improvement in stem cell functionality can also be an improvement in the differentiation ability of stem cells.

[0037] "Improvement of stem cell functionality," "suspension culture of stem cells," "water-soluble polymer," and "albumins" are as described above. In the culture method of the present invention, the water-soluble polymer may be pre-contained together with other components in the culture medium used in the culture method of the present invention, or may be added separately to the basal medium as a water-soluble polymer or an additive containing the water-soluble polymer. The culture method of the present invention also includes treatment with albumins, if desired. The albumins may be pre-contained together with other components in the culture medium used in the culture method of the present invention, or may be added separately to the basal medium during culture. Whether pre-contained in the medium or separately added to the basal medium, the objective of the present invention, "suspension culture of stem cells while improving stem cell functionality," can be achieved by contacting the stem cells with the water-soluble polymer and, if desired, albumins in the medium. "Treating stem cells with a water-soluble polymer" means "a step of contacting stem cells with a water-soluble polymer in the medium," and "treating stem cells with albumins" means "a step of contacting stem cells with albumins in the medium."

[0038] In the present invention, the amount of the water-soluble polymer is adjusted appropriately depending on the type of water-soluble polymer used, but the water-soluble polymer is typically contained / added to the medium so as to give a final concentration during culture of 0.1 μg / mL to 10 mg / mL, preferably 1 μg / mL to 8 mg / mL, more preferably 10 μg / mL to 7 mg / mL, even more preferably 100 μg / mL to 6 mg / mL, and even more preferably 200 μg / mL to 5 mg / mL.

[0039] In the present invention, albumins are preferably contained / added to the medium so that the final concentration during culture is usually 0.5 to 30 g / L, preferably 0.5 to 20 g / L, and more preferably 0.5 to 15 g / L.

[0040] The time for treating stem cells with a water-soluble polymer and, optionally, with albumins varies depending on the type of stem cell functionality to be improved, but typically spans the entire suspension culture period. When the stem cell functionality is "proliferative ability" and "maintenance of undifferentiated potential," the treatment time can be the period for suspension culture for maintenance culture of the stem cells.

[0041] In the culture method of the present invention, suspension culture of stem cells can be performed according to conventional suspension culture methods. Specifically, stem cells are seeded in the medium of the present invention using culture equipment or devices such as cell culture plates, cell culture flasks, and bioreactors, depending on the culture scale. The cells are then cultured typically at 25°C to 39°C, preferably 33°C to 39°C, in the presence of typically 4% to 10% by volume, preferably 4% to 6% by volume, carbon dioxide, and typically 1% to 25% by volume, preferably 4% to 20% by volume, oxygen, for typically 1 to 30 days, preferably 2 to 14 days, and more preferably 3 to 10 days. The medium is replaced every 1 to 3 days. Medium replacement can be achieved by separating the animal cells and medium by sedimentation, centrifugation, or filtration, followed by adding fresh medium to the animal cells. Alternatively, the animal cells can be appropriately concentrated by sedimentation, centrifugation, or filtration, followed by adding fresh medium to the concentrated cell solution. The medium may be replaced in its entirety or in part (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total amount of medium in use). The gravitational acceleration (G) during the centrifugation is typically 50 G to 1,000 G, preferably 100 G to 500 G, and the pore size of the filter used for filtration is typically 10 μm to 200 μm.

[0042] The culture method of the present invention can be carried out by stirring, shaking, reflux, aeration, etc. Stirring can be performed using a bioreactor, a culture tank with agitator blades, etc. Stirring is typically performed at a stirring speed of 10 rpm to 2,000 rpm, preferably 40 rpm to 1,000 rpm. Shaking can be performed using a shaker or a shaking culture machine. Shaking is typically performed at a shaking speed of 10 rpm to 500 rpm, preferably 50 rpm to 250 rpm. Reflux can be performed using a peristaltic pump, a tubing pump, etc. Reflux tubes include peristaltic pump tubes and tubing pump tubes made of silicone, neoprene (chloroprene rubber), marprene (polypropylene-ethylene propylene rubber), etc. Reflux is typically performed at a flow rate of 10 μL / min to 1,000 mL / min, preferably 1 mL / min to 100 mL / min. Gas aeration can be carried out using various spargers such as a microsparger or a filter sparger. Gas aeration can be carried out at an aeration rate of usually 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, it is preferable to carry out suspension culture of animal cells with stirring or shaking.

