阅读说明：本技术 含有核黄素衍生物的培养基 (Culture medium containing riboflavin derivatives ) 是由 米山和也 古关琴衣 横山水穗 冈元训 于 2018-06-27 设计创作，主要内容包括：本发明的课题在于,提供一种用于对iPS细胞、ES细胞等多能干细胞进行增殖培养的培养基,特别是,提供一种不会发生完全培养基的保存导致的劣化的细胞增殖用培养基,并且提供制造该培养基的方法。本发明提供一种含有核黄素衍生物的干细胞增殖用培养基等。(The present invention addresses the problem of providing a culture medium for the growth culture of pluripotent stem cells such as iPS cells and ES cells, in particular, a culture medium for cell growth that does not undergo deterioration due to complete medium storage, and a method for producing the culture medium. The present invention provides a culture medium for stem cell proliferation containing a riboflavin derivative.)

1. A culture medium for stem cell proliferation, which contains a riboflavin derivative.

2. The medium according to claim 1, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, their salts, and their hydrates.

3. The medium according to claim 1 or 2, wherein the riboflavin derivative is flavin adenine dinucleotide or a salt thereof, or a hydrate thereof.

4. The culture medium according to any one of claims 1 to 3, wherein the stem cells are ES cells or iPS cells.

5. The medium according to any one of claims 1 to 4, which is free of riboflavin.

6. A culture medium for stem cell proliferation, which is a culture medium obtained by mixing a basal medium containing amino acids, vitamins, minerals and buffers and one or more supplements, wherein the basal medium contains a riboflavin derivative.

7. The medium according to claim 6, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, their salts, and their hydrates.

8. The medium according to claim 6 or 7, wherein the riboflavin derivative is flavin adenine dinucleotide or a salt thereof, or a hydrate thereof.

9. The culture medium according to any one of claims 6 to 8, wherein the stem cells are ES cells or iPS cells.

10. The culture medium according to any one of claims 6 to 9, which is free of riboflavin.

11. A stabilizer for a medium for stem cell proliferation, which contains a riboflavin derivative.

12. The stabilizer according to claim 11, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, their salts, and their hydrates.

13. A stabilizer according to claim 11 or 12, wherein the riboflavin derivative is flavin adenine dinucleotide or a salt thereof, or a hydrate thereof.

14. A stabiliser according to any of claims 11 to 13, wherein the stem cells are ES cells or iPS cells.

15. A method for stabilizing a medium for stem cell proliferation, which comprises adding a riboflavin derivative.

16. The method according to claim 15, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, their salts, and their hydrates.

17. The method according to claim 15 or 16, wherein the riboflavin derivative is flavin adenine dinucleotide or a salt thereof, or a hydrate thereof.

18. The method of any one of claims 15 to 17, wherein the stem cell is an ES cell or an iPS cell.

19. A method for producing a culture medium for stem cell growth, characterized by adding a riboflavin derivative.

20. The method of claim 19, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, salts thereof, and hydrates thereof.

21. The method according to claim 19 or 20, wherein the riboflavin derivative is flavin adenine dinucleotide or a salt thereof, or a hydrate thereof.

22. The method of any one of claims 19 to 21, wherein the stem cell is an ES cell or an iPS cell.

23. A method for producing a culture medium for stem cell growth, characterized by mixing a basal medium containing a riboflavin derivative, an amino acid, a vitamin, a mineral, and a buffer, and one or more supplements.

24. The method of claim 23, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, salts thereof, and hydrates thereof.

25. The method according to claim 23 or 24, wherein the riboflavin derivative is flavin adenine dinucleotide or a salt thereof, or a hydrate thereof.

26. The method of any one of claims 23 to 25, wherein the stem cell is an ES cell or an iPS cell.

27. A method for culturing stem cells, which comprises culturing the stem cells in the medium according to any one of claims 1 to 10.

28. A method for producing stem cells, which comprises culturing the stem cells in the medium according to any one of claims 1 to 10.

Technical Field

The present invention relates to a culture medium for growing stem cells, particularly pluripotent stem cells, which has excellent storage stability, particularly excellent stability when stored in a liquid form as a complete medium, and a method for producing the same.

