Cell culture method

Abstract

The purpose of the present invention is to provide a method capable of sufficiently proliferating cells, in particular immunocompetent cells, even in a serum-free medium or a low-serum medium, and a medium additive and a medium composition to be used in the method. By containing boric acid or a salt thereof, a comparable effect of maintaining and promoting cell proliferation can be obtained even in a serum-free medium or a low-serum medium compared to that achieved by culturing in a serum-containing medium.

Description

Technical Field

[0001] The present invention relates to a method for culturing / proliferating cells in a serum-free medium or a low-serum medium, and a medium additive and a medium composition used in the method. The present invention relates to a method for producing a cell population in which the number of desired cells is expanded in a serum-free or low-serum medium, and a medicament containing the cell population obtained by the method. Background Art

[0002] Conventionally, cell culture is carried out using a culture medium containing serum (hereinafter also referred to as a "serum-containing culture medium"). For example, fetal bovine serum (FBS) is widely used in cell culture as an important additive for cell proliferation. However, when culturing cells or their derivatives for medical purposes, heterologous components may become a source of infection for blood-borne pathogens or heterologous antigens. In addition, differences between serum batches may cause variations in culture results. For this reason, in recent years, it has become mainstream to reduce the amount of serum to be used as much as possible and to culture cells using a culture medium with a clear chemical composition (chemically defined culture medium), and the development of serum-free culture medium and low-serum culture medium is underway.

[0003] Recently, chimeric antigen receptor (CAR) T cell (hereinafter referred to as CAR-T cell) therapy has been developed as one of the immunotherapy for cancer patients, wherein the T cell receptor (TCR) of cytotoxic T cells (CTL) is genetically modified so that CTL directly and selectively recognizes tumor cells, and plays an anti-tumor effect. Cancer therapy using CAR-T cells kills cancer cells based on a mechanism different from conventional anticancer drugs and radiotherapy, and therefore it is expected to be effective for intractable or refractory cancers. Current CAR-T cell therapy is generally carried out using autologous CAR-T cell therapy, wherein the CAR gene is introduced into T cells collected from patients in vitro, and then the resulting CAR-T cells are administered to patients, such as Kymriah (trade name) and Yescarta (trade name) approved in the United States. However, this method requires multiple steps over a long period of time, such as activation / proliferation of T cells, preparation of viral vectors, and gene transfer to T cells, and therefore there is a problem of high manufacturing cost due to the cost required for cell culture and preparation of viral vectors. Therefore, iPS cell-derived CAR-T cells that can achieve allogeneic CAR-T therapy have been developed. In this method, in the process of generating iPS cell-derived CAR-T cells, especially in the expansion culture process, a serum-containing culture medium is also used to achieve a sufficient cell proliferation effect. However, from the point of view of GMP (good manufacturing practice), the culture medium is preferably xeno-free, that is, it does not contain xenogeneic components, and is preferably serum-free or low-serum. In addition, considering that T cells derived from iPS cells are particularly susceptible to washing pressure compared to primary T cells, there is a strong demand for serum-free or low-serum culture media that do not require a large amount of washing.

[0004] There are many reports about maintaining the desired cell proliferation effect or promoting the cell proliferation effect in cell culture. For example, there are also reports about boric acid exerting cell proliferation effect via its transporter NaBC1 (non-patent literature 1). However, the culture medium used here is a serum-containing culture medium, and it is unclear whether the same cell proliferation effect can be achieved in a serum-free culture medium or a low-serum culture medium. Patent Document 1 describes a serum-free culture medium containing borates and suitable for culturing diploid cells. In addition, non-patent literature 2 describes that taurine transporter Taut is important for the recall response of T cells.

[0005] [Citation list]

[0006] [Patent Document]

[0007] [Patent Document 1]

[0008] Chinese Patent No. 105462912

[0009] [Non-patent literature]

[0010] [Non-patent document 1]

[0011] Park, Meeyoung et al., Mol Cell. 2004 Nov 5;16(3):331-41.

[0012] [Non-patent document 2]

[0013] Kaesler, Susanne et al., Eur. J. Immunol. 2012 42:831-841 Summary of the invention

[0014] [Technical issues]

[0015] The present invention aims to provide a method for culturing and fully proliferating cells, especially immunocompetent cells, in a serum-free medium or a low-serum medium, as well as a medium additive and a medium composition to be used in the method. In addition, the present invention aims to provide a method for producing a cell population having a desired number of expanded cells, especially immunocompetent cells, in a serum-free medium or a low-serum medium, and a medicament containing the cell population obtained by the method.

