Cell culture medium composition

Abstract

The present invention provides a cell culture medium composition containing 150 ng / mL or more of basic fibroblast growth factor (bFGF).

Description

[Technical Field]

[0001] The present invention relates to a medium composition for cells, and more particularly to a medium composition for high-density suspension culture of cells. [Background technology]

[0002] Cell-based regenerative medicine technology has attracted considerable attention in recent years as a potential means of treating various diseases and injuries that have been difficult to treat until now. Because regenerative medicine requires large numbers of cells, the development of efficient cell culture methods has been actively pursued. For example, Patent Document 1 reports a method for culturing stem cells, which involves treating stem cells in a culture medium with a ROCK inhibitor. Furthermore, in 2018, the present inventors also reported a method for high-density culture of animal cells by adding glucose and / or specific amino acids to the culture medium (Patent Document 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-099662 [Patent Document 2] Patent application No. 2018-184352 Summary of the Invention [Problem to be solved by the invention]

[0004] The method described in Patent Document 2 is one of the most excellent methods for high-density culture of animal cells, including pluripotent stem cells. However, the present inventors have reexamined this method and attempted to develop an even more preferable method for high-density culture of cells. [Means for solving the problem]

[0005] As a result of extensive research into the above-mentioned problems, the present inventors have found that, in high-density cell suspension culture, the addition of basic fibroblast growth factor (bFGF) to the medium in amounts far exceeding those hitherto recommended in the art significantly promotes cell proliferation. Furthermore, they have also found that, in the case of pluripotent stem cells, the addition of a large amount of bFGF can effectively maintain the undifferentiated state of pluripotent stem cells cultured at high density, and that pluripotent stem cells prepared by high-density culture in a medium supplemented with a large amount of bFGF have excellent differentiation potential. Based on these findings, they have conducted further research and have completed the present invention. That is, the present invention is as follows.

[0006] [1] A cell culture medium composition comprising basic fibroblast growth factor (bFGF) at a concentration of 150 ng / mL or more, or a stabilized bFGF at a concentration that exerts an effect equivalent to that of bFGF at said concentration. [2] The medium composition according to [1], which is for suspension culture. [3] The medium composition according to [1] or [2], which is for high-density culture. [4] The cell density of suspension cultured cells is 6.0 × 10 5 [3] The medium composition according to [3], wherein the concentration of the medium composition is 1000 cells / mL or more. [5] The medium composition according to any one of [1] to [4], wherein the cells are pluripotent stem cells, adult stem cells, or progenitor cells. [6] A method for culturing cells, comprising culturing cells in suspension in the medium composition according to [1]. [7] The method described in [6], wherein bFGF is added to the culture medium at 10 to 1000 ng / mL / day, or a stabilized bFGF at a concentration that has an effect equivalent to that of bFGF at said concentration. [8] The cell density of pluripotent stem cells cultured in suspension is 6.0 × 10 5 The method according to [6] or [7], wherein the IgG antibody is IgG4-dependent ... [9] The method according to any one of [6] to [8], wherein the cells are pluripotent stem cells, adult stem cells, or progenitor cells. [Effects of the Invention]

[0007] According to the present invention, cells can be prepared extremely efficiently. Furthermore, according to the present invention, high-quality pluripotent stem cells that are well maintained in an undifferentiated state and have good differentiation potential can be prepared very efficiently. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the effect of bFGF concentration on the proliferation of iPS cells (n=1). [Figure 2] FIG. 2 shows the effect of the amount of bFGF added on the expression of CD30 in iPS cells (n=1). [Figure 3] FIG. 3 shows that the desired effects of the present invention can be achieved even when stabilized bFGF is used. [Figure 4] FIG. 4 shows the change over time in cell number (VCD: Viable Cell Density) when iPS cells are cultured in a medium containing a high concentration of bFGF. [Figure 5] FIG. 5 shows the expression level of CD30 in iPS cells cultured in a medium containing a high concentration of bFGF. [Figure 6] Figure 6 shows that iPS cells cultured in a medium containing a high concentration of bFGF were able to differentiate into paraxial mesoderm (PM) with high efficiency at a high density of over 1.0 × 10 cells / mL. [Figure 7] FIG. 7 shows that cells induced to differentiate from iPS cells cultured in a medium containing a high concentration of bFGF highly express DLL1 (PM marker). DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention will be described in detail below.

[0010] definition As used herein, "suspension culture" refers to a cell culture method in which cells are not attached to a culture vessel. In the present invention, suspension culture may or may not involve external pressure or vibration applied to the liquid medium, or shaking or rotation in the liquid medium.

[0011] As used herein, "high-density culture" refers to culture at a cell density higher than that expected in general cell culture. The standard for high density may vary depending on the culture method (contact culture / suspension culture, etc.) and the type of cell, but for example, when iPS cells are cultured in suspension, as used herein, a high-density culture is defined as 6 × 10 5 cells / mL or more (preferably 2 x 10 6 High-density culture is defined as a culture at a density of 1000 cells / mL or more.