Background

In regenerative medicine using stem cells such as iPS cells and ES cells, it is assumed that these cells are efficiently proliferated and then differentiated into target tissues to be transplanted. Various culture media have been reported or commercially available for the purpose of cell proliferation (non-patent documents 1 to 3). Most of them are constituted by a plurality of bottles in which various components are put, and they are mixed for use. The mixed material is generally referred to as "complete medium".

The various bottles include, for example, a basic medium composed of amino acids, vitamins, minerals, buffers, etc., a supplement composed of proteins, etc., and the supplement is further contained in 2 to 2 or more bottles, respectively, depending on the kind. They are supplied as refrigerated or frozen products.

In general, a culture medium for stem cell proliferation is prepared by mixing a basal medium and a supplement(s) at the time of use to prepare a complete medium, and then storing the complete medium in a liquid form. Complete media can usually be stored for about 2 weeks. However, in practice, when a complete medium stored for 2 weeks is used, the cells may not grow, and the reason for this is not clear.

In regenerative medicine, a system capable of stably culturing and supplying cells is indispensable, and development of a corresponding culture medium and a method for producing the culture medium are required.

Disclosure of Invention

Problems to be solved by the invention

The present invention aims to provide a culture medium for propagation culture of cells, particularly pluripotent stem cells such as iPS cells and ES cells, and particularly aims to provide a culture medium for propagation of cells which is highly stable in a liquid state, a method for producing the same, and the like.

Means for solving the problems

In order to solve the above problems, the present inventors have variously modified the composition of the medium, and have studied in detail what kind of components are responsible for the deterioration of the storage stability of the medium. As a result, it was confirmed that Riboflavin (RFV) is essential for the growth of cells, but is also a cause of deterioration of the medium during liquid storage. As a result of intensive studies on alternative components to RFV, it was found that a riboflavin derivative (RFV derivative) such as Flavin Adenine Dinucleotide (FAD), Flavin Mononucleotide (FMN), and Riboflavin Tetrabutyrate (RTB) can be stored without deteriorating the medium without using RFV in the medium composition, and the present invention was completed.

Namely, the present invention is as follows.

[1] A culture medium for stem cell proliferation, which contains a riboflavin derivative.

[2] The medium according to [1] above, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, a salt thereof and a hydrate thereof.

[3] The medium according to [1] or [2], wherein the riboflavin derivative is flavin adenine dinucleotide, a salt thereof, or a hydrate thereof (preferably flavin adenine dinucleotide disodium hydrate).

[4] The medium according to any one of [1] to [3], wherein the stem cell is an ES cell or an iPS cell.

[5] The medium according to any one of the above [1] to [4], which does not contain riboflavin.

[6] A culture medium for stem cell proliferation, which is a culture medium obtained by mixing a basal medium containing amino acids, vitamins, minerals and buffers and one or more supplements, wherein a riboflavin derivative is contained in the basal medium.

[7] The medium according to [6] above, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, a salt thereof and a hydrate thereof.

[8] The medium according to [6] or [7], wherein the riboflavin derivative is flavin adenine dinucleotide, a salt thereof, or a hydrate thereof (preferably flavin adenine dinucleotide disodium hydrate).

[9] The medium according to any one of [6] to [8], wherein the stem cell is an ES cell or an iPS cell.

[10] The medium according to any one of [6] to [9] above, which does not contain riboflavin.

[11] A stabilizer for a medium for stem cell proliferation, which contains a riboflavin derivative.

[12] The stabilizer according to [11] above, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, a salt thereof and a hydrate thereof.

[13] The stabilizer according to [11] or [12], wherein the riboflavin derivative is flavin adenine dinucleotide, a salt thereof, or a hydrate thereof (preferably flavin adenine dinucleotide disodium hydrate).

[14] The stabilizer according to any one of [11] to [13], wherein the stem cell is an ES cell or an iPS cell.

[15] A method for stabilizing a medium for stem cell proliferation, which comprises adding a riboflavin derivative.

[16] The method according to [15], wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, salts thereof and hydrates thereof.

[17] The method according to [15] or [16], wherein the riboflavin derivative is flavin adenine dinucleotide, a salt thereof, or a hydrate thereof (preferably flavin adenine dinucleotide disodium hydrate).