[0043] Cultured animal cells can be recovered by sedimentation, centrifugation, or filtration using a filter. Centrifugation is performed at 50 to 1,000 G, preferably 100 to 500 G, for about 1 to 10 minutes. Filtration can be performed using a filter with pores of about 10 to 200 μm.

[0044] The cultured stem cells are preferably stored in liquid nitrogen or in a deep freezer at −150° C. using a freezing medium containing a cryoprotectant such as STEM-CELLBANKER (Nippon Zenyaku Kogyo Co., Ltd.).

[0045] The culture method of the present invention can improve the rate at which undifferentiated states are maintained during maintenance culture of stem cells.

[0046] Furthermore, when water-soluble polymers were used, the size of cell aggregates in suspension culture tended to decrease. It has been reported that the size of aggregates affects the efficiency of differentiation induction in stem cells (Sebastien Sart et al., Process Biochemistry 2017, 59:276-288). Therefore, the use of water-soluble polymers is expected to control the size of stem cell aggregates formed in suspension culture and improve differentiation induction efficiency. Therefore, the culture method of the present invention can improve the differentiation induction ability of stem cells, thereby enabling the efficient acquisition of differentiated cells.

[0047] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present invention in any way.

[0048] Example 1: Experimental procedure of human pluripotent stem cells 1.1 Adherent culture iMatrix-511 (Nippi) at 0.5 μg / cm ２ 1.5 mL of StemFit (registered trademark) AK03N containing 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well of a 6-well plate (CORNING) coated with 5% CO. 8,000 or 13,000 iPS cells were seeded into each well. ２ / CO set at 37°C ２ The cells were cultured statically in an incubator, and after 24 hours, the medium was replaced with StemFit® AK03N without Y-27632. Thereafter, the medium was replaced no more than every two days, and every seven days, the cells were detached using 50% CTS™ TrypLE™ Select (Thermo Fisher Scientific) or Accutase (Innovative Cell Technologies) and replated onto a new 6-well plate.

[0049] 1.2 Pre-suspension culture The cells maintained in the experimental procedure 1.1 were cultured with iMatrix-511 at 0.25 μg / cm ２ A density of 3.0 to 4.5 × 10 was applied to a 6-well plate coated with ３ cells / cm ２The cells were seeded at 100°C. StemFit® AK03N supplemented with 10 μM Y-27632 was used as the medium at the time of seeding, and the entire medium was replaced with StemFit® AK03N without Y-27632 the day after seeding, 2 days later, and 4 days later. Five days later, the culture supernatant was removed, and the culture surface was washed with D-PBS(-) (Nacalai Tesque). Accutase was added, and the cells were incubated at 37°C for 4 minutes. After that, the cells were dissociated into single cells by pipetting and resuspended in StemFit® AK03N containing 10 μM Y-27632. The viable cell concentration of the resulting cell suspension was measured using a ViCELLXR viable cell analyzer (Beckman Coulter).

[0050] 1.3 Suspension Culture The cell suspension obtained in Experimental Procedure 1.2 was seeded at 300,000 cells / well or 1,000,000 cells / flask into a 6-well plate or a 5 mL spinner flask that had previously been filled with 2 mL / well or 5 mL / flask of medium containing 10 μM Y-27632. ２ The cells were placed on an orbital shaker installed in an incubator and rotated horizontally at a rotation speed of 70 rpm, or placed on the base of a flask and shaken at 80 rpm to initiate suspension culture.

[0051] 1.4 Medium exchange for suspension culture On the day after seeding and 3 days later, 1 mL of culture supernatant was removed from each well or 2.5 mL from each flask, and 1 mL or 2.5 mL of fresh medium without Y-27632 was added to perform a 50% medium exchange.