[0012] As used herein, the term "pluripotent stem cells" refers to cells that have the ability to differentiate into all tissues and cells that constitute a living organism. Examples of pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells).

[0013] As used herein, "adult stem cells (also called somatic stem cells)" refers to cells that have the ability to differentiate into cells that constitute a specific tissue (organ). Examples of adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, germline stem cells, intestinal stem cells, epidermal stem cells, and mesenchymal stem cells.

[0014] As used herein, the term "precursor cells" refers to cells that are at an intermediate stage in the differentiation of the aforementioned pluripotent stem cells or adult stem cells into specific somatic cells or germ cells.

[0015] 1. Medium Composition The present invention provides a cell culture medium composition (hereinafter, sometimes referred to as the "medium composition of the present invention") that contains basic fibroblast growth factor (bFGF) at a concentration of 150 ng / mL or more, or stabilized bFGF at a concentration that has an effect equivalent to that of bFGF at that concentration.

[0016] The medium composition of the present invention can be prepared by adding a very high concentration of bFGF to a basal medium for general cells.

[0017] The cell culture medium used to prepare the medium composition of the present invention may be prepared by a method known per se in accordance with the cells to be cultured, or may be a commercially available product.

[0018] Examples of commercially available media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A Medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, and Fischer's Medium.

[0019] Furthermore, examples of media particularly for stem cell culture include STEMPRO (registered trademark) hESC SFM medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), TeSR-E8 medium (STEMCELL Technologies), Essential 8 medium (Life Technologies), HEScGRO (trademark) Serum-Free Medium for hES cells (Millipore), PluriSTEM (trademark) Human ES / iPS Medium (EMD Millipore), NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), NutriStem (trademark) XF / FF Culture Medium (Stemgent), AF NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), S-medium (DS Pharma Biomedical Co., Ltd.), and StemFit (registered trademark). Examples include AK03N medium (Ajinomoto Co., Inc.), hESF9 medium, hESF-FX medium, CDM medium, DEF-CS 500 Xeno-Free 3D Spheroid Culture Medium (Cellartis), and StemFlex medium (Thermo Fisher Scientific).

[0020] As of the filing date of this application, the amount of bFGF contained in a medium for culturing cells is at most about 100 ng / mL. This is because it is believed that even if bFGF is added at a concentration higher than this, the effect on cell proliferation and / or undifferentiation is constant. However, the lower limit of the bFGF concentration contained in the medium composition of the present invention may be generally 150 ng / mL, preferably 200 ng / mL, more preferably 250 ng / mL, even more preferably 275 ng / mL, and particularly preferably 300 ng / mL, or even higher. Furthermore, the upper limit is not particularly limited, but from the standpoint of cost, etc., it may be generally 1500 ng / mL, preferably 1000 ng / mL, more preferably 800 ng / mL, even more preferably 600 ng / mL, and particularly preferably 500 ng / mL. In one embodiment, the concentration of bFGF in the medium composition of the present invention can generally be 150 to 1500 ng / mL, preferably 200 to 1000 ng / mL, more preferably 250 to 800 ng / mL, even more preferably 275 to 600 ng / mL, and particularly preferably 300 to 500 ng / mL.

[0021] In one embodiment, the bFGF added to the medium composition of the present invention may be stabilized. The stabilized bFGF that can be added to the medium composition of the present invention may be produced by a method known per se, or may be a commercially available product. Suitable commercially available products include, but are not limited to, Heat Stable Recombinant Human bFGF (manufactured by Thermo Fisher Scientific). Another example of stabilized bFGF is "FGF2-G3." FGF2-G3 is a mutant bFGF protein that has nine amino acid mutations (R31L, V52T, E54D, H59F, S94I, L92Y, C96N, S109E, and T121P) compared to wild-type bFGF. Without wishing to be bound by theory, it has been reported that FGF2-G3 exhibits equivalent activity to unstabilized wild-type bFGF at approximately 40% of the dosage (Hui-Hsuan Kuo et al., Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture).

[0022] In this embodiment, the preferred concentration range of stabilized bFGF can be a concentration that provides the same cell proliferation-promoting effect and / or effect of maintaining undifferentiation as unstabilized bFGF. A preferred concentration range when using stabilized bFGF can be determined appropriately by those skilled in the art, for example, by referring to the above-mentioned concentration range and confirming the difference in effect between unstabilized bFGF and stabilized bFGF using a method known per se. The concentration of stabilized bFGF in the medium composition can vary depending on the means and degree of bFGF stabilization, but typically, a concentration range that provides the same effect as the desired effect of the present invention (cell proliferation-promoting effect and / or effect of maintaining undifferentiation) achieved when the above-mentioned concentration range of unstabilized bFGF is used may be appropriately determined. Specifically, the concentration range of stabilized bFGF may be determined as follows: (1) Quantifying the effect (i.e., the effect of promoting cell proliferation and / or the effect of maintaining undifferentiated cells) of using a medium composition containing unstabilized bFGF (the effect can be quantified using the method used in the Examples of the present application described later), (2) Perform a test similar to that in (1) using stabilized bFGF instead of unstabilized bFGF to determine the concentration of stabilized bFGF that achieves an effect equivalent to that quantified in (1).

