Undifferentiated maintenance medium additive

Abstract

To provide a medium for efficiently proliferating pluripotent stem cells while keeping them undifferentiated, and a method for proliferating pluripotent stem cells using the medium.SOLUTION: A serum-free medium additive for promoting the proliferation of human pluripotent stem cells comprises L-tryptophan or a L-tryptophan derivative at a concentration equal to or higher than 176 μM, wherein the L-tryptophan derivative is selected from dipeptides formed by peptide bonding between L-kynurenine, kynurenic acid or L-tryptophan, and amino acids.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a medium for efficiently growing pluripotent stem cells while maintaining them in an undifferentiated state, and a method for growing pluripotent stem cells using the medium. [Background technology]

[0002] Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), are expected to be used in regenerative medicine due to their excellent proliferation and differentiation potential. iPS cells, in particular, are considered to be an excellent source of regenerative medicine because they are relatively easy to produce and obtain, there are few ethical restrictions on their production, and there is no risk of rejection during transplantation.

[0003] When using pluripotent stem cells for regenerative medicine, a large amount of pluripotent stem cells is required for the treatment of diseases and the development and research of treatment methods. Therefore, it is important to develop and improve pluripotent stem cell culture methods that enable the supply of a large amount of pluripotent stem cells. The key to this is the improvement of the culture medium. A large amount of culture medium is required to culture a large amount of cells. For example, 6 10 iPS cells were cultured 10 To generate cardiomyocytes of this size and transplant them into a single patient, 100 liters of culture medium would be required per patient, and the cost of the culture medium would reach at least $100,000. One way to reduce culture medium costs is to increase the number of cells that can be cultured per unit volume of medium. In other words, there is a need for more efficient, low-cost, and effective culture media for pluripotent stem cells.

[0004] Culture media typically contain essential amino acids. For example, stem cell culture media such as mTeSR1 medium (Non-Patent Documents 1, 2, 3) and Essential-8 medium (Non-Patent Document 4) use Dulbecco's Modified Eagle Medium (DMEM) / F12 medium as the basal medium, and are supplemented with several factors such as bFGF and insulin. The amino acid content of this DMEM / F12 medium is determined based on the amount of free amino acids in blood, and it contains 9.0200 mg / L of L-tryptophan. Although many culture media have been developed, the amino acid composition of these media has not been significantly improved. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-135672 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-228815 [Patent Document 3] US2010 / 0317104A1 [Patent Document 4] WO2011 / 100286A2 [Non-patent literature]

[0006] [Non-Patent Document 1] Ludwig TE et.al.Nat. Methods 3(8):637-46;2006 [Non-patent document 2] Ludwig TE et.al.Nat.Biotechnol 24(2):185-7;2006 [Non-patent document 3] Masters et.al.Human Cell Culture.Dordrecht:Springer Netherlands;2007 [Non-patent document 4] Chen G et.al.Nat.Methods 8(5)424-9;2011 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a medium capable of efficiently growing pluripotent stem cells, and a method for efficiently growing pluripotent stem cells using the medium. [Means for solving the problem]

[0008] To achieve the above-mentioned objectives, the present inventors focused on changes in the amount of amino acids in the culture medium during the culture of pluripotent stem cells. They found that the amount of L-tryptophan in the culture medium rapidly decreased during the culture of pluripotent stem cells, and that L-tryptophan was the amino acid that was depleted most rapidly among all amino acids contained in the culture medium. Here, L-tryptophan, an essential amino acid, is formulated at a lower concentration in the culture medium than other amino acids (Non-Patent Documents 1, 2, 3, 4). It was predicted that L-tryptophan would quickly become depleted during mass proliferation of pluripotent stem cells, limiting cell proliferation. The present inventors then discovered that the proliferation of pluripotent stem cells could be promoted by increasing the amount of L-tryptophan in the culture medium or by adding L-tryptophan during the culture to replenish the consumed L-tryptophan, leading to the completion of the present invention.

[0009] That is, the present invention is as follows. [1] A medium for culturing pluripotent stem cells, comprising L-tryptophan or an L-tryptophan derivative at a concentration of 176 μM or more. [2] The medium according to [1], containing L-tryptophan or an L-tryptophan derivative in a concentration of 176 μM to 1408 μM. [3] The medium according to [1] or [2], which is a serum-free medium. [4] The medium according to any one of [1] to [3], wherein the pluripotent stem cells are induced pluripotent stem cells. [5] A medium according to any one of [1] to [4], wherein the tryptophan derivative is a dipeptide formed by a peptide bond between tryptophan and an amino acid. [6] The medium of [5], wherein the dipeptide is L-alanyl-L-tryptophan. [7] A method for culturing pluripotent stem cells, comprising culturing pluripotent stem cells in the medium of any one of [1] to [6]. [8] The method of [7], which is a method for expanding pluripotent stem cells. [9] A pluripotent stem cell culture preparation comprising a medium according to any one of [1] to [6] and pluripotent stem cells.

[10] A method for culturing pluripotent stem cells, comprising the following steps: (1) culturing pluripotent stem cells in a medium containing L-tryptophan or an L-tryptophan derivative; (2) adding L-tryptophan or an L-tryptophan derivative to the resulting pluripotent stem cell culture to partially or completely replace the L-tryptophan or L-tryptophan derivative in the medium consumed in (1); and (3) The pluripotent stem cell culture to which L-tryptophan or an L-tryptophan derivative has been added is subsequently cultured.

[11] The method according to

[10] , wherein the medium containing L-tryptophan or an L-tryptophan derivative is a serum-free medium.

[12] The method according to

[10] or

[11] , wherein the tryptophan derivative is a dipeptide in which tryptophan and an amino acid are peptide-bonded.

[13] The method of

[12] , wherein the dipeptide is L-alanyl-L-tryptophan.

[14] A culture medium additive for promoting the proliferation of pluripotent stem cells, comprising L-tryptophan or an L-tryptophan derivative.

[15] A culture medium additive according to

[14] , wherein the tryptophan derivative is a dipeptide formed by a peptide bond between tryptophan and an amino acid.