[18] The method according to any one of [15] to [17], wherein the stem cell is an ES cell or an iPS cell.

[19] A method for producing a culture medium for stem cell growth, characterized by adding a riboflavin derivative.

[20] The method according to [19] above, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, a salt thereof and a hydrate thereof.

[21] The method according to [19] or [20], wherein the riboflavin derivative is flavin adenine dinucleotide, a salt thereof, or a hydrate thereof (preferably flavin adenine dinucleotide disodium hydrate).

[22] The method according to any one of [19] to [21], wherein the stem cell is an ES cell or an iPS cell.

[23] A method for producing a culture medium for stem cell growth, characterized by mixing a basal medium containing a riboflavin derivative, an amino acid, a vitamin, a mineral, and a buffer, and one or more supplements.

[24] The method according to [23] above, wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, a salt thereof and a hydrate thereof.

[25] The method according to [23] or [24], wherein the riboflavin derivative is flavin adenine dinucleotide, a salt thereof, or a hydrate thereof (preferably flavin adenine dinucleotide disodium hydrate).

[26] The method according to any one of [23] to [25], wherein the stem cell is an ES cell or an iPS cell.

[27] A method for culturing stem cells, which comprises culturing the stem cells in the medium according to any one of the above [1] to [10 ].

[28] A method for producing stem cells, which comprises culturing the stem cells in the medium according to any one of the above [1] to [10 ].

[29] A culture medium for stem cell proliferation comprising a riboflavin derivative, amino acids, vitamins, minerals, buffers and one or more supplements.

[30] The medium according to [29], wherein the riboflavin derivative is at least 1 selected from the group consisting of flavin adenine dinucleotide, flavin mononucleotide, riboflavin tetrabutyrate, a salt thereof and a hydrate thereof.

[31] The medium according to [29] or [30], wherein the riboflavin derivative is flavin adenine dinucleotide, a salt thereof, or a hydrate thereof (preferably flavin adenine dinucleotide disodium hydrate).

[32] The medium according to any one of [29] to [31], wherein the stem cell is an ES cell or an iPS cell.

[33] The medium according to any one of [29] to [32], which does not contain riboflavin.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, it is possible to provide a medium for cell growth in which the performance in cell growth does not decrease even when the complete medium is stored in a liquid form. Therefore, more cells, particularly pluripotent stem cells such as ES cells and iPS cells, can be efficiently obtained, and a large amount of these cells can be supplied for use in research, medical treatment, and the like.

Drawings

FIG. 1 is a bar graph showing the results of studying the effectiveness of RFV in culturing iPS cells (Fe)²⁺Content ratio, Zn²⁺The content was 25 mol% of the basal medium). The vertical axis represents cell coverage (%), and the horizontal axis represents RFV concentration.

FIG. 2 is a bar graph showing the results of studying the effectiveness of RFV in culturing iPS cells (Fe)²⁺Content ratio, Zn²⁺The content is 100 mol% of the basal medium). The vertical axis represents cell coverage (%), and the horizontal axis represents RFV concentration.

FIG. 3 is a bar graph showing the results of measurement of cell growth capacity (cell coverage) when iPS cells were cultured using various media stored under refrigeration (2-8 ℃ C., 2 weeks or 5 weeks). The results are shown in the upper part of the medium after 2 weeks of cold storage and in the lower part of the medium after 5 weeks of cold storage. DMEM/F-12 containing RFV as the basal medium (solution A) or Modified DMEM/F-12 containing FMN in place of RFV was used as supplements (solutions B and C), and either Essential8 supplement (solution B only) or TeSR-E8 supplement (solutions B and C) was used for each medium.

FIG. 4 shows the effect on Fe²⁺、Zn²⁺And a histogram of the results of the study on the extent to which riboflavin affects the storage stability of the medium. The cell growth ability (cell coverage) of iPS cells cultured using each medium stored refrigerated for 2 weeks was investigated.