[0023] In one embodiment, the lower limit of the concentration of stabilized bFGF contained in the medium composition of the present invention is usually 75 ng / mL, preferably 100 ng / mL, more preferably 125 ng / mL, even more preferably 138 ng / mL, and particularly preferably 150 ng / mL, or may be higher. The upper limit is not particularly limited, but from the standpoint of cost, etc., it is usually 750 ng / mL, preferably 500 ng / mL, more preferably 400 ng / mL, even more preferably 300 ng / mL, and particularly preferably 250 ng / mL. In one embodiment, the concentration of bFGF in the medium composition of the present invention is usually 75 to 750 ng / mL, preferably 100 to 500 ng / mL, more preferably 125 to 400 ng / mL, even more preferably 138 to 300 ng / mL, and particularly preferably 150 to 250 ng / mL.

[0024] In one embodiment, the lower limit of the concentration of stabilized bFGF contained in the medium composition of the present invention is usually 50 ng / mL, preferably 67 ng / mL, more preferably 84 ng / mL, even more preferably 92 ng / mL, and particularly preferably 100 ng / mL, or may be higher. The upper limit is not particularly limited, but from the viewpoint of cost, etc., it is usually 500 ng / mL, preferably 334 ng / mL, more preferably 267 ng / mL, even more preferably 200 ng / mL, and particularly preferably 167 ng / mL. In one embodiment, the concentration of bFGF in the medium composition of the present invention is usually 50 to 500 ng / mL, preferably 67 to 334 ng / mL, more preferably 84 to 267 ng / mL, even more preferably 92 to 200 ng / mL, and particularly preferably 100 to 167 ng / mL.

[0025] In one embodiment, the lower limit of the concentration of stabilized bFGF contained in the medium composition of the present invention is usually 38 ng / mL, preferably 50 ng / mL, more preferably 63 ng / mL, even more preferably 69 ng / mL, and particularly preferably 75 ng / mL, or may be higher. The upper limit is not particularly limited, but from the standpoint of cost, etc., it is usually 375 ng / mL, preferably 250 ng / mL, more preferably 200 ng / mL, even more preferably 150 ng / mL, and particularly preferably 125 ng / mL. In one embodiment, the concentration of bFGF in the medium composition of the present invention is usually 38 to 375 ng / mL, preferably 50 to 250 ng / mL, more preferably 63 to 200 ng / mL, even more preferably 69 to 150 ng / mL, and particularly preferably 75 to 125 ng / mL.

[0026] Alternatively, bFGF can be stabilized by separately adding a compound that contributes to stabilization of bFGF to the medium. One example of such an embodiment is the addition of a sulfated compound (e.g., sulfated polysaccharides such as dextran sulfate sodium, sulfated polymers such as sulfogroup-containing polyvinyl alcohol, sulfated derivatives of sugar lactones such as gluconolactone-SO3NA, etc.) (see WO2013 / 147264 for details). When stabilizing bFGF using such a method, a person skilled in the art can appropriately determine the concentration range of bFGF, taking into consideration the concentration range of unstabilized bFGF, the degree of bFGF stabilization, and the effect to be achieved.

[0027] In addition to the above, the medium composition of the present invention can also contain components favorable for cell growth. Such components include, for example, sugars such as glucose, fructose, sucrose, and maltose; amino acids such as asparagine, aspartic acid, glutamine, and glutamic acid; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soybean peptide; serum; vitamins such as choline, vitamin A, B vitamins (thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, and nicotinamide), vitamin C, and vitamin E; fatty acids such as oleic acid, arachidonic acid, and linoleic acid; lipids such as cholesterol; inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate; trace elements such as zinc, copper, and selenium; and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid). buffers such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin; Type I collagen, Type II collagen Examples of suitable components include cell adhesion factors and extracellular matrix components such as collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine; cytokines and growth factors such as interleukin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), and activin A; and hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin. Appropriate components can be selected and used depending on the type of cells to be cultured.