[16] The medium additive according to

[15] , wherein the dipeptide is L-alanyl-L-tryptophan. [Effects of the Invention]

[0010] According to the present invention, it is possible to promote the proliferation of pluripotent stem cells, thereby enabling efficient mass cultivation of pluripotent stem cells. Specific effects of using this medium include the ability to obtain the target cell number in a shorter time than with conventional media, and the ability to obtain the target cell number using existing culture equipment without changing to specialized culture equipment for mass cultivation. Therefore, the human and financial costs associated with culturing pluripotent stem cells can be significantly reduced. [Brief explanation of the drawings]

[0011] [Figure 1] Cell coverage (%) of human induced pluripotent stem cells 201B7 in Essential-8 medium supplemented with L-tryptophan at final concentrations of 44 μM, 176 μM, 352 μM, 704 μM, and 1408 μM. 201B7 cells were seeded at single cell density at 13,000 cells per well in a 6-well plate and cultured for 6 days. The time at which L-tryptophan was added to the above concentrations was defined as time 0, and cell coverage was measured 24, 48, 72, 96, and 120 hours later. Results indicated a concentration-dependent growth-promoting effect of L-tryptophan. [Figure 2] Cell coverage (%) of human induced pluripotent stem cells 201B7 in mTeSR1 medium supplemented with L-tryptophan to final concentrations of 44 μM, 176 μM, 352 μM, 704 μM, and 1408 μM. 201B7 cells were seeded at single cell density at 13,000 cells per well in a 6-well plate and cultured for 6 days. The time at which L-tryptophan was added to the above concentrations was defined as time 0, and cell coverage was measured 24, 48, 72, 96, and 120 hours later. Results indicated a concentration-dependent growth-promoting effect of L-tryptophan. [Figure 3]Cell coverage (%) of human induced pluripotent stem cells 201B7 in TeSR2 medium supplemented with L-tryptophan at final concentrations of 44 μM, 176 μM, 352 μM, 704 μM, and 1408 μM. 201B7 cells were seeded at single cell density at 13,000 cells per well in a 6-well plate and cultured for 6 days. The time at which L-tryptophan was added to the above concentrations was defined as time 0, and cell coverage was measured 24, 48, 72, 96, and 120 hours later. Results indicated a concentration-dependent growth-promoting effect of L-tryptophan. [Figure 4] Cell coverage (%) of human induced pluripotent stem cells 253G4 in mTeSR1 medium supplemented with L-tryptophan to final concentrations of 44 μM, 176 μM, 352 μM, 704 μM, and 1408 μM. 40,000 253G4 cells were seeded at single cell level per well in a 6-well plate and cultured for 6 days. The time at which L-tryptophan was added to the above concentrations was defined as time 0, and cell coverage was measured 24, 48, 72, 96, and 120 hours later. Results showed a proliferation-promoting effect dependent on L-tryptophan concentration. [Figure 5] Cell coverage (%) of human embryonic stem cells H9 in mTeSR1 medium supplemented with L-tryptophan to final concentrations of 44 μM, 176 μM, 352 μM, 704 μM, and 1408 μM. 10,000 H9 cells were seeded as single cells per well in a 6-well plate and cultured for 6 days. The time when L-tryptophan was added to the above concentrations was defined as 0 hours, and cell coverage was measured 24, 48, 72, 96, and 120 hours later. Results showed a proliferation-promoting effect dependent on L-tryptophan concentration. [Figure 6]Cell coverage (%) of human embryonic kidney-derived 293T cells in DMEM medium supplemented with 10% FCS and added with L-tryptophan to final concentrations of 44 μM, 176 μM, 352 μM, 704 μM, and 1408 μM. 293T cells were seeded at single cell density at 10,000 cells per well in a 6-well plate and cultured for 6 days. The time at which L-tryptophan was added to the above concentrations was defined as time 0, and cell coverage was measured 24, 48, 72, 96, and 120 hours later. No results were obtained that indicated a concentration-dependent growth-promoting effect of L-tryptophan. [Figure 7] Cell coverage (%) of human induced pluripotent stem cells 201B7 in mTeSR1 medium supplemented with L-kynurenine to final concentrations of 50μM, 100μM, 200μM, 500μM, and 1000μM. 20,000 201B7 cells were seeded as single cells per well in a 6-well plate and cultured for 6 days. The time of L-kynurenine addition was set as 0, and cell coverage was measured after 0, 24, 48, 72, 96, and 120 hours. Results showed a proliferation-promoting effect in the L-kynurenine addition groups of 50-500μM. [Figure 8] Cell coverage (%) of human induced pluripotent stem cells 201B7 in mTeSR1 medium supplemented with kynurenic acid to final concentrations of 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM. 20,000 201B7 cells were seeded as single cells per well in a 6-well plate and cultured for 6 days. The time point at which kynurenic acid was added was set as 0, and cell coverage was measured after 0, 24, 48, 72, 96, and 120 hours. Results indicated a proliferation-promoting effect in the 50-500 μM kynurenic acid addition groups. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a culture medium that promotes cell proliferation (hereinafter also referred to as the culture medium of the present invention), a method for promoting cell proliferation (hereinafter also referred to as the method of the present invention), and a culture medium additive for promoting cell proliferation.

[0013] (1) L-tryptophan or L-tryptophan derivatives L-tryptophan (2-amino-3-(indolyl)propionic acid) is an essential amino acid that makes up proteins. In this specification, L-tryptophan includes salts of L-tryptophan. Examples of salts of L-tryptophan include, but are not limited to, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate; organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate; inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and ammonium salt; organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt, and diisopropylammonium salt; and amino acid salts such as arginine, aspartic acid, and glutamic acid. As the salt of L-tryptophan, it is preferable to use the hydrochloride, sodium salt or potassium salt.

[0014] L-tryptophan can be obtained by a method known per se, for example, methods for producing L-tryptophan include, but are not limited to, those described in JP 2012-223092 A, JP 2012-100537 A, JP 2011-167071 A, JP 2010-263790 A, and JP 2010-110217 A. Alternatively, commercially available L-tryptophan may be used, including, but not limited to, Wako Pure Chemical Industries, Ltd. (product number: 038-23581), Tokyo Chemical Industry Co., Ltd. (product number: T0541), Nacalai Tesque, Inc. (product number: 13043-92), MP Biomedicals, Inc. (product number: ICN1031505), and Sigma-Aldrich Co., Ltd. (product number: T8941).