FIG. 5 is a bar graph showing the results of the study of cell proliferation ability when iPS cells were cultured in a commercially available L-15 medium (FMN-containing medium). Complete media using L-15 as basal media (liquid a) and Essential8 supplement as supplement (liquid B) were prepared, and the day of preparation the proliferative capacity (cell coverage) of iPS cells was investigated. The upper part shows the results when matrigel was used as support material and the lower part shows the results when iMatrix-511 was used as support material. As a positive control, DMEM/F-12 was used as the basal medium (solution A).

Detailed Description

The present invention will be explained below. Unless otherwise defined, all terms used in the present specification have the meanings commonly used in the art.

In the present specification, "stem cell" means an immature cell having a self-replicating ability and a differentiation/proliferation ability. Among the stem cells, there are subgroups of pluripotent stem cells (pluripotent stem cells), unipotent stem cells (unipotent stem cells) and the like, depending on the differentiation ability. The pluripotent stem cells mean cells having the ability to differentiate into all tissues and cells constituting an organism. By pluripotent stem cells is meant cells that have the ability to differentiate into multiple tissues, cells, but not all species. By a unipotent stem cell is meant a cell that has the ability to differentiate into a particular tissue, cell.

Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), artificial pluripotent stem cells (iPS cells), and pluripotent stem cells induced and selected by stress or cell stimulation. Stem cells established by culturing primary embryos prepared by nuclear transfer of somatic cell nuclei are also preferable 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))。

examples of the pluripotent stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and somatic stem cells such as germ stem cells. The pluripotent stem cell is preferably a mesenchymal stem cell, more preferably a bone marrow mesenchymal stem cell. In a broad sense, a mesenchymal stem cell means a collection of stem cells or progenitor cells thereof that can differentiate into all or some of mesenchymal cells such as osteoblasts, chondroblasts, and adipoblasts.

The stem cell to be the subject of the present invention is preferably a pluripotent stem cell, more preferably an ES cell and an iPS cell, and particularly preferably an iPS cell.

The stem cells to be used in the present invention may be any animal-derived stem cells that can be proliferated. The stem cells that can be cultured using the medium of the present invention are derived from, for example, rodents such as mice, rats, hamsters, and guinea pigs, primates such as rabbits, ungulates such as pigs, cows, goats, horses, and sheep, felines such as dogs and cats, primates such as humans, monkeys, rhesus, apes, chimpanzees, and preferably human-derived stem cells.

1. Method for producing culture medium for stem cell proliferation

The present invention provides a medium for stem cell proliferation containing a riboflavin derivative (hereinafter also referred to as the medium of the present invention) and a method for producing the same.

The culture medium for stem cell growth of the present invention is usually prepared and manufactured by mixing a basic culture medium composed of amino acids, vitamins, minerals, buffers, and the like with auxiliary components (also referred to as supplements) such as proteins to prepare a complete culture medium. As an embodiment of the present invention, a complete medium obtained by mixing one or more supplements in a basal medium can be cited. When a plurality of supplements are used, the supplements may adversely affect each other depending on the type of the supplement, and need to be stored separately (supplements 1 and 2, etc.). As another embodiment of the present invention, a complete medium obtained by mixing supplements 1 and 2 in a basal medium can be cited.

The medium for stem cell proliferation (complete medium) of the present invention preferably contains riboflavin derivatives, amino acids, vitamins, minerals, buffers, one or more supplements.

The medium for stem cell proliferation (complete medium) of the present invention may be: a basal medium containing amino acids, vitamins, minerals, and buffers, and one or more supplements are enclosed in containers such as different bottles, and provided in the form of a culture medium kit for stem cell growth, which is prepared by combining them, and mixed to prepare a complete culture medium for use.

The basal Medium used in the present invention is characterized by containing a riboflavin derivative, and is prepared by adding a Medium commonly known per se, such as DMEM, DMEM/F-12, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α -MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5-A, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB/1, RL-1066, Williams ' media and Brissoc's thermal, MCDB201, NCTC109, NCTC135, Warmula's MB/1, TMDE-1066, Steele's and Technical's thermal, as well as a basal Medium commonly used by Technical SR 3664, and Technical Medium (Technical SR-3, Technical Medium, and Technical Medium, such as a Medium, which can be used by Technical SR-3, and Technical Medium (Technical SR-3, and Technical Medium, such as a Medium commonly known per se (FER, and Technical Medium, such as a Medium, and Technical Medium, and Te.