[0016] [Solution to the problem]

[0017] In view of the above problems, the present inventors prepared various factors to be added to serum-free or low-serum medium, and studied their influence on cell culture or proliferation. As a result, by adding boric acid or its salt, even when serum-free medium or low-serum medium was used, the effect of maintaining and promoting cell proliferation comparable to that achieved by culturing in serum-containing medium was confirmed. Based on these findings, they found more preferred culture conditions and completed the present invention.

[0018] Therefore, the present invention provides the following.

[0019] [1] A method for culturing cells, comprising the step of culturing the cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.

[0020] [2] The method according to [1] above, wherein the final concentration of the boric acid or its salt in the culture medium is 0.1 mM to 0.9 mM.

[0021] [3] The method according to [1] above, wherein the final concentration of the boric acid or its salt in the culture medium is 0.3 mM to 0.6 mM.

[0022] [4] The method according to any one of [1] to [3] above, wherein the serum-free medium or low-serum medium is a serum-free medium.

[0023] [5] The method according to any one of [1] to [4] above, wherein the cell is an immunocompetent cell.

[0024] [6] The method according to [5] above, wherein the immunocompetent cells are selected from dendritic cells, B cells, T cells and natural killer cells.

[0025] [7] The method according to [6] above, wherein the immunocompetent cells are T cells.

[0026] [8] The method according to any one of [1] to [7] above, wherein the cells are derived from pluripotent stem cells.

[0027] [9] The method according to [8] above, wherein the pluripotent stem cells are iPS cells.

[0028]

[10] The method according to any one of [1] to [9] above, wherein the culture medium further contains taurine.

[0029]

[11] A method for proliferating cells, comprising the step of culturing the cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.

[0030]

[12] The method according to

[11] above, wherein the serum-free medium or low-serum medium is a serum-free medium.

[0031]

[13] The method according to

[11] or

[12] above, wherein the culture medium further contains taurine.

[0032]

[14] A method for producing a cell population in which the number of desired cells is expanded, comprising the step of culturing the cell population containing the desired cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.

[0033]

[15] The method according to

[14] above, wherein the serum-free medium or low-serum medium is a serum-free medium.

[0034]

[16] The method as described in

[14] or

[15] above, wherein the culture medium further contains taurine.

[0035]

[17] A medicament comprising a cell population obtained by the method described in any one of

[14] to

[16] above.

[0036]

[18] The drug according to

[17] above, which is used for preventing and / or treating cancer.

[0037]

[19] A culture medium additive comprising boric acid or a salt thereof, wherein the culture medium is a serum-free medium or a low-serum medium.

[0038]

[20] The additive according to

[19] above, wherein the serum-free medium or low-serum medium is a serum-free medium.

[0039]

[21] The additive according to

[19] or

[20] above, wherein the culture medium further contains taurine.

[0040]

[22] A medium composition comprising boric acid or a salt thereof, wherein the composition is obtained by adding the medium additive described in any one of

[19] to

[21] above to a serum-free medium or a low-serum medium.

[0041]

[23] The medium composition according to

[22] above, wherein the serum-free medium or low-serum medium is a serum-free medium.

[0042]

[24] The culture medium composition according to

[22] above, further comprising taurine.

[0043]

[25] A method for preventing and / or treating cancer, comprising administering a cell population obtained by the method of any one of

[14] to

[16] above to a subject in need thereof.

[0044]

[26] A cell population obtained by the method described in any one of

[14] to

[16] above, wherein the cell population is used for preventing and / or treating cancer.

[0045]

[27] Use of a cell population obtained by the method described in any one of

[14] to

[16] above in producing a drug for preventing and / or treating cancer.

[0046] [Advantageous Effects of the Invention]

[0047] According to the present invention, even when a serum-free medium or a low-serum medium is used, an effect of maintaining and promoting cell proliferation comparable to that of culturing in a serum-containing medium can be obtained. Specifically, for cells that are affected by stress due to operations such as cell washing, it is preferred to culture in a serum-free medium or a low-serum medium that does not require a large amount of washing, and the cells are suitable for culturing under such conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] [ Figure 1 ]

[0049] Figure 1is a graph showing the results of a cell proliferation test of iPS cell-derived CAR-T cells, which verifies the effect of adding 0.3 mM boric acid to a culture medium containing 1% FBS. The upper row shows the cell proliferation fold on day 0 of the self-amplification culture, and the lower row shows the measurement results of cell viability. w / o; without, w / ; with