[0052] 1.5 Passage of Cells in Suspension Cell aggregates were photographed using an inverted microscope or an all-in-one fluorescence microscope. Particle size distribution was analyzed using a BZ-X800 Analyzer (KEYENCE). Four days after the start of suspension culture, the culture medium was collected in a centrifuge tube, and cell aggregates were isolated by centrifugation. Accutase was added to the cell aggregates, and the mixture was heated at 37°C for 15 minutes, followed by thorough pipetting to dissociate them into single cells. The cells were resuspended in medium containing 10 μM Y-27632, and the viable cell concentration of the suspension was measured using a ViCELL XR viable cell analyzer. When continuing the culture by subculturing, the cell suspension was seeded at 300,000 cells / well into a 6-well plate previously filled with 2 mL / well of medium containing 10 μM Y-27632, and the culture was restarted on an orbital shaker.

[0053] 1.6 Evaluation of undifferentiated rate (CD30) using flow cytometry 1.0 × 10 cells were collected from the cell suspension obtained in Experimental Procedure 1.5. ５ The cells were placed in a 1.5 mL Eppendorf tube and centrifuged at 5,000 rpm for 2 minutes to precipitate the cells, after which the supernatant was removed. The cells were suspended in a pre-prepared PE Mouse Anti-Human CD30 (Cat. No. 550041; BD Biosciences) or PE Mouse IgG1,κ Isotype Control (Cat. No. 555749; dashed line histogram) antibody staining solution, allowed to stand for 20 minutes, and then washed with FACS buffer. The cells were filtered through a 40 μm filter and measured using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The CD30 positivity rate, which is an index of the undifferentiated state of pluripotent stem cells, was evaluated by appropriate gating using an isotype control.

[0054] Example 2: Experimental procedure of animal-derived stem cells 2.1 Cultivation of animal-derived stem cells 10 cm ２ 10 mL of medium was added to the dish, and 200,000 Wagyu beef-derived stem cells were seeded in each dish. ２ / CO set at 37°C２ The cells were statically cultured in an incubator for 3 days. Every 3 days, the cells were detached using 0.5 g / L-Trypsin / 0.53 mmol / L-EDTA, and new 10 cm ２ The cells were seeded onto a dish. Fibronectin-coated carriers were used for suspension culture. The fibronectin-coated carriers were prepared by adding fibronectin to a carrier-containing solution and incubating the mixture at 37°C for 1 hour.

[0055] 2.2 Suspension culture of animal-derived stem cells The carriers prepared in Experiment 2.1 were cultured in a 5 mL spinner flask at a density of 4,000 cells / cm. ２ The cell suspension was added so that the number of cells was 72,000 per flask. ２ The flasks were placed on the bases of flasks placed in an incubator, and suspension culture was initiated with shaking at 70 rpm. Three days after seeding, 2.5 mL of culture supernatant was removed from each flask, and 2.5 mL of fresh medium was added to perform a 50% medium exchange.

[0056] 2.3 Cell counting of animal-derived stem cells Six days after the start of suspension culture, the culture medium was removed from the flask, 3 mL of 0.5 g / L-Trypsin / 0.53 mmol / L-EDTA was added, and the mixture was stirred at 70 rpm for 10 minutes at 37°C. The cells were then detached from the carrier by thorough pipetting. The cells were passed through a 70 μm cell strainer, collected in a centrifuge tube, and centrifuged at 460 g for 3 minutes. The supernatant was removed, the cells were suspended in medium, and the viable cell concentration of the suspension was measured using a Countess II automated cell counter (Thermo Fisher Scientific).