Examples of the riboflavin derivative used in the present invention include, but are not limited to, Flavin Mononucleotide (FMN), Flavin Adenine Dinucleotide (FAD), riboflavin butyrate (e.g., riboflavin tetrabutyrate), salts thereof, and hydrates thereof. Hereinafter, unless otherwise specified, FMN is used to mean including flavin mononucleotide, a salt thereof, a hydrate thereof, FAD is used to mean including flavin adenine dinucleotide, a salt thereof, a hydrate thereof, and RTB is used to mean including riboflavin butyrate (riboflavin tetrabutyrate), a salt thereof, and a hydrate thereof. FAD is preferred as the riboflavin derivative used in the present invention. The salt form includes acid addition salts, salts with bases, and the like, and preferably salts which do not exhibit cytotoxicity and are acceptable as medical supplies. Examples of the acid to form such a salt include inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid, and phosphoric acid, organic acids such as acetic acid, lactic acid, citric acid, tartaric acid, maleic acid, fumaric acid, methanesulfonic acid, and monomethyl sulfate, and examples of the base to form such a salt include metal hydroxides or carbonates such as sodium, potassium, and calcium, inorganic bases such as ammonia, and organic bases such as ethylenediamine, propylenediamine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. The salt may be a hydrate (hydrous salt). As FAD, flavin adenine dinucleotide disodium hydrate is preferably used. As FMN, riboflavin 5 '-sodium monophosphate dihydrate (riboflavin 5' -sodium phosphate dihydrate) is preferably used.

Riboflavin derivatives (including salts thereof, hydrates thereof) are commercially available and, in addition, may be prepared according to the literature.

In the present invention, the concentration of the riboflavin derivative in the basal medium (the concentration in terms of anhydrous free body in the case of salts and/or hydrates) is not particularly limited as long as deterioration of the complete medium is suppressed, and is usually 5nM to 500. mu.M, preferably 5nM to 100. mu.M, and particularly preferably 5nM to 20. mu.M, in the medium for growing stem cells of the present invention (complete medium).

In the case where the riboflavin derivative is FMN (anhydrous free body converted to FMN in the case of salt and/or hydrate), the concentration of the riboflavin derivative in the culture medium for stem cell growth (complete culture medium) of the present invention is preferably 5nM to 3 μ M; in the case of FAD (in the case of salts and/or hydrates, the free anhydrous body is converted to FAD), the concentration of the compound in the medium for stem cell growth of the present invention (complete medium) is preferably 5nM to 20 μ M; in the case of RTB (anhydrous free body converted to RTB in the case of salts and/or hydrates), the medium for stem cell growth (complete medium) of the present invention is preferably 5nM to 0.5. mu.M.

In the medium of the present invention, the basic medium contains iron (Fe)²⁺) 0.083-0.125 mg/L (preferably about 0.104mg/L) of compound (such as ferrous sulfate heptahydrate) and Zn (Zn)²⁺) When the compound (e.g., zinc sulfate heptahydrate) is 0.086 to 0.13mg/L (preferably about 0.108mg/L), the culture medium for stem cell growth of the present invention (complete culture medium) is particularly preferably 5nM to 2.57 μ M in the case where the riboflavin derivative is FMN (in the case of salt and/or hydrate, as an anhydrous free form of FMN); in the case of FAD (in the case of salts and/or hydrates, the free anhydrous body converted to FAD), the culture medium for stem cell proliferation (complete culture medium) of the present invention is particularly preferably 5nM to 15.2 μ M; in the case of RTB (anhydrous free body converted to RTB in the case of salt and/or hydrate), the medium for stem cell growth (complete medium) of the present invention is particularly preferably 5nM to 0.47. mu.M.