[0015] The L-tryptophan derivative is not particularly limited as long as it provides L-tryptophan in the medium, and examples thereof include substances that provide L-tryptophan upon hydrolysis when added to the medium. Examples of L-tryptophan derivatives include, but are not limited to, dipeptides in which tryptophan is bound to an amino acid by a peptide bond, C1-6 alkyl esters of tryptophan, and N-acetyltryptophan. An example of a dipeptide consisting of tryptophan and an amino acid is L-alanyl-L-tryptophan. Tryptophan, L-arginyl-L-tryptophan, L-asparaginyl-L-tryptophan, L-aspartic acid-L-tryptophan, L-cysteinyl-L-tryptophan, L-glutaminyl-L-tryptophan, L-glutamic acid-L-tryptophan, glycyl-L-tryptophan, L-histidinyl-L-tryptophan, L-isoleucyl-L-tryptophan, L-leucyl-L-tryptophan, L-lysyl-L-tryptophan, L-methionyl-L-tryptophan, L-phenylalanyl-L-tryptophan, L-prolyl-L-tryptophan, L-seryl-L-tryptophan, L-threonyl-L-tryptophan, L-tyrosyl-L-tryptophan, L-valyl-L-tryptophan, L-tryptophanyl-L-tryptophan, L-tryptophanyl-L-tryptophan L-tryptophanyl-L-alanine, L-tryptophanyl-L-arginine, L-tryptophanyl-L-asparagine, L-tryptophanyl-L-aspartic acid, L-tryptophanyl-L-cysteine, L-tryptophanyl-L-glutamine, L-tryptophanyl-L-glutamic acid, L-tryptophanyl-glycine, L-tryptophanyl-L-histidine, L-tryptophanyl-L-isoleucine, L-tryptophanyl-L-leucine, L-tryptophanyl-L-lysine, L-tryptophanyl-L-methionine, L-tryptophanyl-L-phenylalanine, L-tryptophanyl-L-proline, L-tryptophanyl-L-serine, L-tryptophanyl-L-threonine, L-tryptophanyl-L-tyrosine, and L-tryptophanyl-L-valine. C1-6 alkyl esters of tryptophan include, but are not limited to, L-tryptophan methyl ester, L-tryptophan ethyl ester, and the like. In the present specification, the L-tryptophan derivative includes a salt of the L-tryptophan derivative. Examples of the salt of the L-tryptophan derivative include those salts of L-tryptophan described above. When used in culturing pluripotent stem cells, the L-tryptophan derivative is preferably L-alanyl-L-tryptophan or glycyl-L-tryptophan. In the present specification, the term "L-tryptophan derivative" includes metabolites of L-tryptophan or salts thereof. When used in the culture of pluripotent stem cells, the L-tryptophan metabolites are preferably L-kynurenine or kynurenic acid.

[0016] L-tryptophan derivatives can be obtained by known methods. For example, methods for producing L-tryptophan dipeptides include, but are not limited to, general solid-phase synthesis methods. Alternatively, commercially available L-tryptophan derivatives may be used, including, but not limited to, Wako Pure Chemical Industries, Ltd. (product number: 038-23581), Tokyo Chemical Industry Co., Ltd. (product number: T0541), Nacalai Tesque, Inc. (product number: 13043-92), MP Biomedicals, Inc. (product number: ICN1031505), and Sigma-Aldrich Co., Ltd. (product number: T8941).

[0017] (2) Pluripotent stem cells As used herein, pluripotent stem cells refer to immature cells that have the ability to self-replicate and differentiate / proliferate, and that have the ability to differentiate into all tissues and cells that constitute a living body. Examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) (Takahashi K et al., Cell. 2007 Nov 30;131(5):861-72), spermatogonial stem cells (mGS cells) (Kanatsu-Shinohara M et al., Biol Reprod.2007 Jan;76(1):55-62), embryonic germ cells (Matsui Y et al., Cell.1992 Sep 4;70(5):841-7).

[0018] Pluripotent stem cells can be obtained by methods known per se. For example, embryonic stem cells (ES cells) The present invention provides a method for culturing the inner cell mass in mammalian blastocysts (e.g., Manipulating These include, but are not limited to, the method described in the Mouse Embryo: A Laboratory Manual, Fourth Edition 2014, Cold Spring Harbor Laboratory Press), and a method for culturing early embryos produced by somatic cell nuclear transfer (Wilmut et al., Nature. 1997 Feb 27; 385(6619):810-3; Wakayama et al., Nature. 1998 Jul 23; 394(6691):369-74; Wakayama T et al., Science. 2001 Apr 27; 292(5517):740-3).

[0019] Furthermore, embryonic stem cells can be obtained from designated institutions. For example, human ES cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University.

[0020] An example of a method for obtaining induced pluripotent stem cells is a method for introducing nuclear reprogramming substances (e.g., Oct3 / 4, Sox2, c-Myc, Klf4, etc.) into somatic cells (Takahashi K et al., 2011). et al., Cell. 2006 Aug 25; 126(4):663-76, WO2007 / 069666 International Publication). In addition, induced pluripotent stem cells can be derived from various methods, including, but not limited to, Takahashi K et al., Nat Protoc. 2007; 2(12):3081-9, Aoi et al., Science. 2008 Aug 1; 321(5889):699-702, Takahashi, K et al. al.,Cell.2007 Nov 30;131(5):861-72, Yu,J et al., Science. 2007 Dec 21; 318(5858): 1917-20, Nakagawa, M et al., Nat Biotechnol. 2008 Jan; 26(1): 101-6, etc., but are not limited to these.

[0021] Furthermore, induced pluripotent stem cells can also be obtained from designated institutions. For example, human iPS cells, 253G1 cells and 201B7 cells, can be purchased from iPS Academia Japan, Inc.

[0022] Embryonic germ cells can be induced by isolating primordial germ cells according to conventional methods and culturing them in the presence of LIF, bFGF, and SCF. mGS cells can also be produced from testicular cells according to the method described in WO2005 / 100548.

[0023] The pluripotent stem cells used in the present invention are preferably embryonic stem cells or induced pluripotent stem cells, more preferably induced pluripotent stem cells.

[0024] In the present invention, pluripotent stem cells derived from mammals are generally used. Examples of mammals include, but are not limited to, rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. In the present invention, pluripotent stem cells derived from rodents such as mice or primates such as humans are preferably used, and more preferably, pluripotent stem cells derived from humans are used.

[0025] In the present invention, human induced pluripotent stem cells are most preferably used.

[0026] (3) Culture medium for pluripotent stem cells In one embodiment of the present invention, the present invention provides a method for producing a high concentration of L-tryptophan or L-tryptophan. A medium for culturing pluripotent stem cells (herein also referred to as the medium of the present invention) containing a fan derivative is provided. Use of the medium of the present invention enables efficient proliferation of pluripotent stem cells. In particular, the medium of the present invention is useful for proliferating and maintaining pluripotent stem cells while maintaining their undifferentiated state.