[0057] Example 3: Culture medium preparation method and water-soluble polymer StemFit (registered trademark) AK03N was prepared according to the product protocol. Other media were prepared by adding a predetermined amount of albumin or water-soluble polymer to albumin-free, 2x concentrated StemFit (registered trademark) AK03N (hereinafter referred to as 03NΔAlb) and diluting it to 1:1 with Otsuka distilled water (Otsuka Pharmaceuticals). Alternatively, water-soluble polymer was added directly to StemFit (registered trademark) AK03N. Alternatively, for the medium for expanding animal-derived stem cells, Essential 8 ＴＭVarious proteins or water-soluble polymers were added to culture media (Thermo Fisher Scientific). Recombinant human serum albumin (rHA) and bovine serum albumin (BSA) were used as albumins (supplied by Fujifilm Wako Pure Chemical Industries, Ltd., etc.). Water-soluble polymers included polyvinyl alcohol (PVA), Kolliphor®-P188 (KP188), sodium hyaluronate (Hya), poly-γ-glutamic acid (PGA), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), methylcellulose (MC), carboxymethyl cellulose (CMC), and hydroxypropyl methylcellulose (HPMC) (supplied by Nippon Synthetic Chemical Industry, Sigma-Aldrich, Fujifilm Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., etc.). Other polymers used include γ-cyclodextrin (γ-CD) (supplied by Fujifilm Wako Pure Chemical Industries, Ltd., etc.), gellan gum (GG) (supplied by Fujifilm Wako Pure Chemical Industries, Ltd., etc.), dextran (Dex) (supplied by Nacalai Tesque Inc., etc.), and dextran sulfate sodium salt (DSS) (supplied by Fujifilm Wako Pure Chemical Industries, Ltd., etc.). rHA from different suppliers is referred to as rHA(i), rHA(ii), and rHA(iii), respectively. HEC from different suppliers or with different product numbers from the same supplier is referred to as HEC(i), HEC(ii), HEC(iii), HEC(iv), and HEC(v), respectively. HPC from different suppliers or with different product numbers from the same supplier is referred to as HPC(i), HPC(ii), HPC(iii), HPC(iv), and HPC(v), respectively. Dex with different product numbers will be referred to as Dex(i) and Dex(ii), respectively.

[0058] Example 4: Differences in Cell Responses Between iPS Cells in Suspension Culture and Cell Lines The CD30 positivity rates when iPS cells (cell lines: 1210B2, 1383D2, 201B7, and 253G4) were cultured in suspension using StemFit® AK03N are shown in Figures 1 to 4. The 1383D2 line showed a positivity rate of ~97%, while the 1210B2 line showed a CD30 positivity rate of ~77%, the 201B7 line showed a positivity rate of ~46%, and the 253G4 line showed a positivity rate of ~60%. Histogram peaks, which appear to represent cell populations, were also observed in the negative compartment. These results confirmed that there are differences in the ease with which pluripotent stem cells maintain an undifferentiated state depending on the cell line.

[0059] Example 5: Effect of increasing the amount of albumin on improving cell quality A medium was prepared by adding 2.5 g / L or 10 g / L of albumin (rHA(i), BSA) to 03NΔAlb and further adding distilled water. The CD30 positivity rate when iPS cells (cell line: 201B7) were cultured in suspension using this medium is shown in Figure 5 (rHA(i)) and Figure 6 (BSA). As predicted from prior literature, it was confirmed that increasing the amount of albumin improved the CD30 positivity rate.

[0060] Example 6: Effect of adding water-soluble polymers (albumin-free) on improving cell quality A medium containing a water-soluble polymer instead of albumin was prepared by adding water-soluble polymers (PVA, KP188, Hya, PGA, HEC(i)) and distilled water to 03NΔAlb. For comparison, a medium supplemented with albumin (rHA(i)) was also prepared. The CD30 positivity rate when iPS cells (cell line: 201B7) were cultured in suspension using these media is shown in Figure 7. As with the addition of albumin, an improvement in the CD30 positivity rate was observed when HEC(i), KP188, or Hya was added.

[0061] Example 7: Effect of adding HEC (co-addition of albumin) on improving cell quality. Culture media containing albumin and a water-soluble polymer, either alone or in combination, were prepared by adding rHA(i), KP188, HEC(i), or rHA, KP188, and HEC(i) to 03NΔAlb, followed by the addition of distilled water. The CD30 positivity rate when iPS cells (cell line: 201B7) were cultured in suspension using these media is shown in Figure 8. A stronger effect of improving the CD30 positivity rate was observed in media co-added with water-soluble polymer (KP188 or HEC) and albumin (rHA) compared to media containing albumin or a water-soluble polymer alone.