[0028] In one embodiment, the medium composition of the present invention may contain choline (or a salt thereof) at the following concentrations (in the case of a choline salt, the amount is converted to the free form): [1]1~100mg / L, 10~100mg / L, 20~100mg / L, 30~100mg / L, 40~100mg / L, 50~100mg / L, 60~100mg / L, 70~100mg / L, 80~100mg / L, 90~100mg / L; [2]1~90mg / L, 10~90mg / L, 20~90mg / L, 30~90mg / L, 40~90mg / L, 50~90mg / L, 60~90mg / L, 70~90mg / L, 80~90mg / L; [3]1~80mg / L, 10~80mg / L, 20~80mg / L, 30~80mg / L, 40~80mg / L, 50~80mg / L, 60~80mg / L, 70~80mg / L; [4]1~70mg / L, 10~70mg / L, 20~70mg / L, 30~70mg / L, 40~70mg / L, 50~70mg / L, 60~70mg / L; [5]1~60mg / L, 10~60mg / L, 20~60mg / L, 30~60mg / L, 40~60mg / L, 50~60mg / L; [6]1~50mg / L, 10~50mg / L, 20~50mg / L, 30~50mg / L, 40~50mg / L; [7]1~40mg / L / , 10~40mg / L, 20~40mg / L, 30~40mg / L; [8]1~30mg / L / , 10~30mg / L / , 20~30mg / L; [9] 1-20mg / L, 10-20mg / L).

[0029] In a preferred embodiment, D-glucose and five amino acids (tryptophan, serine, cysteine ​​(or cystine), methionine, and arginine) may be added to the medium composition of the present invention. The amounts of these components to be added may be appropriately determined based on the purpose of the culture and various culture conditions (e.g., cell density, frequency of medium change), etc., with reference to the corresponding descriptions in "2. Cell Culture Method" below.

[0030] In one embodiment, the cells can be, but are not limited to, pluripotent stem cells, adult stem cells, or progenitor cells.

[0031] In one embodiment, the pluripotent stem cells may be embryonic stem cells (ES cells) or iPS cells, preferably iPS cells. Furthermore, the adult stem cells may be, but are not limited to, hematopoietic stem cells, neural stem cells, germline stem cells, intestinal stem cells, epidermal stem cells, or mesenchymal stem cells. The origins of the pluripotent stem cells, adult stem cells, and progenitor cells are not particularly limited, but are preferably derived from mammals, and more preferably from humans.

[0032] In one embodiment, the medium composition of the present invention can be provided as a medium for suspension culture of pluripotent stem cells. In another embodiment, the medium composition of the present invention can also be provided as a medium for suspension culture and high-density culture of pluripotent stem cells. The medium composition of the present invention has the function of maintaining high-density suspension culture of pluripotent stem cells in a favorable state, thereby achieving excellent cell proliferation of pluripotent stem cells. At the same time, pluripotent stem cells grown using the medium composition of the present invention maintain an undifferentiated state well. In other words, pluripotent stem cells grown using the medium composition of the present invention are of high quality and therefore suitable for use in, for example, regenerative medicine. In consideration of this, the medium composition of the present invention can be said to be a medium composition for maintaining the undifferentiated state of pluripotent stem cells in culture, or a medium composition for maintaining the undifferentiated state of pluripotent stem cells and promoting their proliferation. The undifferentiated state of pluripotent stem cells can be confirmed using known methods. For example, one skilled in the art can easily confirm this by checking the expression of known markers indicative of an undifferentiated state (e.g., CD30, Oct3 / 4, Nanog, etc.).

[0033] The medium of the present invention may be provided in a liquid state, or may be prepared in a more concentrated state than the concentration at the time of use, or in a solid state such as a freeze-dried powder, which is diluted with a solvent such as water, or dissolved or dispersed in a solvent such as water, at the time of use. Furthermore, because bFGF is easily thermally decomposed, it can be added to the medium immediately before use.

[0034] 2. Cell culture method The present invention also provides a method for culturing cells (hereinafter sometimes referred to as "the method of the present invention"), which comprises culturing cells in suspension using the medium composition of the present invention.

[0035] In one embodiment of the method of the present invention, bFGF is additionally added to the medium during culture. The amount of bFGF to be additionally added is not particularly limited as long as the desired effects of the present invention are obtained, but it is generally 10 to 1000 ng / mL / day, preferably 30 to 800 ng / mL / day, more preferably 50 to 600 ng / mL / day, even more preferably 70 to 500 ng / mL / day, and particularly preferably 105 to 420 ng / mL / day.

[0036] In one embodiment of the present invention, stabilized bFGF can also be added. The amount of stabilized bFGF to be added can vary depending on the means and degree of stabilization of bFGF, but typically, an amount to be added can be appropriately determined so as to achieve the desired effect of the present invention (the effect of promoting cell proliferation and / or the effect of maintaining undifferentiated cells) equivalent to that achieved when the amount of unstabilized bFGF added is used.

[0037] In another embodiment of the method of the present invention, stabilized bFGF is additionally added to the culture medium during culture. The amount of stabilized bFGF to be additionally added is not particularly limited as long as the desired effects of the present invention are obtained, but it is generally 5 to 500 ng / mL / day, preferably 15 to 400 ng / mL / day, more preferably 25 to 300 ng / mL / day, even more preferably 35 to 250 ng / mL / day, and particularly preferably 53 to 210 ng / mL / day.