[0027] The medium of the present invention is characterized by containing a high concentration of L-tryptophan or an L-tryptophan derivative. "High concentration" means a concentration exceeding the L-tryptophan concentration (44 μM) equivalent to the free L-tryptophan concentration in human blood. When pluripotent stem cells are cultured using a conventional medium containing L-tryptophan equivalent to the free L-tryptophan concentration in human blood, the proliferation of the pluripotent stem cells is limited due to the early depletion of L-tryptophan in the medium. Because the medium of the present invention contains a high concentration of L-tryptophan or an L-tryptophan derivative, L-tryptophan depletion is unlikely to occur during pluripotent stem cell culture, enabling high proliferation rates and long-term proliferation of pluripotent stem cells.

[0028] The concentration of L-tryptophan or an L-tryptophan derivative in the medium of the present invention is not particularly limited as long as it can promote the proliferation of pluripotent stem cells, but for example, the L-tryptophan concentration in the medium of the present invention is 176 μM or more, preferably 352 μM or more, and more preferably 704 μM or more. The upper limit of the L-tryptophan or L-tryptophan derivative concentration in the medium of the present invention is theoretically the saturation concentration of L-tryptophan or an L-tryptophan derivative, but from the viewpoints of the solubility of L-tryptophan or an L-tryptophan derivative in the medium and cost, the L-tryptophan or L-tryptophan derivative concentration in the medium is preferably 1408 μM or less.

[0029] The medium of the present invention has the effect of promoting the proliferation of pluripotent stem cells. "Promoting the proliferation of pluripotent stem cells" means that the proliferation of pluripotent stem cells is promoted when cultured in the medium of the present invention compared to when cultured in a control medium having the same composition as the medium of the present invention except that the L-tryptophan concentration is equivalent to the free L-tryptophan concentration in human blood (44 μM).

[0030] The components other than L-tryptophan or L-tryptophan derivatives contained in the medium of the present invention are not particularly limited as long as they can achieve the effect of promoting the proliferation of pluripotent stem cells, and compositions used in the maintenance culture of conventional pluripotent stem cells can be appropriately adopted.

[0031] The medium of the present invention can be prepared by adding L-tryptophan or an L-tryptophan derivative to a medium suitable for the maintenance culture of pluripotent stem cells at the above-mentioned concentrations. To prepare the medium, one type of L-tryptophan or L-tryptophan derivative may be used, or multiple types of L-tryptophan and / or L-tryptophan derivatives may be used in combination.

[0032] The medium of the present invention may be prepared using a medium commonly used for culturing mammalian cells as the basal medium. The basal medium is not particularly limited as long as it can achieve the desired effect, such as promoting the proliferation of pluripotent stem cells. Examples of the basal medium include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof, which can be used for culturing animal cells. Alternatively, a medium commonly used for culturing pluripotent stem cells may be prepared as the basal medium. Commercially available basal media for stem cell culture include StemFit® AK medium (Ajinomoto Co., Inc.), Essentia Examples of suitable medium include l8 medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), RHB medium (StemCells, Inc.), TeSR™-E6 (STEMCELL Technologies), hESF-GRO medium (Nipro Corporation), HESF-DIF medium (Nipro Corporation), CSTI-7 (Cell Science Institute, Inc.), and Essential 6 medium (Life Technologies).

[0033] The medium of the present invention is preferably a chemically defined medium (CDM) whose components are chemically defined, in order to avoid contamination with chemically undefined components. The medium of the present invention is preferably a serum-free medium, in order to avoid contamination with chemically undefined components. In the present invention, "serum-free medium" refers to a medium that does not contain unconditioned or unpurified serum. In the present invention, media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors such as bFGF) are also included in the serum-free medium, as long as they do not contain unconditioned or unpurified serum.

[0034] The serum-free medium may contain a serum substitute. Examples of serum substitutes include those that appropriately contain serum albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679. Commercially available serum substitutes may also be used. Examples of such commercially available serum substitutes include Knockout TM Examples of such a surfactant include, but are not limited to, Serum Replacement (Life Technologies: hereinafter sometimes referred to as KSR), Chemically-defined Lipid concentrated (Life Technologies), Glutamax™ (Life Technologies), B27 (Life Technologies), and N2 (Life Technologies).

[0035] Typically, the medium of the present invention contains, in addition to L-tryptophan, all essential amino acids other than L-tryptophan (L-leucine, L-lysine, L-phenylalanine, L-isoleucine, L-threonine, L-histidine, L-methionine, and L-valine).

[0036] The medium of the present invention preferably contains all non-essential amino acids (L-alanine, L-arginine, L-asparagine, L-aspartic acid, glycine, L-glutamine, L-glutamic acid, L-cysteine, L-serine, L-tyrosine, and L-proline), although L-alanyl-L-glutamine may be used instead of L-glutamine.

[0037] The medium of the present invention may contain natural amino acids such as L-cystine in addition to the 20 amino acids mentioned above.

[0038] The medium of the present invention may further contain medium additives. Examples of medium additives include, but are not limited to, ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitors such as Y-27632, antibiotics such as penicillin-streptomycin, vitamins, L-ascorbic acid, L-ascorbyl magnesium phosphate, sodium pyruvate, 2-aminoethanol, glucose, sodium bicarbonate, HEPES, insulin, progesterone, sodium selenate, and putrescine. The additives are preferably contained within a concentration range known per se.

[0039] The medium of the present invention may contain a fatty acid. Examples of fatty acids include, but are not limited to, leic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, palmitic acid, stearic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, butyric acid, acetic acid, palmitoleic acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, pentadecylic acid, margaric acid, hexenic acid, eleostearic acid, arachidic acid, 8,11-eicosadienoic acid, 5,8,11-eicosatrienoic acid, behenic acid, lignoceric acid, nervonic acid, cerotic acid, montanic acid, and melissic acid. The fatty acids contained in the medium of the present invention may be saturated or unsaturated.

[0040] The medium of the present invention may have a composition used in known cell cultures, depending on the intended use. For example, when the objective is to proliferate pluripotent stem cells while maintaining their undifferentiated state, the medium of the present invention preferably does not contain a substance that promotes the differentiation of pluripotent stem cells, and preferably contains a substance that inhibits the differentiation of pluripotent stem cells. Examples of substances that inhibit the differentiation of pluripotent stem cells include FGF2 for human pluripotent stem cells and leukemia inhibitory factor (LIF) for mouse pluripotent stem cells.