[0062] Example 8: Investigation of the amount of albumin and water-soluble polymer added Various amounts of albumin (rHA(i)), water-soluble polymer (HEC(i)), and distilled water were added to 03NΔAlb to prepare media with different final concentrations (rHA(i): 0-5.0 g / L, HEC: 0-2.5 g / L). Using these media, iPS cells (cell line: 201B7) were subjected to suspension culture for two consecutive passages. The CD30 positivity rate and cumulative increase fold are shown in Figure 9 and Figure 10, respectively. The cumulative increase fold was calculated by dividing the total number of viable cells obtained by converting the aggregates recovered after the end of culture into single cells by the number of cells seeded at the start of culture, and then multiplying the increase fold for that culture cycle. An improvement in the CD30 positivity rate was observed when rHA(i) and HEC(i) were co-added. In particular, significant improvement was observed when rHA(i) was 1.0 g / L and HEC(i) was 2.5 g / L, rHA(i) was 2.5 g / L and HEC(i) was 1.5 g / L, rHA was 2.5 g / L and HEC(i) was 2.5 g / L, and rHA(i) was 5.0 g / L and HEC(i) was 1.5 g / L. Furthermore, when the amount of rHA(i) was 2.5 g / L, a higher proliferation rate was observed when the amount of HEC(i) was 1.5 g / L compared to 2.5 g / L. These results confirmed the improvement effect of co-addition over a wide range of concentrations. It was suggested that there is an optimum value for albumin and water-soluble polymers in terms of proliferation performance.

[0063] Example 9: Evaluation of culture using multiple types of albumin Culture media were prepared using multiple types of albumin. rHA (rHA(ii), rHA(iii)) and BSA from different suppliers were used. rHA(ii), rHA(iii), or BSA was added to r03NΔAlb, or rHA(ii), rHA(iii), or BSA was co-added with HEC(i), and distilled water was further added to prepare media. The CD30 positivity rates when iPS cells (cell line: 201B7) were cultured in suspension using these media are shown in Figures 11 and 12. It was confirmed that the co-addition of a water-soluble polymer had an effect of improving the CD30 positivity rate, regardless of the type of albumin.

[0064] Example 10: Culture Evaluation Using Multiple Types of HEC Culture media were prepared using multiple types of HEC. HECs (HEC(i), HEC(ii), HEC(iii), HEC(iv), and HEC(v)) differed in physical properties, such as source, average molecular weight, and viscosity in aqueous solution at the same weight percent concentration. rHA(i) was used as albumin. Regarding the physical properties of HEC, HEC(i) had a viscosity of 200-300 mPa·s at 20°C when 2% by weight of HEC(ii), a viscosity of 800-1500 mPa·s at 20°C when 2% by weight of HEC(iii), and a viscosity of 4500-6500 mPa·s at 25°C when 2% by weight of HEC(iii) were used. HEC(iv) had an average molecular weight of approximately 380,000, and HEC(v) had an average molecular weight of approximately 1,300,000. To 03NΔAlb, rHA(i) was added, or rHA(i) and HEC (any of HEC(i) to HEC(v)) were co-added, and distilled water was further added to prepare media. The CD30 positivity rate when iPS cells (cell line: 201B7) were cultured in suspension using these media is shown in Figure 13. The improvement in the CD30 positivity rate when rHA and HEC were co-added was confirmed, regardless of the supplier or physical properties of the HEC.

[0065] Example 11: Culture evaluation using multiple iPS cell lines. A medium was prepared by adding albumin rHA to 03NΔAlb, or by adding rHA and HEC together, and then adding distilled water. The CD30 positivity rate when iPS cells (cell line: 253G4) were cultured in suspension using the prepared medium is shown in Figure 14. The improvement in CD30 positivity rate, as previously confirmed with the 201B7 cell line, was also confirmed with the 253G4 line.