In the medium of the present invention, the basic medium contains iron (Fe)²⁺) 0.332-0.5 mg/L (preferably about 0.417mg/L) of compound (such as ferrous sulfate heptahydrate) and zinc (Zn)²⁺) When the compound (e.g., zinc sulfate heptahydrate) is 0.344 to 0.52mg/L (preferably about 0.432mg/L), the culture medium for stem cell growth of the present invention (complete culture medium) is particularly preferably 5nM to 0.47 μ M in the case where the riboflavin derivative is FMN (in the case of salt and/or hydrate, as an anhydrous free form of FMN); in the case of FAD (in the case of salts and/or hydrates, the free anhydrous body converted to FAD), the culture medium for stem cell proliferation (complete culture medium) of the present invention is particularly preferably 5nM to 2.57 μ M; in the case of RTB (anhydrous free body converted to RTB in the case of salt and/or hydrate), the medium for stem cell growth (complete medium) of the present invention is particularly preferably 5nM to 0.47. mu.M.

Examples of the amino acids contained in the basal medium include glycine, L-alanine, L-arginine, L-asparagine (e.g., L-asparagine (monohydrate)), L-asparaginic acid, L-cysteine, L-cystine (e.g., L-cystine dihydrochloride), L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine (e.g., L-lysine hydrochloride), L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. It is preferred to contain each amino acid in a concentration range known per se.

For example, the amino acid content in the basic culture medium is about 1mg to 100g per 1L of the basic culture medium.

Examples of the vitamins contained in the basal medium include inositol (e.g., myo-inositol), choline (e.g., choline chloride), vitamin a, vitamin B1 (thiamine (e.g., thiamine hydrochloride)), vitamin B3, vitamin B4, vitamin B5 (pantothenic acid (e.g., D-calcium pantothenate)), vitamin B6 (pyridoxine (e.g., pyridoxine hydrochloride)), vitamin B7 (biotin (e.g., D-biotin)), vitamin B12, vitamin B13, vitamin B15, vitamin B17, vitamin Bh, vitamin Bt, vitamin Bx, vitamin D, vitamin E, vitamin F, vitamin K, vitamin M (folic acid), vitamin P, and nicotinamide, which are vitamins. The vitamins are preferably contained in a concentration range known per se, and the basic medium used in the present invention does not contain riboflavin, or if it contains riboflavin, its concentration is usually 6 μ M or less, preferably 0.6 μ M or less, and more preferably 0.6nM or less in the complete medium. That is, the concentration of riboflavin in the basal medium is 0 to 6. mu.M, preferably 0 to 0.6. mu.M, and more preferably 0 to 0.6 nM. It is particularly preferred that the basal medium of the invention is free of riboflavin.

For example, the content of vitamins in the basic medium is about 0.0001 to 100mg per 1L of the basic medium.

Examples of the mineral substance contained in the basal medium include calcium chloride (e.g., calcium chloride (anhydrous)), copper sulfate (e.g., copper sulfate pentahydrate), iron (III) nitrate (e.g., iron nitrate nonahydrate), iron sulfate (e.g., ferrous sulfate heptahydrate), magnesium chloride (e.g., magnesium chloride hexahydrate), magnesium sulfate (e.g., magnesium sulfate (anhydrous)), potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate (e.g., sodium dihydrogen phosphate (anhydrous)), and zinc sulfate (e.g., zinc sulfate heptahydrate). Various minerals may be contained in concentration ranges known per se.

For example, the content of the mineral in the basic medium is about 0.0001 to 10000mg relative to 1L of the basic medium.

The culture medium of the invention, preferably in a basal medium, preferably contains at least iron and/or zinc, such as iron sulfate or its hydrates (e.g. ferrous sulfate heptahydrate), and/or zinc sulfate or its hydrates (e.g. zinc sulfate heptahydrate).

The content of ferric sulfate or a hydrate thereof in the culture medium of the present invention is preferably 5 to 500 mol%, more preferably 10 to 150 mol%, and even more preferably 20 to 30 mol% with respect to the content of ferric sulfate or a hydrate thereof in DMEM/F-12.

The content of zinc sulfate or a hydrate thereof in the culture medium of the present invention is preferably 5 to 500 mol%, more preferably 10 to 150 mol%, and still more preferably 20 to 30 mol% with respect to the content of zinc sulfate or a hydrate thereof in DMEM/F-12.

The content of ferric sulfate or a hydrate thereof in the culture medium of the present invention is preferably 0.021 to 2.085mg, more preferably 0.042 to 0.626mg, and further preferably 0.083 to 0.125mgL, based on 1L of the basic culture medium, in the case of ferrous sulfate heptahydrate.