[0038] In another embodiment of the method of the present invention, the amount of stabilized bFGF to be additionally added is not particularly limited as long as the desired effects of the present invention are obtained, but may typically be 4 to 334 ng / mL / day, preferably 10 to 267 ng / mL / day, more preferably 17 to 200 ng / mL / day, even more preferably 24 to 167 ng / mL / day, and particularly preferably 35 to 140 ng / mL / day.

[0039] In another embodiment of the method of the present invention, the amount of stabilized bFGF to be added is not particularly limited as long as the desired effects of the present invention are obtained, but may typically be 3 to 250 ng / mL / day, preferably 8 to 200 ng / mL / day, more preferably 13 to 150 ng / mL / day, even more preferably 18 to 125 ng / mL / day, and particularly preferably 27 to 105 ng / mL / day.

[0040] In one embodiment of the method of the present invention, the addition of the above-mentioned bFGF or stabilized bFGF can be performed using the medium of the present invention in accordance with the timing of medium replacement. The frequency of medium replacement can be determined appropriately based on the cell density, cell type, etc., and is not particularly limited. However, to prevent the concentration of active bFGF in the medium from decreasing to a level that adversely affects the proliferation and / or maintenance of the undifferentiated state of pluripotent stem cells, the medium replacement is performed at least once a day (preferably at least twice a day), and 50 to 100% (preferably 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, or 100%) of the medium in use can be replaced with the medium composition of the present invention during the medium replacement. In one embodiment, the medium replacement frequency is once or more times a day (e.g., once, twice, or three times a day), and 70 to 100% of the medium in use can be replaced with the medium composition of the present invention during the medium replacement.

[0041] In one embodiment, D-glucose and five amino acids (tryptophan, serine, cysteine ​​(or cystine), methionine, and arginine) may be additionally added to the medium of the present invention.

[0042] Glucose (or a salt thereof) can be added to the medium of the present invention so that the glucose concentration is usually 0.1 g / L / day to 900 g / L / day, preferably 1 g / L / day to 200 g / L / day, and more preferably 1 g / L / day to 20 g / L / day.

[0043] The five amino acids (tryptophan, serine, cysteine ​​(cystine), methionine, and arginine) are added to the medium in such a manner that the tryptophan concentration (concentration converted to free tryptophan) is usually 0.1 mg / L / day to 11,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, and more preferably 1 mg / L / day to 100 mg / L / day, the serine concentration (concentration converted to free serine) is usually 0.1 mg / L / day to 425,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, and more preferably 1 mg / L / day to 100 mg / L / day, and the cysteine ​​or cystine concentration (concentration converted to free cysteine) is usually 0.1 mg / L / day to 425,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, and more preferably 1 mg / L / day to 100 mg / L / day. / L / day to 280,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, more preferably 1 mg / L / day to 100 mg / L / day, methionine concentration (concentration converted to free methionine) is usually 0.1 mg / L / day to 55,000 mg / L / day, preferably 1 mg / L / day to 1,000 mg / L / day, more preferably 1 mg / L / day to 100 mg / L / day, arginine concentration (concentration converted to free arginine) is usually 0.1 mg / L / day to 150,000 mg / L / day, preferably 1 mg / L / day to 2,000 mg / L / day, more preferably 1 mg / L / day to 200 mg / L / day.

[0044] In one embodiment, choline (e.g., choline chloride) may be added to the medium of the present invention. In the method of the present invention, the amount of choline added to the medium (content converted into free form) is usually 0.01 to 1,000,000 mg / L / day, preferably 0.1 to 1,000 mg / L / day, more preferably 1 to 100 mg / L / day (e.g., [1]1~100mg / L / day, 10~100mg / L / day, 20~100mg / L / day, 30~100mg / L / day, 40~100mg / L / day , 50~100mg / L / day, 60~100mg / L / day, 70~100mg / L / day, 80~100mg / L / day, 90~100mg / L / day; [2]1~90mg / L / day, 10~90mg / L / day, 20~90mg / L / day, 30~90mg / L / day, 40~90mg / L / day, 50~90mg / L / day, 60~90mg / L / day, 70~90mg / L / day, 80~90mg / L / day; [3]1~80mg / L / day, 10~80mg / L / day, 20~80mg / L / day, 30~80mg / L / day, 40~80mg / L / day, 50~80mg / L / day, 60~80mg / L / day, 70~80mg / L / day; [4]1~70mg / L / day, 10~70mg / L / day, 20~70mg / L / day, 30~70mg / L / day, 40~70mg / L / day, 50~70mg / L / day, 60~70mg / L / day; [5]1~60mg / L / day, 10~60mg / L / day, 20~60mg / L / day, 30~60mg / L / day, 40~60mg / L / day, 50~60mg / L / day; [6]1~50mg / L / day, 10~50mg / L / day, 20~50mg / L / day, 30~50mg / L / day, 40~50mg / L / day; [7]1~40mg / L / day, 10~40mg / L / day, 20~40mg / L / day, 30~40mg / L / day; [8]1~30mg / L / day, 10~30mg / L / day, 20~30mg / L / day; [9]1~20mg / L / day, 10~20mg / L / day) It can be said that:

[0045] In one embodiment, the pluripotent stem cells may be embryonic stem cells (ES cells) or iPS cells, preferably iPS cells.