[0066] Example 12: Evaluation of culture using HEC analogs Media were prepared by adding rHA to 03NΔAlb, or by co-adding rHA and HEC or HPC (HPC(i), HPC(ii), HPC(iii)) at concentrations of 1.5 to 2.5 g / L, followed by the addition of distilled water. The CD30 positivity rate when iPS cells (cell line: 201B7) were cultured in suspension using these media is shown in Figure 15. Here, HPC(i), HPC(ii), and HPC(iii) have different viscosities in aqueous solutions at the same wt% concentration. Specifically, HPC(i) has a viscosity of 2.0-2.9 mPa·s when 2% by weight of aqueous solution at 20°C, HPC(ii) has a viscosity of 6.0-10.0 mPa·s when 2% by weight of aqueous solution at 20°C, and HPC(iii) has a viscosity of 150-400 mPa·s when 2% by weight of aqueous solution at 20°C. The improvement in CD30 positivity rate seen with the co-addition of rHA and HEC was also confirmed in a medium co-added with HPC, an analog of HEC, and rHA. These results suggest that the effects of the present invention can be obtained by adding hydroxyalkyl cellulose.

[0067] Example 13 Appearance of Aggregates in Medium Supplemented with Hya Figure 16 shows a bright field image of the aggregates in the Hya-containing medium obtained in Example 5. The size of the aggregates could be controlled to be small by adding a water-soluble polymer at an appropriate concentration.

[0068] Example 14: Effect of adding HEC to StemFit (registered trademark) AK03N A medium was prepared by adding HEC(i) to StemFit (registered trademark) AK03N at a concentration of 1.5 g / L. The CD30 positivity rate when iPS cells (cell line: 201B7) were cultured in suspension using this medium is shown in Figure 17. The improvement in CD30 positivity rate observed with the addition of HEC(i) to 03NΔAlb was also confirmed when StemFit (registered trademark) AK03N was used.

[0069] Example 15: Effect of Adding HEC in a Culture System Using a 5 mL Spinner Flask A medium was prepared by adding HEC(i) to StemFit® AK03N at a concentration of 1.5 g / L. Using this medium, iPS cells (cell line: 201B7) were cultured in suspension in a spinner flask. The CD30 positivity rate when this medium was used is shown in Figure 18. The improvement in CD30 positivity rate observed in shaking suspension culture with the addition of HEC(i) to StemFit® AK03N was also confirmed in spinner flask culture. These results suggest that the effects of the present invention can be obtained regardless of the culture vessel.

[0070] Example 16: Evaluation of Culture Using Multiple Polymers (MC) StemFit® AK03N was supplemented with MC at a concentration of 1.5 g / L or 2.5 g / L to prepare a medium. iPS cells (cell line: 201B7) were cultured in suspension using this medium, and the CD30 positivity rate is shown in Figure 19 . The improvement in CD30 positivity rate observed with the addition of HEC(i) to StemFit® AK03N was also confirmed with the addition of MC. In particular, a greater improvement in CD30 positivity rate was observed when MC was added at 2.5 g / L.

[0071] Example 17: Evaluation of Culture Using Multiple Polymers (CMC) StemFit® AK03N was supplemented with CMC at concentrations of 2.5 g / L or 5.0 g / L to prepare a medium. The CD30 positivity rate observed when iPS cells (cell line: 201B7) were cultured in suspension using this medium is shown in Figure 20 . The improvement in CD30 positivity rate observed with the addition of HEC(i) to StemFit® AK03N was also confirmed with the CMC-supplemented medium. In particular, a greater improvement in CD30 positivity rate was observed when CMC was added at 5.0 g / L.

[0072] Example 18: Evaluation of Culture Using Multiple Polymers (HPMC) A medium was prepared by adding HPMC to StemFit® AK03N at a concentration of 2.5 to 5.0 g / L. Figure 21 shows the CD30 positivity rate when iPS cells (cell line: 201B7) were cultured in suspension using this medium. The improvement in CD30 positivity rate observed with the addition of HEC(i) to StemFit® AK03N was also confirmed with HPMC-supplemented medium. This result, along with the results obtained in Examples 14, 16, and 17, strongly suggests that the effects of the present invention can be achieved by adding celluloses composed only of β-glycosidic bonds.