[0041] More specifically, media for promoting proliferation while maintaining the undifferentiated state of pluripotent stem cells include a medium containing L-ascorbic acid, selenium, transferrin, NaHCO3, insulin, FGF2, and TGFβ1 in DMEM / F-12 medium (Chen G et al., Nat Methods. 2011 May;8(5):424-429), and a medium containing non-essential amino acids, L-glutamine, β-mercaptoethanol, insulin, transferrin, cholesterol, serum albumin, pipecolic acid, lithium chloride, FGF2, and TGFβ1 in DMEM / F-12 medium (Ludwig TE et al., Nat Methods. 2006 Aug;3(8):637-46), a serum-free medium for maintaining mouse embryonic stem cells supplemented with leukemia inhibitory factor, polyvinyl alcohol, L-glutamine, insulin, transferrin, selenium, 2-mercaptoethanol, and antibiotics (Japanese Patent Laid-Open No. 2007-228815), a serum-free medium comprising a mixture of pannexin, bFGF, PDGF, EGF, and vitamin C (Japanese Patent Laid-Open No. 2008-148643), and a medium for maintaining the pluripotency of mesenchymal stem cells characterized by containing TGF-β (Japanese Patent Laid-Open No. 2010-094062). The medium of the present invention can be prepared with reference to these compositions.

[0042] For example, the medium of the present invention can be prepared by adding L-tryptophan or an L-tryptophan derivative to a basal medium containing L-ascorbic acid, selenium, transferrin, insulin, FGF2, and TGFβ1 to a final concentration of 176 μM or more, but is not limited to this.

[0043] The pH of the medium of the present invention is preferably adjusted to about 6.0 to about 8.5, more preferably about 7.0 to about 7.5. The medium is preferably sterilized by filtration sterilization using a membrane filter or the like.

[0044] The medium of the present invention can be used for any culture method, such as adherent culture, suspension culture, embedded culture, and tissue culture. Furthermore, the form of the culture medium of the present invention is not particularly limited as long as the desired effects of the present invention can be obtained, and it can be prepared, for example, in the form of a liquid culture medium, a semi-liquid culture medium, or a solid culture medium. The culture medium of the present invention may also be prepared in powder form. Preparing it in powder form can greatly facilitate transportation and storage. Furthermore, by adding sterilized water and / or agar, etc. at the time of use, liquid, semi-liquid, or solid culture media can be easily prepared.

[0045] (4) Pluripotent stem cell culture method 1 The present invention provides a method for culturing pluripotent stem cells (herein also referred to as method 1 of the present invention), which comprises culturing pluripotent stem cells in the above-described medium of the present invention.

[0046] Use of the medium of the present invention enables efficient proliferation of pluripotent stem cells. In particular, the medium of the present invention is useful for proliferating and maintaining pluripotent stem cells while maintaining their undifferentiated state. Therefore, method 1 of the present invention is preferably a method for proliferating pluripotent stem cells, and more preferably a method for proliferating or maintaining pluripotent stem cells while maintaining their undifferentiated state.

[0047] In the method 1 of the present invention, the concentration of pluripotent stem cells in the medium is not particularly limited as long as the desired effect is achieved. 0 ~10 7 pieces / cm 3 , preferably 10 1 ~10 6 pieces / cm 3 , more preferably 10 2 ~10 5 pieces / cm 3 is.

[0048] Pluripotent stem cells may be cultured by seeding the stem cells in the medium of the present invention, in which the concentration of L-tryptophan or an L-tryptophan derivative has been adjusted to the desired concentration in advance, or by adding L-tryptophan or an L-tryptophan derivative to the medium after initiating cell culture, adjusting the concentration of L-tryptophan or an L-tryptophan derivative to the concentration required by the medium of the present invention, and then continuing the culture. When L-tryptophan or an L-tryptophan derivative is added after the initiation of cell culture, the medium additive of the present invention, which will be described in detail below, may be used.

[0049] The culture conditions in Method 1 of the present invention are not particularly limited as long as the desired effect, such as promoting the proliferation of pluripotent stem cells, can be achieved, except that the culture medium of the present invention is used. Culture conditions typically used for culturing pluripotent stem cells can be appropriately adopted depending on the purpose of the culture.

[0050] For example, methods for culturing pluripotent stem cells while maintaining their undifferentiated state include those described in Experimental Medicine Special Edition: Cell Culture Protocols Selectable by Purpose (Yodosha). Pluripotent stem cells may be cultured using feeder cells such as mouse embryonic fibroblasts (MEF) or mouse fibroblast cell line (STO), or in a feeder-free environment.

[0051] In Method 1 of the present invention, the incubator used for cell culture is not particularly limited as long as it is capable of culturing cells, and examples thereof include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles.

[0052] The culture vessel used for culturing cells may be either cell-adhesive or non-cell-adhesive, and is appropriately selected depending on the purpose. The cell-adhesive culture vessel can be coated with any cell-supporting substrate, such as an extracellular matrix (ECM), or an artificial substance that mimics its function, to improve cell adhesion to the surface of the culture vessel. The cell-supporting substrate can be any material intended for the attachment of stem cells or feeder cells (if used).

[0053] Other culture conditions can be set appropriately. For example, the culture temperature is not particularly limited as long as the desired effect, such as promoting cell proliferation, can be achieved, but is about 30 to 40°C, preferably about 37°C. The CO2 concentration is about 1 to 10%, preferably about 2 to 5%. The oxygen concentration is usually 1 to 40%, but is selected appropriately depending on the culture conditions, etc.

[0054] In Method 1 of the present invention, pluripotent stem cells can be cultured by methods known per se, such as adherent culture, suspension culture, and tissue culture.

[0055] Although there are no particular limitations as long as the desired effect, such as promotion of cell proliferation, can be achieved, the period for culturing pluripotent stem cells in Method 1 of the present invention is usually 2 days or more, preferably 4 days or more, and more preferably 7 days or more, and theoretically the culture can be continued indefinitely. By recovering the pluripotent stem cells cultured in the medium of the present invention, and passage a portion or all of them in fresh medium of the present invention, and continuing the culture, the pluripotent stem cells can be proliferated or maintained over a long period of time while maintaining their undifferentiated state.

[0056] (5) Pluripotent stem cell culture preparation The present invention provides a pluripotent stem cell culture preparation (culture preparation of the present invention) comprising the above-described medium of the present invention and pluripotent stem cells.

[0057] The pluripotent stem cells in the culture preparations of the present invention are viable, proliferating cells.

[0058] The pluripotent stem cells in the culture preparation of the present invention are preferably isolated. "Isolated" means that the cells have been subjected to a procedure to remove target components and factors other than cells, and are no longer in a naturally occurring state. The purity of "isolated pluripotent stem cells" (the percentage of pluripotent stem cells in the total number of cells) is usually 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 99% or more, and most preferably 100%.

[0059] In the culture preparation of the present invention, the pluripotent stem cells are present in, for example, a liquid or semi-liquid medium of the present invention. In one embodiment, the culture preparation of the present invention is a suspension of the pluripotent stem cells in the medium of the present invention. The culture preparation of the present invention may be sealed in a suitable container.

[0060] The culture preparation of the present invention is useful for carrying out the above-mentioned Method 1 of the present invention.

[0061] (6) Pluripotent stem cell culture method 2 A method for culturing pluripotent stem cells (culturing method 2 of the present invention), comprising the following steps: (1) culturing pluripotent stem cells in a medium containing L-tryptophan or an L-tryptophan derivative; (2) adding L-tryptophan or an L-tryptophan derivative to the resulting pluripotent stem cell culture to partially or completely replace the L-tryptophan or L-tryptophan derivative in the medium consumed in (1); and (3) The pluripotent stem cell culture to which L-tryptophan or an L-tryptophan derivative has been added is subsequently cultured.

[0062] The medium of the present invention may be used in step (1). However, the concentration of L-tryptophan or an L-tryptophan derivative in the medium used in step (1) is sufficient as long as it is a concentration capable of growing pluripotent stem cells (preferably a concentration capable of growing and maintaining pluripotent stem cells while maintaining an undifferentiated state), and does not need to be as "high" as the medium of the present invention. The concentration of L-tryptophan or an L-tryptophan derivative in the medium used in step (1) is, for example, 10 μM or more, preferably 15 μM or more, and more preferably 44 μM or more at the start of culture.

[0063] The composition of the medium used in step (1) is the same as that of the medium of the present invention, except that the concentration of L-tryptophan or an L-tryptophan derivative does not need to be "high."

[0064] The culture conditions in step (1) are the same as those in the above-mentioned method 1 of the present invention, except that the concentration of L-tryptophan or an L-tryptophan derivative in the medium does not need to be "high."

[0065] As a result of the culture in step (1), the pluripotent stem cells proliferate (preferably while maintaining an undifferentiated state), and as a result, L-tryptophan or an L-tryptophan derivative in the culture medium is consumed, resulting in a decrease in its concentration in the culture medium.

[0066] In step (2), the timing of adding L-tryptophan or an L-tryptophan derivative to the pluripotent stem cell culture obtained in step (1) is not particularly limited as long as the desired effect, such as promoting pluripotent stem cell proliferation, can be achieved, and addition can be made at any time. For example, in step (1), L-tryptophan or an L-tryptophan derivative is added when the L-tryptophan or L-tryptophan derivative concentration in the medium has decreased to less than 10 μM, preferably less than 15 μM, and more preferably less than 44 μM. Alternatively, in step (1), L-tryptophan or an L-tryptophan derivative is added when the L-tryptophan or L-tryptophan derivative concentration in the medium has decreased to 50% or less, preferably 25% or less, of the concentration at the start of culture (100%). For example, L-tryptophan or an L-tryptophan derivative can be added 2 to 5 days, preferably 3 to 5 days, and more preferably 4 to 5 days, after the start of culture in step (1).

[0067] The L-tryptophan and / or L-tryptophan derivative to be added to the medium may be one type of L-tryptophan or L-tryptophan derivative, or a combination of multiple types of L-tryptophan and / or L-tryptophan derivatives.

[0068] The amount of L-tryptophan or an L-tryptophan derivative added to the medium is not particularly limited as long as the desired effect, such as promoting pluripotent stem cell proliferation, can be achieved. The L-tryptophan or L-tryptophan derivative is added to the medium at a concentration capable of proliferating pluripotent stem cells (preferably, at a concentration capable of proliferating and maintaining pluripotent stem cells while maintaining their undifferentiated state). For example, the L-tryptophan or L-tryptophan derivative is added to the medium at a concentration of 176 μM or higher, preferably 352 μM or higher. The L-tryptophan or L-tryptophan derivative may be added to the medium at a "high concentration" similar to that of the medium of the present invention. In one embodiment, the L-tryptophan or L-tryptophan derivative is added to the medium at a concentration of 176 μM or higher, preferably 352 μM or higher, more preferably 704 μM or higher. Theoretically, the upper limit of the concentration of L-tryptophan or an L-tryptophan derivative in the medium after addition is the saturation concentration of L-tryptophan or an L-tryptophan derivative. However, from the viewpoint of the solubility of L-tryptophan or an L-tryptophan derivative in the medium and the cost, the concentration of L-tryptophan or an L-tryptophan derivative in the medium is preferably 1408 μM or less. In this specification, "replenishing part or all of the L-tryptophan or L-tryptophan derivative in the medium consumed in (1)" includes not only replenishing part or all of the amount of L-tryptophan or L-tryptophan derivative added at the beginning of the culture in step (1), but also adding to the medium an amount of L-tryptophan or L-tryptophan derivative equal to or greater than the amount added at the beginning of the culture.

[0069] Here, the feature of Method 2 of the present invention is that, during the culture of pluripotent stem cells, part or all of L-tryptophan, which is the amino acid contained in the culture medium that is consumed and depleted the fastest, is replenished by adding exogenous L-tryptophan or an L-tryptophan derivative, and therefore amino acids other than L-tryptophan may or may not be added together with L-tryptophan or an L-tryptophan derivative.

[0070] In one embodiment, in step (2), only L-tryptophan or an L-tryptophan derivative is added as an amino acid, and no other amino acids are added.

[0071] In another embodiment, in step (2), amino acids other than L-tryptophan (L-leucine, L-lysine, L-phenylalanine, L-isoleucine, L-threonine, L-histidine, L-methionine, L-valine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, glycine, L-glutamine, L-glutamic acid, L-cysteine, L-serine, L-tyrosine, L-proline) or derivatives thereof may or may not be added together with L-tryptophan or an L-tryptophan derivative. The amino acids added may be one type or multiple types.

[0072] Then, in step (3), the pluripotent stem cell culture to which L-tryptophan or an L-tryptophan derivative has been added is subsequently cultured. The culture conditions in step (3) may be the same as those in step (1), or may be changed as long as the desired effect of the present invention is obtained. In a preferred embodiment of the present invention, the culture conditions in step (3) are the same as those in step (1). Since the addition of L-tryptophan or an L-tryptophan derivative prevents L-tryptophan depletion, the pluripotent stem cells can continue to proliferate (preferably while maintaining an undifferentiated state).

[0073] In method 2 of the present invention, the proliferation of pluripotent stem cells can be maintained by adding only L-tryptophan or an L-tryptophan derivative as an amino acid, or only a portion of amino acids containing L-tryptophan or an L-tryptophan derivative, without the need to replace the entire culture medium, thereby enabling pluripotent stem cells to be proliferated efficiently and at low cost.

[0074] (7) Culture medium additives The present invention provides a medium additive containing L-tryptophan or an L-tryptophan derivative (herein also referred to as the medium additive of the present invention). The medium additive of the present invention can be used when adding L-tryptophan or an L-tryptophan derivative in the above-mentioned Method 1 or 2 of the present invention.

[0075] The L-tryptophan and / or L-tryptophan derivative contained in the culture medium additive of the present invention may be a single type of L-tryptophan or L-tryptophan derivative, or a combination of multiple types of L-tryptophan and / or L-tryptophan derivatives.

[0076] The medium additive of the present invention, when added to a culture medium for pluripotent stem cells, has the effect of promoting the proliferation of pluripotent stem cells. The medium additive of the present invention is preferably used for promoting the proliferation of pluripotent stem cells.

[0077] The medium additive of the present invention may further contain, in addition to L-tryptophan and / or an L-tryptophan derivative, a serum substitute, a medium additive, or a fatty acid depending on the intended use, as long as the desired effect is not impaired. These serum substitutes, medium additives, and fatty acids are as described above, and each is preferably contained within a known concentration range. The medium additive of the present invention may also contain, in addition to L-tryptophan and / or an L-tryptophan derivative, additives that have traditionally been used in cell culture, as appropriate, as long as the desired effect is not impaired.

[0078] In one embodiment, the medium additive of the present invention contains only L-tryptophan or an L-tryptophan derivative as an amino acid, and does not contain any other amino acids.

[0079] In one embodiment, the medium additive of the present invention contains, in addition to L-tryptophan or an L-tryptophan derivative, one, two, three, four, five, or six amino acids selected from the group consisting of L-glutamine, L-arginine, L-cysteine, L-aspartic acid, L-serine, and L-methionine. In this case, amino acids other than those listed above as amino acids contained in the medium additive of the present invention may or may not be contained in the medium additive of the present invention.

[0080] The medium additive of the present invention may contain, in addition to L-tryptophan and / or an L-tryptophan derivative, any additive, such as a stabilizer, a tonicity agent, a pH adjuster, etc., in an appropriate amount, as long as the desired effect is not impaired.

[0081] The medium additive of the present invention may be in any dosage form as long as the desired effect can be obtained, such as a solution, solid, powder, etc. When it is in solid or powder form, it can be dissolved in an appropriate buffer solution or the like to a desired concentration and used. When the medium additive is in the form of a solution, the pH of the solution is preferably adjusted to about 5.0 to about 8.5, more preferably about 6.0 to about 8.0. When the medium additive is in the form of a solution, the solution is preferably subjected to a sterilization treatment such as filtration sterilization using a membrane filter or the like.

[0082] The present invention will be explained in more detail below by showing Reference Examples and Examples, but these are not intended to limit the scope of the present invention.

[0083] (Reference example) The pluripotent stem cell line 201B7 (iPS Academia Japan) was cultured for 5 days using a medium in which the amount of each amino acid in the medium had been previously determined, and the amount of each amino acid in the medium was measured. The pluripotent stem cells were cultured in a 100 mm tissue culture dish (353003, Becton Dickinson Japan) coated with Matrigel (354277, Corning) at 1.0 00,000 cells were seeded and cultured at 5% CO2 / 37°C. The amount of amino acids was measured by the following method. Quantitative analysis of amino acids was performed according to the method described by Shinbo et al. (Anal Chem. 2009 Jul 1;81(13):5172-9. Multifunctional and highly sensitive precolumn re agents for amino acids in liquid chromatography / tandem mass spectrometry. Shimbo K, Y Ahashi A, Hirayama K, Nakazawa M, Miyano H. The analysis was carried out using the LC-MS / MS system reported in [1]. The samples were placed in L-type tubes and stored at -80°C until measurement. After protein removal, the samples were derivatized with APDS reagent and then analyzed. The amino acid concentrations in each sample were calculated using a calibration curve. As a result, L-tryptophan, which was present at a concentration of 44 μM at the start of culture, was depleted by the fourth day of culture. On the other hand, the other amino acids remained in the medium even after five days of culture, with the lowest remaining amount being approximately 20%. Therefore, it was found that L-tryptophan is the amino acid that is depleted the fastest in pluripotent stem cell culture. [Example]

[0084] Example 1: Growth-promoting effect of L-tryptophan in three commercially available media First, we evaluated the effect of L-tryptophan on the proliferation of induced pluripotent stem cells (iPS cells) using three commercially available media. Human iPS cells, the 201B7 strain purchased from iPS Academia Japan, were cultured under conditions of 5% CO2 / 37°C. L-tryptophan (Sigma-Aldrich: T8941) is an essential The compounds were added to the media containing 8 (Life Technologies: A1517001), mTeSR1 (Stem Cell Technologies: 05850), and TeSR2 (Stem Cell Technologies: 05860) at predetermined concentrations, and the effects were examined by culturing the media.

[0085] L-tryptophan (Sigma-Aldrich: T8941) was added to Essential-8, mTeSR1, and TeSR2 media at final concentrations of 44, 176, 352, 704, and 1408 μM to prepare medium for the study of the proliferation-promoting effect of L-tryptophan. Six-well plates coated with Matrigel (Becton Dickinson Japan) were prepared as a basement membrane matrix, and 13,000 cells were seeded at single cell density per well. The day after seeding, the cells were evaluated using the medium prepared above. The culture period was 6 days, and the addition of L-tryptophan was set as 0, with cell coverage measured using an IncuCyte™ microscope at 24, 48, 72, 96, and 120 hours. The medium used for seeding was medium without Y-27632 at a final concentration of 10 μM. The results of a series of experiments performed for each medium are shown in Figures 1, 2, and 3. The results showed that L-tryptophan concentration-dependently promoted proliferation. Furthermore, the expression of undifferentiated markers (OCT, Nanog, alkaline phosphatase) was not affected by the addition of high concentrations of L-tryptophan (data not shown).

[0086] Example 2: Growth-promoting effect of L-tryptophan - Culture results using another human induced pluripotent stem cell line Next, we evaluated the proliferation-promoting effect of L-tryptophan using 253G4, a different human induced pluripotent stem cell (iPS cell) line, and the H9 human embryonic stem cell line. Culture was performed in mTeSR1 medium (Stem Cell Technologies, Inc.: 05850) at 5% CO2 and 37°C. L-tryptophan (Sigma-Aldrich: T8941) was added to mTeSR1 (Stem Cell Technologies: 05850) medium at a predetermined concentration, and the medium was used for culture to examine its effect.

[0087] The proliferation-promoting effect of L-tryptophan was examined by adding L-tryptophan (Sigma-Aldrich: T8941) to mTeSR1 medium at final concentrations of 44, 176, 352, 704, and 1408 μM. Six-well plates were prepared and coated with Matrigel (Becton Dickinson Japan) as a basement membrane matrix. 253G4 cells were seeded at 40,000 cells per well, and H9 cells were seeded at 10,000 cells per well. The day after seeding, the medium was replaced with the above-prepared medium. The culture period was 6 days. The addition of L-tryptophan was set as 0, and cell coverage was measured using an IncuCyte™ system at 24, 48, 72, 96, and 120 hours. The medium used for seeding was medium without Y-27632 at a final concentration of 10 μM. The experiment was performed in quintuplicate for each cell type, and the results are shown in Figures 4 and 5. The concentration-dependent growth-promoting effect of L-tryptophan was also confirmed in other human pluripotent stem cells.

[0088] Example 3: Growth-promoting effect of L-tryptophan - Culture results using human embryonic kidney-derived cell line HEK293T Next, we evaluated the growth-promoting effect of L-tryptophan on HEK293T cells, a human embryonic kidney cell line. The cells were cultured in DMEM medium (Thermo Fisher Scientific: 11965) supplemented with 10% fetal bovine serum at 5% CO2 and 37°C. L-tryptophan (Sigma-Aldrich: T8941) was added to DMEM medium (Thermo Fisher Scientific: 11965) supplemented with 10% fetal bovine serum to a predetermined concentration, and its effects were examined by culturing the medium.

[0089] The proliferation-promoting effect of L-tryptophan was examined by adding L-tryptophan (Sigma-Aldrich: T8941) to DMEM medium (Thermo Fisher Scientific: 11965) supplemented with 10% fetal bovine serum at final concentrations of 44, 176, 352, 704, and 1408 μM. Six-well plates were prepared and 10,000 cells were seeded at single cell density per well. The day after seeding, the medium was replaced with the medium prepared above. The culture period was 6 days, and cell coverage was measured using an IncuCyte™ microscope after 24, 48, 72, 96, and 120 hours, with the addition of L-tryptophan set as 0. The results of five replicate experiments are shown in Figure 6. The concentration-dependent growth-promoting effect of L-tryptophan was not confirmed in HEK293 cells, a human embryonic kidney-derived cell line.

[0090] Example 4: Growth-promoting effect of L-kynurenine in commercially available medium L-kynurenine (Sigma-Aldrich: K8625) was added to mTeSR1 medium at final concentrations of 50, 100, 200, 500, and 1000 μM to prepare media for the study of proliferation promotion. Y-27632 was added at a final concentration of 10 μM for two days after seeding. Six-well plates coated with Matrigel (Becton Dickinson Japan) were prepared as a basement membrane matrix, and 20,000 cells were seeded at single cell density per well. Two days after cell seeding, evaluation was performed using the medium prepared as described above but without Y-27632. The time point at which L-kynurenine was added was set as zero, and cell coverage was measured using an IncuCyte™ microscope at 0, 24, 48, 72, 96, and 120 hours after addition. The results are shown in Figure 7. As shown in Figure 7, the addition of L-kynurenine at 50-500 μM demonstrated a cell proliferation promotion effect.

[0091] Example 5: Growth-promoting effect of kynurenic acid in a commercial medium Kynurenic acid (Sigma-Aldrich: K3375) was added to mTeSR1 medium to final concentrations of 50, 100, 200, 500, and 1000 μM to prepare media for examining the growth-promoting effect. Y-27632 was added at a final concentration of 10 μM for two days after seeding. Six-well plates were prepared, coated with Matrigel (Becton Dickinson Japan) as a basement membrane matrix, and 20,000 cells were seeded per well as single cells. Two days after cell seeding, evaluation was performed using the medium prepared as described above but without Y-27632. The addition of kynurenic acid was set as 0, and cell coverage was measured using IncuCyte after 0, 24, 48, 72, 96, and 120 hours. The results are shown in Figure 8. As shown in Figure 8, the addition of 50-500 μM kynurenic acid demonstrated a cell proliferation-promoting effect. [Industrial Applicability]

[0092] According to the present invention, it is possible to promote the proliferation of pluripotent stem cells, thereby reducing the human and financial costs involved in culturing pluripotent stem cells.

[0093] This application is based on patent application No. 2017-063842 filed in Japan (filing date: March 28, 2017), the contents of which are incorporated in their entirety herein.

Claims

1. (1) L-tryptophan, or (2) A serum-free medium additive for promoting the proliferation of human pluripotent stem cells, comprising L-tryptophan or an L-tryptophan derivative, for adding to a medium an L-tryptophan derivative selected from L-kynurenine, kynurenic acid, and L-tryptophan and a dipeptide formed by peptide-bonding an amino acid to the medium at a concentration of 176 μM to 1408 μM in the medium.

2. 2. The medium additive according to claim 1, wherein the L-tryptophan derivative is a dipeptide in which L-tryptophan and an amino acid are bound by a peptide bond.

3. 3. The medium additive according to claim 2, wherein the dipeptide is L-alanyl-L-tryptophan.

4. The serum-free medium additive according to any one of claims 1 to 3, wherein the medium additive is a medium additive for promoting the proliferation of human pluripotent stem cells while maintaining their undifferentiated state, and the medium comprises a substance that has the effect of suppressing the differentiation of pluripotent stem cells.

5. A serum-free medium additive for promoting the proliferation of human pluripotent stem cells, comprising L-tryptophan, for adding L-tryptophan to a medium to a concentration of 176 μM to 1408 μM in the medium.

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