[0046] As described above, pluripotent stem cells cultured in suspension using the medium composition of the present invention not only proliferate efficiently but also maintain a favorable undifferentiated state. Therefore, the method of the present invention can also be described as a method for promoting the proliferation and / or maintaining the undifferentiated state of pluripotent stem cells.

[0047] In the method of the present invention, the culture conditions are not particularly limited, and a method known per se may be selected depending on the cell type, cell density, culture method (adherent culture / suspension culture, etc.), etc. For example, the culture temperature may usually be 25°C to 39°C, preferably 33°C to 39°C. The carbon dioxide concentration may usually be 4% to 10% by volume, preferably 4% to 6% by volume. The oxygen concentration may usually be 1% to 25% by volume, preferably 4% to 20% by volume.

[0048] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way. [Example]

[0049] Example In the following examples, the proliferation effect of bFGF on induced pluripotent stem cells (iPS cells) and their ability to maintain an undifferentiated state were evaluated. The iPS cells used were the 1210B2 strain purchased from iPS Academia Japan. Commercially available iPS cell culture media were StemFit AK03N (Ajinomoto Co.) or StemFit Basic03 (Ajinomoto Co.).

[0050] [Example 1] Proliferation-promoting effect of iPS cells by enhancing bFGF in a suspension culture system Using a 30 mL single-use bioreactor for iPS cells (ABLE: BWV-S03A), 6 × 10 iPS cell line 1210B2 cells were cultured in StemFit AK03N + 10 μM Y-27632 (Wako: 034-24024). 5 Cells were seeded at a cell density of 100 cells / mL and cultured in a CO2 incubator at 37°C, 5% CO2, and 120 rpm. On day 2, 70% of the medium was replaced with StemFit AK03N. On day 3, 10 mL of the cell suspension was resuspended in fresh StemFit Basic03 + 100 ng / mL bFGF (Peprotech) or StemFit Basic03 + 300 ng / mL bFGF and transferred to the ambr15 bioreactor (sartorius: 001-0881). Culture was continued at 37°C, pH 7.2, 20% dissolved oxygen, and 300 rpm. Seventy percent of the medium was replaced twice daily, and both groups were supplemented with 40 mg / L / day Trp (Ajinomoto Co.), 40 mg / L / day Ser (Ajinomoto Co.), 40 mg / L / day Cys (Nippon Protein), 40 mg / L / day Met (Ajinomoto Co.), 160 mg / L / day Arg (Ajinomoto Co.), and 4 g / L / day D-glucose (Nacalai Tesque: 16806-25). After the fifth day of culture, viable cell counts were measured using the Vi-CELL viability analyzer. TM Measurements were performed using a StemFit Basic 03 (Beckman Coulter) XR. Note that StemFit Basic 03 does not contain bFGF.

[0051] The effect of bFGF concentration on iPS cell proliferation was examined in a series of experiments, and the results are shown in Figure 1. Increasing the bFGF concentration in the medium from 100 ng / mL to 300 ng / mL promoted iPS cell proliferation.

[0052] [Example 2] Effect of strengthening bFGF on maintaining undifferentiated iPS cells in a suspension culture system Using a 30 mL single-use bioreactor for iPS cells, 6 × 10 1210B2 iPS cells were cultured in StemFit AK03N + 10 μM Y-27632. 5Cells were seeded at a cell density of 1000 cells / mL and cultured in a CO2 incubator at 37°C, 5% CO2, and 120 rpm. On day 2, 70% of the medium was replaced with StemFit AK03N. On day 3, 10 mL of the cell suspension was resuspended in fresh StemFit Basic03 + 100 ng / mL bFGF (Peprotech) or StemFit Basic03 + 150 ng / mL bFGF and transferred to the ambr15 bioreactor. Culture was continued at 37°C, pH 7.2, 20% dissolved oxygen, and 300 rpm. Seventy percent of the medium was replaced daily, and both groups were supplemented with 40 mg / L / day Trp, 40 mg / L / day Ser, 40 mg / L / day Cys, 40 mg / L / day Met, 160 mg / L / day Arg, and 4 g / L / day D-glucose. Cells were harvested on day 10 of culture and stained for the iPS cell marker CD30 as follows:

[0053] For CD30 staining, 2 x 10 5 The cells were collected in a 1.5 mL Eppendorf tube. After centrifugation (400 × g, 4°C, 5 min), the supernatant was removed. 20 μL of PBS(-) (Nacalai Tesque: 14249-24) containing 0.2% BSA (Nacalai Tesque: 01281-84) (hereafter referred to as 0.2% BSA-PBS) supplemented with 20% PE Mouse anti-CD30 (BD Biosciences: 550041) was added. The mixture was homogenized by pipetting 5–10 times and allowed to stand at 4°C for 20 min in the dark. 0.5 mL of 0.2% BSA-PBS was added, followed by centrifugation (400 × g, 4°C, 5 min). The supernatant was removed, and 300 μL of 0.2% BSA-PBS was added. The mixture was homogenized by pipetting 5–10 times and transferred to a 5 mL round tube with a 35 μm cell strainer.