[0073] Example 19: Evaluation of culture using multiple polymers (γ-CD) A medium was prepared by adding γ-CD to StemFit (registered trademark) AK03N at a concentration of 2.5 g / L. iPS cells (cell line: 201B7) were cultured in suspension using this medium, and the CD30 positivity rate is shown in Figure 22. The improvement in CD30 positivity rate observed with the addition of HEC(i) to StemFit (registered trademark) AK03N was not observed in the γ-CD-supplemented medium.

[0074] Example 20: Evaluation of Culture Using Multiple Polymers (GG) StemFit® AK03N was supplemented with GG at a concentration of 0.01 or 0.02% to prepare a medium. Figure 23 shows the CD30 positivity rate when iPS cells (cell line: 201B7) were cultured in suspension using this medium. The improvement in CD30 positivity rate observed with the addition of HEC(i) to StemFit® AK03N was not observed with GG-supplemented medium. GG is a water-soluble polymer composed of β-glycosidic bonds in a 3:1 ratio to α-glycosidic bonds, suggesting that the effects of the invention cannot be achieved by adding a water-soluble polymer composed of 75% β-glycosidic bonds.

[0075] Example 21: Evaluation of culture using multiple polymers (Dex) 03NΔAlb was supplemented with rHA(i), or rHA(i) and Dex(i) or Dex(ii) were co-added at a concentration of 2.5 g / L, and distilled water was added to prepare a medium in which iPS cells (cell line: 201B7) were cultured in suspension. The CD30 positivity rate when cultured is shown in Figure 24. The improvement in CD30 positivity rate observed with the addition of HEC(i) to StemFit (registered trademark) AK03N was not observed in Dex(i)- or Dex(ii)-supplemented medium. Dex is a water-soluble polymer composed only of α-glycosidic bonds, suggesting that the effects of the invention cannot be achieved by adding a water-soluble polymer composed of such a main backbone.

[0076] Example 22: Evaluation of culture using multiple polymers (DSS) rHA(i) was added to 03NΔAlb, or rHA(i) and DSS were both added at a concentration of 2.5 g / L, and distilled water was further added to prepare a medium, and iPS cells (cell line: 201B7) were cultured in suspension in the medium, and the CD30 positivity rate is shown in Figure 25. The improvement in CD30 positivity rate seen with the addition of HEC(i) to StemFit (registered trademark) AK03N was not confirmed in the DSS-supplemented medium.

[0077] Example 23: Effect of adding polymer to animal-derived stem cells When animal-derived stem cells were cultured in suspension, the basal medium (Essential 8 ＴＭVarious water-soluble polymers were added to the culture medium, and the proliferation rates with and without the addition of polymers were compared. The proliferation rates are shown in Figure 26. Culture in a medium without added polymer served as a control. It was confirmed that the addition of a water-soluble polymer with a cellulose backbone increased the number of animal-derived stem cells obtained. These results suggest that the addition of celluloses is also effective in improving the functionality of suspension culture of stem cells other than pluripotent stem cells.

[0078] According to the present invention, it is possible to perform highly versatile (cell line-independent) suspension culture of stem cells, particularly pluripotent stem cells. According to the present invention, it is possible to improve the proliferation ability and / or ability to maintain an undifferentiated state of stem cells, particularly pluripotent stem cells. This application is based on Japanese Patent Application No. 2023-217459 (filing date: December 22, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for culturing stem cells in suspension while improving their functionality, comprising treating the stem cells with a water-soluble polymer.

2. The method of claim 1, further comprising treating with albumins.

3. The method of claim 2, wherein the albumin is serum albumin.

4. The method according to claim 1, wherein the water-soluble polymer is at least one member selected from the group consisting of poloxamers and polysaccharides in which 80% or more of the glycosidic bonds are β-glycosidic bonds.