[0050] [ Figure 2 ]

[0051] Figure 2 is a graph showing the results of a cell proliferation test of iPS cell-derived CAR-T cells, which verifies the effect of adding 0.3 mM boric acid to a serum-free expansion medium. The upper row shows the cell proliferation fold from day 0 of the expansion culture, and the lower row shows the results of measuring cell viability. w / o; without, w / ; with

[0052] [ Figure 3 ]

[0053] Figure 3 : is a graph showing the results of a proliferation test of iPS cell-derived CAR-T cells when boric acid is added to a serum-free expansion medium at different concentrations. The upper row shows the cell proliferation fold from day 0 of the expansion culture, and the lower row shows the measurement results of cell viability.

[0054] w / o; does not contain, w / ; contains

[0055] [ Figure 4 ]

[0056] Figure 4 : is a graph showing the results of cell proliferation test of iPS cell-derived CAR-T cells, which verified the effect of adding ITS-X alone and adding ITS-X in combination with taurine to a serum-free medium containing 0.3 mM boric acid. The upper row shows the cell proliferation fold on day 0 of the self-expansion culture, and the lower row shows the measurement results of cell viability.

[0057] w / o; does not contain, w / ; contains

[0058] [ Figure 5 ]

[0059] Figure 5 : is a graph showing the results of a cell proliferation test of human-derived primary T cell-derived CAR-T cells, which verifies the effect of adding taurine alone to a serum-free medium containing 0.3 mM boric acid. The upper row shows the cell proliferation fold on day 0 of the self-amplification culture, and the lower row shows the measurement results of cell viability. w / o; without, w / ; with DETAILED DESCRIPTION

[0060] The present invention is described below. Unless otherwise specified, the terms used in this specification have the meanings generally used in the relevant field.

[0061] (1) Cell culture method (hereinafter also referred to as "culture method of the present invention"), cell proliferation method (hereinafter also referred to as "proliferation method of the present invention")

[0062] The culture method or proliferation method of the present invention comprises the step of culturing cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.

[0063] 1-1. Cells

[0064] In the culture method and the proliferation method of the present invention, the type of cells used as the culture target is not particularly limited. Examples of cell types include reproductive cells, such as sperm and ovum, somatic cells constituting living organisms, stem cells (pluripotent stem cells, etc.) and cells induced to differentiate from stem cells, progenitor cells, cancer cells isolated from living organisms, cells (cell lines) of cells isolated from living organisms that obtain immortalization ability and stably maintain cells in vitro, cells isolated from living organisms through artificial genetic modification, and cells isolated from living organisms and having artificially exchanged nuclei. Examples of somatic cells constituting a living organism and cells induced to differentiate from stem cells include, but are not limited to, immunocompetent cells (natural killer (NK) cells, macrophages, monocytes, mast cells, dendritic cells, Langerhans cells, neutrophils, eosinophils, basophils, B cells, T cells, etc.), fibroblasts, bone marrow cells, erythrocytes, platelets, osteocytes, pericytes, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic β cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and monocytes. Somatic cells include cells obtained from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eye, brain, or neural tissue.

[0065] Stem cells are cells that have the ability to self-replicate and differentiate into a variety of other cell lineages. Examples include, but are not limited to, embryonic tumor cells, pluripotent stem cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells.

[0066] "Pluripotent stem cells" refer to cells with self-replication and differentiation / proliferation capabilities, and have the ability to differentiate into all tissues and cells constituting a living organism. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and pluripotent stem cells induced and selected by pressure or cell stimulation. Stem cells established by culturing early embryos produced by nuclear transplantation of the nucleus of somatic cells are also preferably used as pluripotent stem cells (Nature, 385, 810 (1997); Science, 280, 1256 (1998); Nature Biotechnology, 17, 456 (1999); Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); Nature Genetics, 24, 109 (2000)). In the present invention, iPS cells are preferably used as pluripotent stem cells. Due to the undifferentiated nature of iPS cells, undifferentiated markers can be used as indicators to identify iPS cells. Examples of undifferentiated markers include alkaline phosphatase, Oct3 / 4, Sox2, Nanog, ERas, Esgl, etc. Methods for detecting these undifferentiated markers include methods for detecting mRNA (using primers and probes), immunological detection methods (using antibodies and labels), etc.

[0067] Cells induced to differentiate from stem cells (differentiation-induced cells) are any cells that have been subjected to a differentiation-inducing treatment that induces the stem cells to differentiate into a specific cell type.