[0054] The expression rate of CD30 stained as described above was measured using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The effect of bFGF on CD30 expression was examined in a series of experiments, and the results are shown in Figure 2. By increasing the bFGF concentration in the medium from 100 ng / mL to 150 ng / mL, CD30 expression in iPS cells was maintained at a high level of approximately 80% by day 10 of culture.

[0055] [Example 3] Effect of stabilized bFGF on maintaining undifferentiated iPS cells Using a 30 mL single-use bioreactor for iPS cells, 6 x 10 iPS cell line 1210B2 was cultured in StemFit AK03N + 10 μM Y-27632. 5 Cells were seeded at a density of 1000 cells / mL and cultured in a CO2 incubator at 37°C, 5% CO2, and 120 rpm. On day 2, 70% of the medium was replaced with StemFit AK03N. On day 3, 10 mL of the cell suspension was resuspended in fresh StemFit Basic03 + 100 ng / mL bFGF (Peprotech) or StemFit Basic03 + 100 ng / mL Heat Stable Recombinant Human bFGF (Thermo Fisher Scientific) (hereafter referred to as stabilized bFGF) and transferred to an ambr15 microbioreactor. Culture was continued at 37°C, pH 7.2, 20% dissolved oxygen, and 300 rpm. The StemFit Basic03 + 100ng / mL bFGF medium was replaced once or twice daily, while the StemFit Basic03 + 100ng / mL stabilized bFGF medium was replaced once daily. Both groups were supplemented with 40mg / L / day Trp, 40mg / L / day Ser, 40mg / L / day Cys, 40mg / L / day Met, 160mg / L / day Arg, and 4g / L / day D-glucose. Cells were harvested on day 10 of culture and stained for the iPS cell marker CD30 as follows: 2x10 5The cells were collected in a 1.5 mL Eppendorf tube and centrifuged (400 × g, 4°C, 5 min). The supernatant was removed, and 20 μL of 0.2% BSA (Nacalai Tesque: 01281-84) in PBS(-) (Nacalai Tesque: 14249-24) (hereafter referred to as 0.2% BSA-PBS) containing 20% ​​PE Mouse anti-CD30 (BD Biosciences: 550041) was added. The mixture was homogenized by pipetting 5–10 times and allowed to stand for 20 minutes at 4°C in the dark. 0.5 mL of 0.2% BSA-PBS was added, and the mixture was centrifuged (400 × g, 4°C, 5 min). The supernatant was removed, and 300 μL of 0.2% BSA-PBS was added. The mixture was homogenized by pipetting 5–10 times and transferred to a 5 mL round tube with a 35 μm cell strainer. The expression rate of CD30 stained as described above was measured using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The effect of stabilized bFGF on CD30 expression was examined in duplicate and the results are shown in Figure 3. It was demonstrated that the use of stabilized bFGF maintained CD30 expression even with a medium change once a day. In other words, the use of stabilized bFGF instead of adding a large amount of bFGF was also shown to be effective.

[0056] [Example 4] Evaluation of the differentiation potential of iPS cells cultured using bFGF-enriched medium 1. High density culture of iPS cells and maintenance of undifferentiated state Using a 30 mL single-use bioreactor for iPS cells (ABLE: BWV-S03A), 6 × 10 iPS cell line 1210B2 cells were cultured in StemFit® AK03N + 10 μM Y-27632 (Wako: 034-24024). 5Cells were seeded at a cell density of 1000 cells / mL and cultured in a CO2 incubator at 37°C, 5% CO2, and 120 rpm. On the second day of seeding, 70% of the medium was replaced with StemFit® AK03N. On the third day of seeding, 10 mL of the cell suspension was resuspended in fresh StemFit® AK03N + 10 mg / L Choline Chloride (Fujifilm Wako Pure Chemical Industries) + 200 ng / mL bFGF (Peprotech) and transferred in duplicate to a microbioreactor, ambr15 (sartorius: 001-0881). Culture was continued at 37°C, pH 7.2, 4% dissolved oxygen, and 300 rpm. Seventy percent of the medium was replaced twice daily with StemFit® AK03N + 10 mg / L Choline Chloride + 200 ng / mL bFGF, and both groups were further supplemented with 40 mg / L / day Trp (Ajinomoto Co., Inc.), 40 mg / L / day Ser (Ajinomoto Co., Inc.), 40 mg / L / day Cys (Nippon Protein), 40 mg / L / day Met (Ajinomoto Co., Inc.), 160 mg / L / day Arg (Ajinomoto Co., Inc.), 18.6 mg / L / day His (Ajinomoto Co., Inc.), 43.7 mg / L / day Ile (Ajinomoto Co., Inc.), 47.3 mg / L / day Leu (Ajinomoto Co., Inc.), 73.1 mg / L / day Lys HCl (Ajinomoto Co., Inc.), 28.4 mg / L / day Phe (Ajinomoto Co., Inc.), 42.3 mg / L / day Val (Ajinomoto Co., Inc.), and 4 g / L / day D-glucose (Nacalai Tesque) was added. On the 7th day of culture, the cells were analyzed by the Vi-CELL live / dead cell autoanalyzer. TM The number of viable cells was quantified using XR (Beckman Coulter), and the expression rate of CD30, an iPS cell marker, was measured.