The content of zinc sulfate or a hydrate thereof in the medium of the present invention is preferably 0.022 to 2.160mg, more preferably 0.043 to 0.648mg, and further preferably 0.086 to 0.130mg, based on 1L of the basal medium, in the case of zinc sulfate heptahydrate.

Examples of the buffer contained in the basic medium include physiological Phosphate Buffer (PBS), citric acid buffer, HEPES, and the like.

For example, the content of the buffer agent in the basal medium is about 0.001 to 10000mg per 1L of the basal medium.

The basic culture medium used in the present invention may contain additives known per se, and examples of the additives include saccharides (e.g., glucose), organic acids (e.g., pyruvic acid (e.g., sodium pyruvate), lactic acid, etc.), reducing agents (e.g., 2-mercaptoethanol, etc.), steroids (e.g., β -estradiol, progesterone, etc.), antibiotics (e.g., streptomycin, penicillin, gentamicin, etc.), purine derivatives (e.g., hypoxanthine), lipoic acid (e.g., DL-lipoic acid), fatty acids (e.g., linoleic acid), pH indicators (e.g., phenol red), polyamines (e.g., 1, 4-butanediamine dihydrochloride), pyrimidine deoxynucleotides (e.g., thymidine), and the like.

The supplements used in the present invention include, for example, proteins such as insulin, bFGF, transferrin, TGF- β, minerals such as selenium and sodium bicarbonate, and vitamins such as L-ascorbic acid, and supplements which do not desirably coexist are prepared separately from each other, that is, in the present invention, the supplements are provided as supplement 1, supplement 1 and supplement 2 as needed, and as the supplements in the present invention, substances which have been reported and commercially available can be used, and for example, a supplement solution such as Essential8(ThermoFisher SCIENTIFIC) and a supplement solution such as TeSR-E8 (STEELL Technologies) can be used.

The amount of the supplement in the culture medium of the present invention is, for example, about 1 to 50 parts by weight per 100 parts by weight of the basic culture medium.

The medium used in the present invention may contain serum. The serum is not particularly limited as long as it is derived from an animal, and is preferably derived from a mammal (e.g., fetal bovine serum, human serum, etc.), as long as it does not inhibit the proliferation of stem cells. The concentration of serum may be within a range of concentrations known per se. However, it is known that the serum component also contains a differentiation factor of human ES cells, and there is a possibility that the culture result fluctuates depending on the difference between serum lots, and therefore, it is preferable that the serum content is lower, and it is most preferable that the serum is not contained. Further, when stem cells after culture are used for medical purposes, it is preferable that they contain no serum because components derived from a foreign species may become infectious agents of blood-borne pathogenic bacteria or foreign antigens. In the absence of Serum, alternative supplements to Serum may be used (e.g., Knockout Serum Replacement (KSR) (Invitrogen), chemical ly-defined Lipid centralized (Gibco), Glutamax (Gibco), B-27 supplement, etc.). These components are typically provided as supplements, separate from the basal medium.

2. Method for stabilizing culture medium for stem cell proliferation

The medium for stem cell proliferation is usually composed of a basal medium and a supplement (one or more supplements), and is mixed at the time of use to prepare a liquid complete medium. The complete medium after storage is deteriorated in its performance in cell growth, but the deterioration of the medium after storage can be prevented by adding a riboflavin derivative to the medium. In the present specification, "deterioration" of the complete medium after storage means that the degree of cell growth obtained when the cells are cultured in the medium before storage (for example, immediately after preparation) is reduced as compared with the degree of cell growth obtained when the cells are cultured in the medium before storage. In the method of stabilizing a medium for stem cell proliferation characterized by adding a riboflavin derivative (hereinafter also referred to as the stabilizing method of the present invention), examples of the riboflavin derivative to be added include the same ones as those used in the medium for stem cell proliferation described in the above 1. The riboflavin derivative may be added to the complete culture medium or to the basal culture medium, preferably to the basal culture medium. The amount of riboflavin derivative to be added in the stabilization method of the present invention is not particularly limited as long as it can suppress deterioration due to liquid storage of the medium for stem cell proliferation, and is usually added in a range of 6nM to 16 μ M, preferably 6nM to 3 μ M, and more preferably 6nM to 0.6 μ M with respect to the basal medium. The amount of riboflavin derivative to be added in the stabilization method of the present invention is 5nM to 500. mu.M, preferably 5nM to 100. mu.M, and particularly preferably 5nM to 20. mu.M in the medium for stem cell proliferation (complete medium). In order to facilitate addition to the basal medium or complete medium, a stabilizer for a medium for stem cell growth (hereinafter also referred to as the stabilizer of the present invention) containing a riboflavin derivative at a predetermined concentration may be prepared in advance. The stabilizer of the present invention may contain or may not contain other components as long as it contains the riboflavin derivative as an active ingredient. Various additives may be contained in the culture medium from the viewpoint of ease of handling, storage stability, and the like, and in addition, the culture medium may be used by adding the culture medium. As various additives, known per se substances can be used, or 1 to 2 or more kinds of medium components can be formulated together.