[0068] Cell lines are cells that have acquired unlimited proliferation capacity outside a living organism by artificial manipulation. Examples include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human embryonic kidney cell), HeLa (human uterine cancer cell line), HepG2 (human liver cancer cell line), UT7 / TPO (human leukemia cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five (trade name), Vero, etc.

[0069] In a preferred embodiment, the cells as the target of culture are immunocompetent cells.Immunocompetent cells refer to cells that participate in various immune responses in vivo.Examples of immunocompetent cells include natural killer (NK) cells, macrophages, monocytes, mast cells, dendritic cells, Langerhans cells, neutrophils, eosinophils, basophils, B cells and T cells, and are preferably selected from dendritic cells, B cells, T cells and natural killer (NK) cells.More preferably, immunocompetent cells are T cells.

[0070] T cells include CD4 positive CD8 negative T cells, CD4 negative CD8 positive T cells, αβ-T cells, γδ-T cells, regulatory T cells and NKT cells. T cells can be subpopulations, such as naive T cells, effector T cells, memory T cells, etc.

[0071] Immunocompetent cells can be cells separated from people (primary cells) or cells obtained by differentiation from stem cells, such as pluripotent stem cells (e.g., iPS cells, ES cells), hematopoietic stem cells and mesenchymal stem cells, and are preferably primary cells and cells obtained by differentiation from pluripotent stem cells (particularly iPS cells). As primary cells, preferably primary T cells derived from people. Differentiation induction from stem cells to immunocompetent cells can be carried out by methods known in the art, specifically depending on the type of stem cell used and the type of immunocompetent cells of interest. In one embodiment, differentiation induction from iPS cells to T cells and differentiation induction from iPS cells to NK cells can be carried out by the method described in the embodiment.

[0072] In addition, the target cell may be an autologous cell or an allogeneic cell. In the present invention, "autologous cells" refer to cells obtained from a subject receiving a cell population (described below) produced by the culture method of the present invention or the method of the present invention, or cells derived from the obtained cells, and "allogeneic cells" refer to cells that are not the above-mentioned "autologous cells".

[0073] In the present invention, the cells as the culture target may be artificially genetically modified cells. Examples of such cells include immunocompetent cells (T cells into which CAR genes have been introduced and natural killer cells into which CAR genes have been introduced), T cells into which exogenous T cell receptors (TCRs) have been introduced, and immunocompetent cells into which genes expressing cytokines and / or chemokines have been introduced.

[0074] CAR is a structure from the N-terminal side to the C-terminal side of the protein, including an extracellular domain specific to the target, a transmembrane domain, and an intracellular signaling domain for the effector function of immune cells, and the CAR gene is a gene encoding such a receptor. The extracellular domain contains an antigen recognition site that exhibits specific binding properties to the target. The transmembrane domain is located between the extracellular domain and the intracellular signaling domain. The intracellular signaling domain transmits the signal necessary for immune cells to exert their effector functions. That is, an intracellular signaling domain that can transmit the signal necessary for activating immune cells when the extracellular domain binds to the target antigen is used. There have been many reports on experiments, clinical studies, etc. using CAR (e.g., Rossig C, et al. Mol Ther 10:5-18, 2004; Dotti G, et al. Hum gene Ther 20:1229-1239, 2009; Ngo MC, et al. Hum Mol Genet 20(R1):R93-99, 2011; Ahmed N, et al. Mol Ther 17:1779-1787, 2009; Pule MA, et al. Nat Med 14:1264-1270, 2008; Louis CU, et al. Blood 118:6050-6056, 2011; Kochenderfer JN, et al. Blood 116:4099-4102, 2010; Kochenderfer JN, et al. Blood 119:2709-2720, 2012; Porter DL, et al. N Engl J Med 365:725-733, 2011; Kalos M, et al. Sci Transl Med 3:95ra73, 2011; Brentjens RJ, et al. Blood 118:4817-4828, 2011; Brentjens RJ, et al. Sci Transl Med 5:177ra38, 2013), and the CAR in the present invention can be constructed by referring to these reports.