[0057] The results for iPS cell proliferation and undifferentiation markers are shown in Figures 4 and 5. As shown in Figures 1 and 2, by culturing iPS cells in a medium containing a high concentration of bFGF, high-density culture of 8.8 to 9.7 x 10^6 cells / mL was achieved while maintaining the undifferentiated state of the iPS cells.

[0058] 2. Differentiation of iPS cells into paraxial mesoderm (PM) Duplicate cell suspensions cultured as described in step 1 were combined into one replicate and induced to differentiate into PM. Specifically, the cells were resuspended in fresh Differentiation 1 Medium (AK02N·A Solution (Ajinomoto Co.) + 20% StemFit For Differentiation (Ajinomoto Co.) + 10 μM CHIR99021 (Fujifilm Wako Pure Chemical Industries) + 30 ng / mL Activin A (Ajinomoto Co.) + 100 ng / mL bFGF + 300 nM LDN-193189 (Fujifilm Wako Pure Chemical Industries)) and cultured at 37°C, pH 7.2, dissolved oxygen concentration 20%, and agitation speed 300 rpm. After 12 hours, 70% of the medium was replaced with differentiation 1 medium, and 6 hours later, 40 mg / L / day Trp, 40 mg / L / day Ser, 40 mg / L / day Cys, 40 mg / L / day Met, 160 mg / L / day Arg, 18.6 mg / L / day His, 43.7 mg / L / day Ile, 47.3 mg / L / day Leu, 73.1 mg / L / day LysHCl, 28.4 mg / L / day Phe, 42.3 mg / L / day Val, and 4 g / L / day D-glucose were added. After 6 hours, 10 mL of the cell suspension was resuspended in fresh Differentiation 2 Medium (AK02N·A solution + 20% StemFit For Differentiation + 5 μM CHIR99021 + 10 μM SB431542 (Stemgent) + 100 ng / mL bFGF + 300 nM LDN-193189) and culture was continued. After 12 hours, 70% of the medium was replaced with differentiation medium 2. Six hours later, 40mg / L / day Trp, 40mg / L / day Ser, 40mg / L / day Cys, 40mg / L / day Met, 160mg / L / day Arg, 18.6mg / L / day His, 43.7mg / L / day Ile, 47.3mg / L / day Leu, 73.1mg / L / day Lys HCl, 28.4mg / L / day Phe, 42.3mg / L / day Val, and 4g / L / day D-glucose were added. After 6 hours, viable cell counts and the expression rate of the PM marker DLL1 were measured.

[0059] The results of cell proliferation and differentiation markers associated with differentiation into PM are shown in Figures 6 and 7. iPS cells cultured in a medium containing a high concentration of bFGF were able to differentiate into PM with high efficiency at a high density of over 1.0 x 10^7 cells / mL. [Industrial Applicability]

[0060] According to the present invention, cells can be prepared extremely efficiently. Furthermore, according to the present invention, high-quality pluripotent stem cells in which the undifferentiated state is well maintained can be prepared extremely efficiently.

[0061] This application is based on patent application No. 2020-003958 filed in Japan (filing date: January 14, 2020), the contents of which are incorporated in their entirety herein.

Claims

1. A cell culture medium composition comprising 200 ng / mL or more of basic fibroblast growth factor (bFGF), The medium composition is for suspension culture of pluripotent stem cells, A medium composition, wherein the suspension culture results in a cell density of 2 x 10 6 cells / mL or more after culture.

2. The medium composition described in claim 1, wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells.

3. A method for culturing pluripotent stem cells, comprising culturing the pluripotent stem cells in suspension using the medium composition according to claim 1, The method, wherein the suspension culture is carried out so that the cell density of the cells after culture is 2 x 10 6 cells / mL or more.

4. The method according to claim 3, wherein bFGF is added to the culture medium at 10 to 1000 ng / mL / day.

5. A method described in claim 3 or 4, wherein the pluripotent stem cells are iPS cells.

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