5. The method of claim 4, wherein the poloxamer is poloxamer-188.

6. The method according to claim 4, wherein the polysaccharide in which 80% or more of the glycosidic bonds are β-glycosidic bonds is at least one selected from the group consisting of hyaluronic acids and celluloses.

7. The method of claim 6, wherein the hyaluronic acid derivative is hyaluronic acid.

8. The method according to claim 6, wherein the cellulose is a cellulose ether.

9. The method of claim 8, wherein the cellulose ether is at least one member selected from the group consisting of hydroxyalkyl cellulose, carboxymethyl cellulose, methyl cellulose and hydroxyalkyl methyl cellulose.

10. The method of claim 9, wherein the hydroxyalkyl cellulose is hydroxyethyl cellulose (HEC) and / or hydroxypropyl cellulose (HPC).

11. The method according to claim 1, wherein the functionality of the stem cells is at least one selected from the group consisting of proliferation ability, ability to maintain an undifferentiated state, and differentiation ability.

12. The method according to claim 1, wherein the functionality of the stem cells is proliferation ability.

13. The method according to claim 1, wherein the functionality of the stem cells is the ability to maintain an undifferentiated state.

14. The method of claim 1, wherein the stem cells are pluripotent stem cells, muscle stem cells or adipose stem cells.

15. The method of claim 14, wherein the stem cells are pluripotent stem cells.

16. The method of claim 14, wherein the stem cells are muscle stem cells or adipose stem cells.

17. A medium for suspension culture of stem cells with improved functionality, comprising a water-soluble polymer.

18. The medium according to claim 17, further comprising an albumin.

19. The medium according to claim 18, wherein the albumin is serum albumin.

20. The medium according to claim 17, wherein the water-soluble polymer is at least one member selected from the group consisting of poloxamers and polysaccharides in which 80% or more of the glycosidic bonds are β-glycosidic bonds.

21. The medium according to claim 20, wherein the poloxamer is poloxamer-188.

22. The medium according to claim 20, wherein the polysaccharide in which 80% or more of the glycosidic bonds are β-glycosidic bonds is at least one selected from the group consisting of hyaluronic acids and celluloses.

23. The medium according to claim 22, wherein the hyaluronic acid derivative is hyaluronic acid.

24. The medium according to claim 22, wherein the cellulose is a cellulose ether.

25. The medium according to claim 24, wherein the cellulose ether is at least one member selected from the group consisting of hydroxyalkyl cellulose, carboxymethyl cellulose, methyl cellulose and hydroxyalkyl methyl cellulose.

26. The medium according to claim 25, wherein the hydroxyalkyl cellulose is hydroxyethyl cellulose (HEC) and / or hydroxypropyl cellulose (HPC).

27. The medium according to claim 17, wherein the functionality of stem cells is at least one selected from the group consisting of proliferation ability, ability to maintain an undifferentiated state, and differentiation ability.

28. The medium according to claim 17, wherein the functionality of stem cells is proliferation ability.

29. The medium according to claim 17, wherein the functionality of stem cells is the ability to maintain an undifferentiated state.

30. The medium according to claim 17, wherein the stem cells are pluripotent stem cells, muscle stem cells or adipose stem cells.

31. The medium of claim 30, wherein the stem cells are pluripotent stem cells.

32. The medium according to claim 30, wherein the stem cells are muscle stem cells or adipose stem cells.

33. A method for producing stem cells with improved functionality, comprising a step of suspension culture in the medium according to any one of claims 17 to 26.

34. The method according to claim 33, wherein the functionality is at least one selected from the group consisting of proliferation ability, ability to maintain undifferentiated state, and differentiation ability.

35. The method of claim 33, wherein the functionality is proliferation ability.

36. The method according to claim 33, wherein the functionality is the ability to maintain undifferentiated state.

37. The method of claim 33, wherein the stem cells are pluripotent stem cells, muscle stem cells or adipose stem cells.

38. The method of claim 37, wherein the stem cells are pluripotent stem cells.

39. The method of claim 37, wherein the stem cells are muscle stem cells or adipose stem cells.