[0075] A preferred embodiment of the cells using the culture method and proliferation method of the present invention includes T cells (CAR-T cells) introduced with CAR genes and NK cells (CAR-NK cells) introduced with CAR genes, more preferably CAR-T cells and CAR-NK cells derived from pluripotent stem cells, further preferably CAR-T cells derived from iPS cells and CAR-NK cells derived from iPS cells, and further more preferably CAR-T cells derived from iPS cells. CAR-T cells and CAR-NK cells can be obtained by introducing CAR genes into T cells and NK cells, or their precursor cells (such as pluripotent stem cells), respectively. For example, CAR-T cells can be obtained by introducing CAR genes into cells obtained by differentiating cells (such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells) into T cells, or by differentiating iPS cells into T cells into which CAR genes have been introduced. Preferably, CAR-T cells are derived from iPS cells and obtained by differentiating iPS cells into T cells introduced with CAR genes, or CAR-T cells are derived from iPS cells and obtained by introducing CAR genes into T cells obtained by differentiating iPS cells, and sometimes these cells are collectively referred to as “CAR-T cells derived from iPS cells”. More preferably, CAR-T cells are derived from iPS cells and obtained by introducing CAR genes into T cells obtained by differentiating iPS cells. For example, in the case of CAR-NK cells, CAR-NK cells may be obtained by introducing CAR genes into cells obtained by differentiating cells (such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells) into NK cells, or by differentiating iPS cells into NK cells introduced with CAR genes. Preferably, CAR-NK cells are derived from iPS cells and obtained by differentiating iPS cells into NK cells introduced with CAR genes, or CAR-NK cells are derived from iPS cells and obtained by introducing CAR genes into NK cells obtained by differentiating iPS cells, and sometimes these cells are collectively referred to as “CAR-NK cells derived from iPS cells”. More preferably, CAR-NK cells are derived from iPS cells and obtained by introducing the CAR gene into NK cells obtained by differentiating iPS cells.

[0076] Inducing iPS cells (in which a CAR gene may be introduced when desired) to differentiate into T cells, or inducing iPS cells (in which a CAR gene may be introduced when desired) to differentiate into NK cells can be performed using known methods, specifically, by the methods described in the Examples.

[0077] CAR gene is generally introduced into the cell using CAR expression vector. CAR expression vector means a nucleic acid molecule capable of transporting a nucleic acid molecule encoding a CAR gene into a cell. It may be DNA or RNA, and there is no particular restriction on its form and source, and various types of vectors may be used. The vector may be a viral vector or a non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated viral vectors, herpes virus vectors, Sendai virus (Sendai viral) vectors and vaccinia virus vectors. Among them, in retroviral vectors, lentiviral vectors and adeno-associated viral vectors, the target gene integrated into the vector is integrated into the host chromosome, and stable and long-term expression can be expected. Each viral vector can be produced according to conventional methods or using commercially available special kits. Examples of non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors and artificial chromosome vectors.

[0078] In one embodiment, the CAR gene can be introduced into the cell by the method described in the Examples.

[0079] 1-2. Culture medium

[0080] The culture medium to be used in the culture method and proliferation method of the present invention is a serum-free medium or a low-serum medium containing boric acid or a salt thereof (hereinafter sometimes collectively referred to as "the culture medium of the present invention").

[0081] In this specification, boric acid is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include orthoboric acid, metaboric acid and tetraboric acid. Of these boric acids, orthoboric acid is preferably used. Boric acid salts are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts; and salts with organic amines such as triethylamine, triethanolamine, morpholine, piperazine and pyrrolidine. In addition, boric acid or its salt may be in the form of a hydrate, such as borax. The concentration of boric acid or its salt in the culture medium is not particularly limited as long as the desired effect is obtained. It is generally 0.1mM to 0.9mM, preferably 0.2mM to 0.8mM, further preferably 0.2mM to 0.7mM, and particularly preferably 0.3mM to 0.6mM. Unless otherwise specified, the concentration of boric acid or its salt in this specification is the concentration converted to boric acid. When the concentration is too high, it affects cell viability, and when the concentration is too low, the effect of promoting cell proliferation is weak.

[0082] In this specification, "serum-free medium" means a medium that is substantially free of, preferably free of, unregulated or unpurified serum, and a medium contaminated by purified blood-derived components or animal tissue-derived components (e.g., growth factors) is considered a serum-free medium. As a culture medium, a culture medium for culturing animal cells (hereinafter also referred to as a basal medium for convenience) or a culture medium for perfusion culture is generally used. "Substantially free" here means that it is completely free of, or even if it is contained, it is below the detection limit.

[0083] Examples of the basal medium include, but are not limited to, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, modified MEM ZincOption medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, F12 medium, and mixed medium thereof (e.g., modified DMEM / F12 medium).