The formulation of the stabilizer of the present invention is not particularly limited, and may be a solution (including a suspension, an emulsion, and the like), a solid (including a powder, and the like), or a semisolid (including a gel, and the like). The stabilizer of the present invention in a solution state is preferably added to the liquid medium easily. The solid or semisolid stabilizer of the present invention is preferable from the viewpoint of ease of handling, storage stability, and the like. The solid or semisolid stabilizer of the present invention may be added directly to the culture medium, or may be dissolved before being added to the culture medium and then used as needed.

The form of the basal medium and supplement of the present invention is not particularly limited, and may be a solution form (including a suspension form, an emulsion form, etc.), a solid form (including a powder form, etc.), or a semisolid form (including a gel form, etc.). The basic culture medium of the present invention in a solution form is a solution-form culture medium prepared by adding desired components of the culture medium in addition to the riboflavin derivative, and when the supplement is a solution, the supplement can be directly mixed to prepare a complete culture medium and used for culturing stem cells. In the case where the supplement is in a solid or semi-solid state, a solution of the supplement may be prepared in advance and then mixed, or the supplement may be directly dissolved in a basic medium in the form of a solution. When the basal medium of the present invention is in a solid or semi-solid state, it contains desired medium components (1 to 2 or more, preferably all) in addition to the riboflavin derivative, and when used, it is dissolved in purified water or the like, and when the supplement is a solution, it can be mixed as it is to prepare a complete medium for culturing stem cells. In the case where the supplement is in a solid or semi-solid state, the supplement may be mixed after a solution of the supplement is prepared in advance, or the supplement may be dissolved directly in a basal medium in a solution state. The pH can be adjusted as necessary and used for culturing cells. Any method is within the scope of the medium for stem cell growth of the present invention.

The present invention provides a method for culturing stem cells (hereinafter also referred to as the method of culturing the present invention).

3. The culture method of the present invention

The culture method of the present invention includes a step of culturing stem cells (preferably iPS cells) with the culture medium of the present invention.

The culture vessel used for culturing the stem cells is not particularly limited as long as it can culture the stem cells, and examples thereof include a culture flask, a tissue culture flask, a culture dish, a petri dish, a tissue culture dish, a Multi-well culture dish, a microplate, a Multi-well plate (Multi plate), a Multi-well plate, a microslide, a chamber slide, a culture bowl (Schale), a test tube, a culture tank, a culture bag, and a spinner flask.

The culture apparatus may be cell-adherent or cell-nonadherent, and is appropriately selected according to the purpose. The cell-adhesive incubator may be a vessel coated with an arbitrary cell supporting substrate such as extracellular matrix (ECM) for the purpose of improving adhesion of the surface of the incubator to cells. The cell-supporting substrate may be any substance for the purpose of adhesion of stem cells or feeder cells (when used).

Other culture conditions may be appropriately set. For example, the culture temperature is not particularly limited, and may be about 30 to 40 ℃ and preferably about 37 ℃. CO 2₂The concentration may be about 1 to about 10%, preferably about 2 to about 5%. The oxygen partial pressure may be 1 to 10%.

Since stem cells can be efficiently proliferated by culturing the stem cells in the medium of the present invention, the present invention can provide a method for efficiently producing stem cells.

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention.