Stem cell culture medium and method for culturing stem cells

Abstract

The stem cell medium according to the present invention contains, as a water soluble polymer compound, carboxymethyl cellulose and / or polyvinyl pyrrolidone. The added amount of carboxymethyl cellulose in the medium is preferably 0.001 µg / mL to 1 mg / mL in terms of the final concentration. Further, the added amount of polyvinyl pyrrolidone in the medium is preferably 0.05 µg / mL to 2 mg / mL in terms of the final concentration.

Description

[Technical field]

[0001] The present invention relates to a medium for stem cells and a method for culturing stem cells. [Background technology]

[0002] Stem cells, such as pluripotent stem cells, are undifferentiated cells with the ability to self-replicate and can differentiate into various types of cells. In recent years, regenerative medicine, in which pluripotent stem cells or cells induced to differentiate from pluripotent stem cells are transplanted into damaged tissues of patients to regenerate their functions, has been actively studied. Since regenerative medicine requires the preparation of large quantities of stem cells, such as pluripotent stem cells and mesenchymal stem cells, and their differentiated cells, methods for efficiently proliferating and efficiently differentiating these stem cells are being investigated.

[0003] Stem cell culture typically involves the use of basal media for stem cells, such as mTeSR1 medium (Non-Patent Document 2), Essential-8 medium (Non-Patent Document 3), or Dulbecco's modified Eagle medium (DMEM) / F12 medium, together with various additives such as cytokines, serum, or its substitutes (e.g., albumin). Patent Document 1 discloses a medium in which the seven kinds of non-essential amino acids used as the additives are reduced. Patent Document 2 discloses the addition of albumin and polyvinyl alcohol to a medium. Patent Document 3 discloses the use of a serum-free medium containing phospholipids and fatty acids together with multiple cytokines for the culture of mesenchymal stem cells, which are a type of stem cell. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication No. 2017 / 0198258 [Patent Document 2] US Patent Application Publication No. 2017 / 0009200 [Patent Document 3] US Patent Application Publication No. 2012 / 0329087 [Non-patent literature]

[0005] [Non-Patent Document 1] Ludwig, et al., Nat. Methods, 2006, vol. 3(8), p.637-46 [Non-Patent Document 2] Chen, et al., Nat. Methods, 2011, vol. 8(5), p.424-9 Summary of the Invention

[0006] However, the techniques described in Patent Documents 1 to 3 and Non-Patent Documents 1 and 2 were not sufficient in terms of the effects on stem cell proliferation, etc., and the development of further improved techniques was therefore required. Therefore, an object of the present invention is to provide a medium for stem cells that has good proliferation performance while maintaining the differentiation potential of stem cells, and a method for culturing stem cells that uses said medium.

[0007] As a result of intensive research to solve the above problems, the inventors discovered that by adding at least one of carboxymethylcellulose and polyvinylpyrrolidone as a water-soluble polymer compound to the culture medium, stem cells can have good proliferation performance while maintaining their differentiation ability, and thus completed the present invention.

[0008] The present invention has been made based on the above findings, and provides the following medium for stem cells and a method for culturing stem cells using said medium. [1] A medium for stem cells, comprising at least one of carboxymethylcellulose and polyvinylpyrrolidone as a water-soluble polymer compound. [2] The stem cell culture medium according to the above [1], wherein the amount of carboxymethylcellulose added to the medium is 0.001 μg / mL to 1 mg / mL as a final concentration. [3] The stem cell culture medium according to the above [1], wherein the amount of polyvinylpyrrolidone added to the medium is 0.05 μg / mL to 2 mg / mL as a final concentration. [4] The medium for stem cells according to any one of [1] to [3] above, wherein the amount of recombinant albumin added to the medium is 0.05 to 1 mg / mL as a final concentration. [5] The medium for stem cells according to any one of [1] to [4] above, further comprising β-nicotinamide mononucleotide. [6] A method for culturing stem cells, comprising using the medium for stem cells according to any one of [1] to [5] above. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a graph showing the results of absorbance measurement by a crystal violet staining method in proliferation performance test I in Examples 1 to 7 and Comparative Example 1. [Diagram 2] FIG. 2 is a graph showing the results of absorbance by the crystal violet staining method in proliferation performance test I in Example 11 and Comparative Example 1. [Diagram 3] FIG. 3(a) is a graph showing the change over time in the cumulative number of divisions in proliferation performance test II in Example 8 and Comparative Example 1, and FIG. 3(b) is a graph showing the change over time in the cumulative number of divisions in proliferation performance test II in Example 13 and Comparative Example 1. [Figure 4] FIG. 4 is a graph showing the results of flow cytometry of differentiation potential test I in Examples 8, 13 and Comparative Example 1. [Diagram 5] FIG. 5 shows images showing the state of stem cells after differentiation induction in Differentiation Potential Test II in Examples 8, 13, and Comparative Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will now be described based on its preferred embodiments (aspects). The medium for stem cells of the present invention is used for culturing stem cells. "Stem cells" are undifferentiated cells that have the ability to self-replicate and differentiate into various cell types. Examples of stem cells include pluripotent stem cells such as ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells), as well as somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, and skin stem cells. Stem cells to be cultured using the stem cell medium according to the present invention are preferably animal-derived stem cells, more preferably mammalian-derived stem cells, and even more preferably human-derived stem cells. From the viewpoint of obtaining better proliferation performance while maintaining differentiation ability, the stem cells cultured using the stem cell medium of the present invention are preferably stem cells capable of differentiating into any one of ectoderm, mesoderm, and endoderm cells, and more preferably stem cells capable of differentiating into mesoderm cells. Examples of such stem cells include mesenchymal stem cells.

[0011] The stem cell culture medium according to the present invention is characterized in that it contains at least one of carboxymethylcellulose and polyvinylpyrrolidone as a water-soluble polymer compound. By including one of these water-soluble polymer compounds in the stem cell culture medium, it is possible to obtain good proliferation performance while maintaining the differentiation ability of stem cells.

[0012] Carboxymethylcellulose (hereinafter referred to as "CMC"; CAS Registry Number 9000-11-7) is a cellulose derivative, a cellulose ether in which some of the hydroxyl groups of the glucopyranose that constitutes cellulose are replaced with carboxymethyl groups. The CMC used in the present invention may be any CMC that has been conventionally used in cell culture media without any particular limitations. Carboxymethylcellulose salts may also be used. From the viewpoint of further improving the growth performance, the CMC preferably has a degree of substitution (degree of etherification) of carboxymethyl ether groups per anhydroglucose unit of 0.6 to 0.8. Such CMC is commercially available, and for example, commercially available products such as sodium carboxymethyl cellulose (cat. no. 07326-95) manufactured by Nacalai Tesque and carboxymethyl cellulose (cat. no. 11676-85) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. can be used.

[0013] Polyvinylpyrrolidone (hereinafter referred to as "PVP"; CAS registration number 9003-39-8) is a water-soluble polymeric compound formed by polymerization of N-vinyl-2-pyrrolidone. The PVP used in the present invention may be any PVP that has been conventionally used in cell culture media, without any particular limitations. From the viewpoint of further improving proliferation performance, it is preferable that the weight-average molecular weight of the PVP is 40,000 to 1,200,000. Such PVP is commercially available, and for example, commercially available products such as polyvinylpyrrolidone K-30 (cat. no. 06306-72) manufactured by Nacalai Tesque, Inc. and polyvinylpyrrolidone K-90 (cat. no. 28315-72) manufactured by Nacalai Tesque, Inc. may be used.

[0014] At least one of CMC and PVP is added to the stem cell culture medium of the present invention. Either one of CMC and PVP may be added to the stem cell culture medium, or both of CMC and PVP may be added to the stem cell culture medium. From the viewpoint of further improving proliferation performance while maintaining the differentiation ability of stem cells, the amount of carboxymethylcellulose added to the stem cell culture medium of the present invention is preferably 0.001 μg / mL to 1 mg / mL as a final concentration, and more preferably 0.02 μg / mL to 0.5 mg / mL as a final concentration. From the same viewpoint as above, the amount of polyvinylpyrrolidone added to the stem cell culture medium of the present invention is preferably 0.05 μg / mL to 2 mg / mL final concentration, more preferably 0.1 μg / mL to 1 mg / mL final concentration.

[0015] Typically, media used for culturing stem cells contain albumin (albumin protein) such as serum albumin. From the viewpoint of further improving the proliferation performance, the medium for stem cells of the present invention preferably contains albumin, more preferably contains mammalian albumin, and even more preferably contains albumin of the same species as the stem cells to be cultured. In addition, the albumin is preferably recombinant albumin. For example, to culture human-derived stem cells, it is preferable to use recombinant albumin derived from human. The stem cell culture medium according to the present invention contains at least one of the above-mentioned carboxymethylcellulose and polyvinylpyrrolidone, which allows for an advantageous reduction in the content of albumin, which is costly. Therefore, the amount of albumin added to the stem cell culture medium according to the present invention can be set to a final concentration in the range of 0.05 to 1 mg / mL, preferably 0.05 to 1 mg / mL, and more preferably 0.1 to 0.5 mg / mL. From the viewpoint of further improving the proliferation performance, it is preferable that the albumin has a reduced amount of fatty acid carried thereon. As such albumin, albumin that has been subjected to a fatty acid removal treatment such as activated carbon treatment can be appropriately used. The fatty acid removal treatment can be performed, for example, by the method described in International Publication No. 2014-192938.

[0016] From the viewpoint of further improving the proliferation ability while maintaining the differentiation ability of stem cells, it is preferable that the stem cell culture medium according to the present invention further contains β-nicotinamide mononucleotide. Nicotinamide mononucleotide (hereinafter also referred to as "NMN"; chemical formula: C 11 H 15 N2O8P) has two optical isomers, α and β, but in the stem cell culture medium of the present invention, it is preferable to use β-NMN (CAS number: 1094-61-7). The structure of β-NMN is shown below.

[0017] [ka]

[0018] β-NMN prepared by any method can be used. For example, β-NMN artificially synthesized and purified by chemical synthesis, enzymatic method, fermentation method, etc. can be used as an active ingredient. For example, as a chemical synthesis method for synthesizing β-NMN, β-NMN can be produced by reacting NAM with L-ribose tetraacetate and phosphorylating the resulting nicotinamide mononucleoside. For example, as an enzymatic method, β-NMN can be produced from NAM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) using nicotinamide phosphoribosyltransferase (NAMPT). For example, as a fermentation method, β-NMN can be produced from NAM using the metabolic system of a microorganism expressing NAMPT. In addition, since β-NMN is a component that is widely present in living organisms, β-NMN obtained by extracting and purifying it from natural raw materials such as animals, plants, microorganisms, etc. can also be used as an active ingredient. Commercially available purified β-NMN can also be used.

[0019] From the viewpoint of further improving proliferation performance while maintaining the differentiation ability of stem cells, the amount of β-NMN added to the stem cell culture medium of the present invention is preferably 0.01 to 5 mM final concentration, more preferably 0.05 to 2 mM final concentration, and even more preferably 0.1 to 1 mM final concentration.

[0020] The basal medium used in the stem cell medium of the present invention may be a general basal medium such as a medium used for maintaining or growing the pluripotency (undifferentiated state) of stem cells or a medium used for culturing animal cells. As such a basal medium, various commercially available culture media for stem cells may be used. Examples of the basal medium include Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), α Eagle's minimum essential medium (αMEM), Iscove's modified Dulbecco's medium (IMDM), F-12 medium, F-10 medium, DMEM / F12 medium, RPMI-1640 medium, mesenchymal cell basal medium (MSCBM), E8 (Essential 8) medium, TeSR-E8 medium, mTeSR1 medium, and mixed media thereof.

[0021] The medium for stem cells according to the present invention may contain, as necessary, active ingredients such as amino acids, inorganic salts, vitamins, antibiotics, etc., in addition to the above-mentioned components, and may contain only one type of these active ingredients or a combination of two or more types. The active ingredients that can be contained in the medium for stem cells according to the present invention are ingredients known to increase the survival efficiency and proliferation efficiency of stem cells, ingredients known to increase the differentiation efficiency, etc. For example, the active ingredients can be appropriately selected from various amino acids, vitamins such as ascorbic acid and α-tocopherol, growth factors such as insulin and transferrin, minerals such as sodium selenite, ethanolamine, Rock inhibitors, physiologically active substances such as valproic acid, dimethyl sulfoxide, dexamethasone, butyric acid, trichostatin A, GSK3 inhibitors, BMP inhibitors, Wnt inhibitors, and cytokines such as PDGF-BB, EGF, VEGF, TGF-β, FGF2 activin, and noggin. The above-mentioned active ingredients are known to increase the survival and proliferation efficiency of stem cells, and to enhance the effect of maintaining the undifferentiated state of stem cells.

[0022] The method for culturing stem cells according to the present invention is a method for efficiently growing stem cells while maintaining the differentiation potential of the stem cells, i.e., a method for stably and efficiently culturing stem cells. The method for culturing stem cells according to the present invention can suitably use the medium for stem cells according to the present invention. The method for culturing stem cells according to the present invention can be carried out in the same manner as a conventional method for culturing stem cells, except that the medium contains at least one of carboxymethylcellulose and polyvinylpyrrolidone as a water-soluble polymer compound. The culture conditions can be the same as those generally used for culturing animal cells, and may be appropriately modified as necessary. For example, the culture temperature is 30 to 40°C, the CO2 concentration is 1 to 10% by volume, and the O2 concentration is 0.1 to 25% by volume. EXAMPLES

[0023] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples in any way. Examples 1 to 7 and Example 11 are reference examples.

[0024] [Preparation of medium for stem cells] A medium for stem cells was prepared. This medium had the same composition as serum-free medium A described in Japanese Patent No. 5804385, except that water-soluble compounds CMC or PVP were added instead of polyvinyl alcohol (PVA). The serum-free medium A was prepared by adding active ingredients such as FGF, PDGF, TGF-β, HGF, EGF, phospholipids, fatty acids, and PVA to a basal medium containing DEME (Sigma, cat. no. D6046) and MCDB201 (Sigma, cat. no. M6770) mixed at a ratio of 1:1 (see Table 1 in Japanese Patent No. 5804385). The amount of albumin added to serum-free medium A was set to a final concentration of 0.2 mg / mL. Specifically, the stem cell media of Examples 1 to 10 were prepared by adding CMC (Nacalai Tesque, sodium carboxymethylcellulose, degree of etherification 0.59-0.85, cat. no. 07326-95) instead of PVA in serum-free medium A, and the stem cell media of Examples 11 to 14 were prepared by adding PVP (Nacalai Tesque, polyvinylpyrrolidone K-90, weight average molecular weight 360,000, cat. no. 28315-72) instead of PVA in serum-free medium A. In addition, β-NMN was further added to the stem cell media of Examples 8 to 10 and Examples 12 to 14. The medium consisting of only the serum-free medium A was used as the medium of Comparative Example 1. The amounts of CMC, PVP, and β-NMN added (final concentrations in the medium) in the stem cell medium of each Example and Comparative Example are as shown in the table below.

[0025] [Table 1]

[0026] [Growth Performance Test I] 96-well cell culture plates at 2.5 μg / cm 2The wells were coated with fibronectin (Corning, product number: 356008). Stem cells were seeded on this 96-well plate at 800 cells / well and at a scale of 100 μL using the stem cell medium of Examples 1 to 7 or Comparative Example 1, and statically cultured under conditions of 37°C and 5% CO2. The stem cells used were human adipose tissue-derived mesenchymal stem cells (Lonza, product number: PT-5006, one donor). The medium was replaced 1 day and 4 days after seeding, and the culture was continued for 6 days from the start of culture. For this culture, the stem cell medium with the above-mentioned CMC content (stem cell medium of Examples 1 to 7 or Comparative Example 1) was used. Next, the proliferation performance of stem cells was evaluated by the crystal violet staining method. First, the culture supernatant in the 96-well plate was removed, and 0.99% paraformaldehyde (PFA) was added to each well and left at 4°C for more than 1 hour. After that, the PFA was removed from each well, and the wells were washed with ultrapure water and 100% ethanol. MilliQ water was used as the ultrapure water. MilliQ water is ultrapure water produced by Millipore's ultrapure water production system MilliQ. After washing, 2.5% crystal violet was added to each well and left at room temperature for 10 minutes. Next, each well was washed with tap water three times, and then the wells were dried and the absorbance at a wavelength of 595 nm was measured using a microplate reader. The above measurements were performed on six independent wells, and the significance was analyzed by T-test. The measurement results are shown in Figure 1.

[0027] Also, 2.5 μg / cm 2 Stem cells were cultured at 5 × 10 3 cells / cm 2 The stem cells were cultured and the proliferation performance of the stem cells was evaluated in the same manner as in Examples 1 to 7 above, except that the cells were seeded in a 3 mL scale using the stem cell medium of Example 11 or Comparative Example 1. The measurement results are shown in Figure 2.

[0028] [Growth Performance Test II] 2.5μg / cm 2The same stem cells as those used in the proliferation performance test I were placed at 5 × 10 3 cells / cm 2 The cells were seeded at a 3 mL scale using the stem cell medium of Examples 8 to 10 or Comparative Example 1 so that the total volume of the cells was 100 mL, and the cells were statically cultured at 37°C under 5% CO2 conditions. One day after seeding, the medium was replaced, and thereafter, the medium was replaced once every 2 or 3 days using each stem cell medium. When the cell density in the culture dish reached approximately 80 to 90% subconfluence, the cells were subcultured. More specifically, the culture supernatant was removed from the culture dish, the cells were washed with phosphate buffered saline [1x PBS(-)], and then 1x Tryple select (Thermo Fisher, product number: 12563011) was added, and the cells were statically cultured at 37°C under 5% CO2 conditions. 2 The cells were then suspended by pipetting to separate the cells that had been detached from the culture dish into single cells. Each stem cell medium was then added, centrifuged (1500 rpm, 5 min), the supernatant was removed, and the cells that had been precipitated were then suspended in each of the above media. The cells in this suspension were counted, and 5 × 10 cells were transferred to a culture dish (60 mm dish) that had been previously coated with fibronectin. 3 cells / cm 2 The medium in the culture dish that had been subcultured in this manner was replaced once every two or three days with each stem cell medium. The above subculture was repeated eight times.

[0029] Next, the cumulative number of divisions of the stem cells obtained through the above passages was calculated and graphed. The cumulative number of divisions was calculated by accumulating the number of divisions of the stem cells based on the initial seeding number of the stem cells and the number of cells counted for each passage. The proliferation performance of the stem cells in each Example was evaluated according to the following evaluation criteria in comparison with the cumulative number of divisions in Comparative Example 1. In the following evaluation criteria, a case in which the cumulative number of divisions is one or more times more than Comparative Example 1 was evaluated as "proliferation performance higher than Comparative Example 1", and a case in which the cumulative number of divisions was the same number as Comparative Example 1 or - (minus) 1 was evaluated as "proliferation performance equivalent to Comparative Example 1". +: Growth performance equivalent to that of Comparative Example 1 was observed. ++: Higher proliferation performance than Comparative Example 1 was observed.

[0030] The evaluation results of this proliferation performance test II using the stem cell media of Examples 8 to 10 and Comparative Example 1 are shown in Table 2. Furthermore, the measurement results of the cumulative number of stem cell divisions in Example 8 and Comparative Example 1 are shown in Figure 3(a). The above-mentioned main proliferation performance test II was carried out using each of the stem cell culture media of Examples 12 to 14 and Comparative Example 1. The evaluation results are shown in Table 3. Furthermore, the measurement results of the cumulative number of stem cell divisions in Example 13 and Comparative Example 1 are shown in Figure 3(b).

[0031] [Table 2]

[0032] [Table 3]

[0033] [Differentiation Potential Test I] 2.5μg / cm 2 The same stem cells as those used in the proliferation performance test I were placed at 5 × 10 3 cells / cm 2 The cells were seeded in 3 mL scale using the stem cell medium of Example 8, Example 13, or Comparative Example 1 so that the cell density in the culture dish became about 80 to 90% subconfluent, and the cells were statically cultured under conditions of 37°C and 5% CO2. When the cell density in the culture dish became about 80 to 90% subconfluent, the same operation as in the above subculture was performed, and the stem cells were detached from the culture dish and 5The collected stem cells were washed with 1xPBS, and the antibodies shown below were added and reacted at 4°C for 30 minutes or more. After that, the cells were washed again with 1xPBS, and flow cytometry analysis was performed using a flow cytometer (Becton Dickinson, model: FACS Calibur) to confirm the expression of cell surface antigens of the collected stem cells. The flow cytometry analysis was performed according to the instruction manual of the flow cytometer.

[0034] The following antibodies were used in this differentiation potential test I. FITC Mouse IgG1, κ isotype Ctrl (BIOLEGEND, product number: 400108) PE Mouse IgG1, κ isotype Ctrl (BIOLEGEND, product number: 400112) PerCP / Cyanine5.5 Mouse IgG1, κ isotype Ctrl (BIOLEGEND, product number: 400150) FITC anti-human CD90(Thy1) (BIOLEGEND, product number: 328108) PE anti-human CD105 (BIOLEGEND, product number: 323206) PerCP / Cyanine5.5 anti-human CD73 (Ecto-5'-nucleotidase) (BIOLEGEND, product number: 344014) FITC anti-human CD235a (BIOLEGEND, product number: 349105) PE anti-human CD45 (BIOLEGEND, product number: 304008) PerCP / Cyanine5.5 anti-human CD31 (BIOLEGEND, product number: 303131) Of the proteins to which the above antibodies bind, CD90, CD105, and CD73 expressed on the cell surface are marker proteins for mesenchymal stem cells, and CD235a, CD45, and CD31 are negative marker proteins for mesenchymal stem cells.

[0035] [Differentiation Potential Test II] The stem cells were cultured four times in the medium for stem cells of Example 8, Example 13, or Comparative Example 1 in the proliferation performance test II, and then cultured in a 48-well cell culture plate at 2×10 4 The cells were seeded at 100 cells / well and cultured until the cell density reached approximately 100% confluence. The medium was then replaced with a differentiation medium for differentiation into osteoblasts, chondrocytes, and adipocytes, and differentiation induction was performed for approximately 3 weeks. The stem cells under differentiation induction were statically cultured at 37°C and 5% CO2.

[0036] In this differentiation potential test II, the following differentiation induction medium was used. -Bone differentiation induction medium To the α-MEM medium, fetal bovine serum was added to a final concentration of 10%, dexamethasone to a final concentration of 10 nM, β-glycerophosphate to a final concentration of 10 mM, ascorbic acid to a final concentration of 50 μg / mL, and penicillin-streptomycin to a final concentration of 0.1%. ·Centrifuge differentiation medium To the α-MEM medium, fetal bovine serum was added to a final concentration of 10%, dexamethasone to a final concentration of 100 nM, ascorbic acid to a final concentration of 25 μg / mL, and pyruvic acid to a final concentration of 1 mM. Adipogenic differentiation medium DMEM-High Glucose medium was supplemented with fetal bovine serum at a final concentration of 10%, IBMX at a final concentration of 0.5 mM, dexamethasone at a final concentration of 1 μM, indomethacin at a final concentration of 200 μM, and penicillin-streptomycin at a final concentration of 0.1%.

[0037] After differentiation induction using each of the above differentiation media, the supernatant was removed from the 48-well cell culture plate, 0.99% PFA was added, and the cells were left to stand for 30 minutes to fix the cells. The PFA was then removed, the plate was washed with water, and a cell staining solution was added and left to stand for 20 minutes to perform cell staining. After cell staining, the plate was washed with MilliQ water at least three times, and the state of cell staining was observed under a microscope. The observed images are shown in Figure 5.

[0038] In this differentiation potential test II, the following staining solutions were used for cell staining. Alizarin S staining solution (Merck Millipore, product number: TMS_008_C) Since the Alizarin S staining solution contains a dye that binds to calcium, differentiation into osteoblasts can be evaluated based on the degree of staining with the staining solution. Alcian blue (Nacalai Tesque, product number: 37154-44) Alcian blue is capable of staining the extracellular matrix of chondrocytes, such as aggrecan and glycosaminoglycan, and differentiation into chondrocytes can be evaluated based on the degree of staining. Oil Red O (Nacalai Tesque, product number: 25633-92) Oil Red O is capable of staining lipid droplets, and differentiation into adipocytes can be evaluated based on the degree of staining.

[0039] As is clear from the results of Proliferation Performance Test I, the stem cell culture media of Examples 1 to 7 to which CMC was added and Example 11 to which PVP was added showed higher proliferation performance than the medium of Comparative Example 1 (see Figures 1 and 2). Such proliferation performance was also shown when stem cells were subcultured in Proliferation Performance Test II (see Tables 2 and 3). Furthermore, the stem cell culture media of Examples 1 to 7 to which CMC was added and Example 11 to which PVP was added showed a higher cumulative number of divisions than the medium of Comparative Example 1 to which CMC or PVP was not added (see Figures 3(a) and (b)).

[0040] As is clear from the results of differentiation potential test I, the stem cells cultured in the stem cell media of Example 8, to which CMC was added, and Example 13, to which PVP was added, maintained an undifferentiated state as well as the stem cells cultured in the medium of Comparative Example 1, to which neither CMC nor PVP was added (see Figure 4). Furthermore, in differentiation potential test II, the stem cells cultured in the stem cell media of Example 8 to which CMC was added and Example 13 to which PVP was added were able to differentiate into osteoblasts, chondrocytes, or adipocytes, similar to the stem cells cultured in the medium of Comparative Example 1 to which neither CMC nor PVP was added (see FIG. 5). In other words, it was shown that the differentiation potential of stem cells is not altered by CMC or PVP. The results of the proliferation performance tests I and II and the differentiation ability tests I and II showed that the stem cell medium containing CMC or PVP according to the present invention does not adversely affect the maintenance and proliferation of stem cells, and can improve proliferation while maintaining the differentiation ability of stem cells. This allows stem cells to be cultured more stably and efficiently. [Industrial Applicability]

[0041] The stem cell medium according to the present invention contains at least one of carboxymethylcellulose and polyvinylpyrrolidone as a water-soluble polymeric compound in a basal medium necessary for culturing stem cells, and thus has good proliferation performance while maintaining the differentiation ability of stem cells. Therefore, by using the stem cell medium, stem cells can be cultured more stably and efficiently.

Claims

1. The water-soluble polymer compound contains at least one of carboxymethyl cellulose and polyvinylpyrrolidone, In the case where the medium contains carboxymethylcellulose, the amount of the carboxymethylcellulose added to the medium is 0.1 mg / mL to 0.5 mg / mL as a final concentration; In the case where the medium contains polyvinylpyrrolidone, the amount of polyvinylpyrrolidone added to the medium is 0.2 mg / mL to 0.6 mg / mL as a final concentration; Further containing β-nicotinamide mononucleotide, Stem cell medium used for culturing mesenchymal stem cells.

2. 2. The medium for stem cells according to claim 1, wherein the amount of recombinant albumin added to the medium is 0.05 to 1 mg / mL as a final concentration.

3. A method for culturing stem cells, comprising using the medium for stem cells according to claim 1 or 2.

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