[0084] Perfusion culture is a culture method that continuously supplies culture medium to a culture system containing cells, while continuously removing an equal amount of cell-free culture supernatant from the culture system to maintain the culture system in a stable state. Since perfusion culture requires a higher cell density than general culture methods, the culture medium used for perfusion culture generally has a higher concentration of nutrient components than the basal culture medium.

[0085] These media are commercially available from Invitrogen, SIGMA, FUJIFILM Wako Pure Chemical Corporation, Sumitomo Pharma, and the like, and media of the same name or the same product name have the same composition regardless of the manufacturer.

[0086] In the present specification, "reduced serum medium" refers to a medium in which serum is added to a basal medium and is characterized in that the serum concentration is reduced compared with a generally used serum-containing medium (serum concentration: 5%-20% (serum concentration is expressed as volume / volume % in the present specification). The serum concentration of the low serum medium may be lower than 5%, lower than 2%, lower than 1%, lower than 0.1%, lower than 0.01% or lower than 0.001%, but is generally 0.1% or higher, or 1% or higher. Specifically, the above-mentioned serum concentration is preferably 0.1% or higher and less than 5%, more preferably 1% or higher and less than 4%.

[0087] The culture medium used in this step may also contain serum substitutes. Examples of serum substitutes include albumin (e.g., lipid-rich albumin), transferrins, fatty acids, collagen precursors, insulin, trace elements (e.g., zinc, selenium, etc.), ITS supplements (a mixture of insulin, transferrins and selenite), B-27 supplements, N2 supplements, knockout serum substitutes, 2-mercaptoethanol or 3'-thioglycerol or its equivalent. These serum substitutes are also commercially available. Knockout serum substitutes can be purchased from Invitrogen. Other serum substitutes can be purchased from Invitrogen, SIGMA, FUJIFILM WakoPure Chemical Corporation, Sumitomo Pharma, etc., and no matter how the manufacturer, the components of the reagents or additives of the same name or trade name are all equivalent.

[0088] The culture medium of the present invention may further contain components that are beneficial to cell proliferation in addition to the basic culture medium. Examples of such components include carbohydrates such as glucose, fructose, sucrose and maltose; amino acids or amino acid derivatives such as glycine, serine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, proline, threonine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, lysine, histidine, ornithine, theanine, citrulline, taurine, betaine, carnitine, creatine and pantothenic acid; proteins such as albumin and transferrin; peptides such as glycylglycerol. aminoacyl glycine and soybean peptides; serum; choline, vitamins such as B vitamins (thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, niacinamide, etc.), vitamin C (ascorbic acid) and vitamin C derivatives; fatty acids such as oleic acid, arachidonic acid and linoleic acid; lipids such as cholesterol; nucleosides such as adenosine, thymidine, guanosine, cytidine, uridine and inosine; inosinic acid; hypoxanthine; inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate and sodium dihydrogen phosphate; trace elements such as zinc, copper, selenium, Vanadium, manganese, nickel, silicon, tin, molybdenum, cadmium, chromium, silver, aluminum, barium, cobalt, germanium, iodine, bromine, fluorine, rubidium and zirconium; buffers such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and N-[tris(hydroxymethyl)methyl]glycine (Tricine); antibiotics such as amphotericin B, kanamycin, gentamicin and streptomycin; cell adhesion factors and extracellular matrix components such as type I collagen, type II collagen, fibronectin, laminin, and thiazolinone. Adhesion protein, poly-L-lysine and poly-D-lysine; cytokines and growth factors, such as interleukins, 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 according to the type of cells to be cultured and used at appropriate concentrations. Specifically, taurine, one of the types of amino acid derivatives, can be added to increase the viability and proliferation of cells, and it can be used at a final concentration of generally 0.5mM to 10mM, preferably 2mM to 5mM. Growth factors can be from animal species other than humans, but are preferably from human sources (which can be recombinant).

[0089] The culture medium may further contain additive factors that support cell survival and proliferation. The additive factor may be a type 1 cytokine family member, a type 2 cytokine family member, a TNF superfamily cytokine, an IL-1 family cytokine or other cytokines (TNF-β, etc.), specifically IL-1 to IL-41, etc., preferably IL-1, IL-2, IL-7, IL-15, IL-18, IL-21, etc. The additive factor may be prepared according to a conventional method, or commercially available products may be used. The additive factor may be from animal species other than humans, but preferably from human sources (which may be recombinant).

[0090] In the present specification, "xeno-free" refers to a state in which components derived from organisms other than the organism of the cell to be cultured are excluded.

[0091] 1-3. Cell Culture

[0092] The present invention also provides a method for culturing cells, characterized by culturing cells in the medium of the present invention (described above) (hereinafter sometimes referred to as "the culturing method of the present invention").

[0093] The incubator to be used for culturing cells is not particularly limited as long as it can culture cells, and examples of the incubator include flasks, tissue culture bottles, culture dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, micro slides, chamber slides, Petri dishes, test tubes, trays, culture bags, roller bottles, and bioreactors. An appropriate incubator is selected according to the target cells.

[0094] The incubator may be cell-adhesive or cell-non-adhesive and may be appropriately selected according to the purpose. A cell-adhesive incubator may be coated with any cell-supporting matrix, such as an extracellular matrix (ECM), to increase the adhesion of the incubator surface to the cells. The cell-supporting matrix may be any substance for cell adhesion.

[0095] Other culture conditions can be appropriately set. For example, the culture temperature is not particularly limited and may be about 30° C. to 40° C., preferably about 37° C. 2 The concentration may be about 1% to 10%, preferably about 2% to 5%. The oxygen partial pressure may be 1% to 21%.

[0096] In cell culture, the frequency of medium replacement and the culture period are generally determined by taking into account various conditions, such as cell density, culture mode (adherent culture / suspension culture), the type of cells to be cultured, medium composition, culture conditions (temperature, gas concentration), the amount of medium to be replaced (total amount / partial amount), medium cost and the lifestyle of the operator. Medium replacement is generally carried out once every 2 to 3 days, once a day or multiple times a day (for example, twice a day), and medium replacement can be carried out at such a frequency in the culture method of the present invention and the proliferation method. In addition, medium exchange can be continuously carried out by perfusion culture. The culture period is generally about 4 days to 4 weeks, preferably about 4 days to 3 weeks, more preferably about 1 week to 2 weeks.

[0097] In one embodiment of the present invention, the cells to be cultured in a serum-free medium or a low serum medium (i.e., the medium of the present invention) containing boric acid or its salt are immunocompetent cells, preferably selected from dendritic cells, B cells, T cells, and natural killer (NK) cells, more preferably T cells and NK cells, and further preferably T cells. In another embodiment of the present invention, the cells to be cultured in the medium of the present invention are primary T cells and T cells derived from pluripotent stem cells (especially iPS cells), more preferably CAR-T cells derived from primary T cells and pluripotent stem cells (especially iPS cells), and more preferably CAR-T cells derived from pluripotent stem cells (especially iPS cells). In another embodiment of the present invention, the cells to be cultured in the medium of the present invention are primary NK cells and NK cells derived from pluripotent stem cells (especially iPS cells), more preferably CAR-NK cells derived from primary NK cells and pluripotent stem cells (especially iPS cells), and more preferably CAR-NK cells derived from pluripotent stem cells (especially iPS cells).

[0098] In one embodiment of the present invention, the culture includes an activation step and an expansion step. When cryopreserved cells are used, a recovery step may be included. In the present invention, when the culture target is cryopreserved cells, the concept of cell culture includes any one, two or all three of the recovery culture consisting of a recovery step, an activation culture consisting of an activation step, and an expansion culture consisting of an expansion step. Preferably, the target is an expansion culture. Another embodiment of the present invention is a culture of CAR-T cells and CAR-NK cells, preferably CAR-T cells derived from pluripotent stem cells (particularly iPS cells) and CAR-NK cells derived from pluripotent stem cells (particularly iPS cells), which includes an activation step and an expansion step, and includes a recovery step (in terms of using cryopreserved cells, etc.) when necessary.

[0099] (Recovery Steps / Recovery Culture)

[0100] This step is performed after thawing the cryopreserved cells. Since cells can be damaged by prolonged exposure to cryoprotectants, the cryoprotectants should be removed from the cells immediately after thawing. Then, the cell damage and cell dysfunction caused by freezing and thawing are repaired. This step / culture method varies depending on the target cells, the cryoprotectants used, the thawing method, etc. Most cells generally recover normally by culturing them in a culture medium without cryoprotectants for several days, preferably 1 to 5 days, and more preferably about 3 days.

[0101] (Activation step / activation culture)

[0102] The method of this step is not particularly limited as long as the desired effect on the cell can be obtained, and for example, it can be performed by contact with a stimulating substance. In general, the cells are cultured in the presence of a stimulating substance, specifically in a culture medium containing the stimulating substance, for several days, preferably 1 to 5 days, more preferably about 3 days. The stimulating substance is a signal molecule that controls cell activity by methods such as autocrine, paracrine or endocrine, and may be a substance that can be secreted by all cells contained in the culture system, or may be a substance added from the outside. Specifically, the step is performed by contacting a ligand-bound matrix, or by culturing in a culture medium containing a ligand, etc.

[0103] For example, CAR-T cells, preferably CAR-T cells derived from pluripotent stem cells (particularly iPS cells) are not particularly limited in the ligand used in the activation culture, as long as it is a molecule that interacts with the surface molecules of CAR-T cells and promotes their activation. Examples include CD3, which is coupled to TCR and is responsible for signal transduction via TCR; and molecules that specifically bind to the surface molecules (such as CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, and CD40L) of co-stimulatory factors known as T cell activation and have the function of transmitting activation signals to T cells. Such molecules may be physiological ligands (or receptors) of the above-mentioned T cell surface molecules or non-physiological ligands (or receptors) with agonistic activity. A preferred example of a non-physiological ligand is an agonist antibody.

[0104] More preferably, the T cell activating ligand to be used in the present invention is an antibody against CD3. The antibody against CD3 may be a complete antibody or a fragment thereof (e.g., Fab, F(ab') 2 , Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc.), as long as it has the ability to specifically bind to CD3 expressed in target T cells induced for activation and stimulate the surface molecules of these T cells to transmit signals into T cells.

[0105] For example, the molecules used in the activation culture of CAR-NK cells, preferably CAR-NK cells derived from pluripotent stem cells (especially iPS cells), are not particularly limited, as long as they are molecules that interact with the surface molecules of CAR-NK cells and promote their activation. For example, it may be a physiological ligand (or receptor) of the above-mentioned NK cell surface molecules or a non-physiological ligand (or receptor) with agonistic activity. It may also be a cytokine. A preferred example of a non-physiological ligand is an antibody against CD3. The antibody may be a complete antibody or a fragment thereof (e.g., Fab, F(ab') 2 , Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc.). Examples of cytokines include IFN-α / β, IFN-γ, IL-2, IL-4, IL-7, IL-12, IL-15, IL-18 and IL-21.

[0106] (Expansion Step / Expansion Culture)

[0107] In this step, the cells activated as described above are proliferated. The method of amplification culture is not particularly limited, as long as the desired effect of the activated cells is obtained, and those skilled in the art can appropriately adjust the method while monitoring the number of cells, etc. When the purpose is to increase the number of cells, cells can be cultured in a culture medium containing the above-mentioned additives, etc., for example, 2 days or longer, preferably 3 days or longer, more preferably 4 days or longer, further preferably 5 days or longer, and further more preferably 6 days or longer. Cells can be cultured and appropriately adjusted while monitoring the number of cells, etc., for example, 7 days or longer, 9 days or longer or 11 days or longer. In addition, by alternately repeating activation culture and amplification culture to continue to cultivate, the number of cells can be increased exponentially. The upper limit of the culture period is not particularly limited, and for example, is 28 days or shorter, preferably 21 days or shorter. For example, for activated CAR-T cells, preferably activated pluripotent stem cells (especially iPS cells) derived CAR-T cells, cultured cells can be expanded in a medium containing cytokines (such as IL-7, IL-15 and IL-2) for 2 days or longer, preferably 3 days or longer, more preferably 4 days or longer, further preferably 5 days or longer, and further more preferably 6 days or longer. When the target cell is a primary T cell (especially CAR-T cell) or NK cell (preferably NK cells (especially CAR-NK cells) derived from pluripotent stem cells (especially iPS cells), and the purpose is to increase the number of cells, the cultured cells can be expanded in a medium containing cytokines (such as IL-7, IL-15, IL-21 and IL-2) for 2 days or longer, preferably 4 days or longer, more preferably 6 days or longer, further preferably 8 days or longer, further preferably 10 days or longer, and even more preferably 11 days or longer.

[0108] (2) Method for producing a cell population (hereinafter also referred to as "the production method of the present invention") and a drug containing the cell population (hereinafter also referred to as "the drug of the present invention")

[0109] The production method of the present invention is a method for producing a cell population in which the number of desired cells is expanded, and the method includes the step of culturing the cell population containing the desired cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.

[0110] The "serum-free medium or low-serum medium containing boric acid or a salt thereof" is the same as described in the culture method of the present invention and the proliferation method of the present invention in (1) above.

[0111] The desired cells are cells intended to proliferate, and include those exemplified in the subsection "1-1. Cells" in (1) above. Preferably, immunocompetent cells are used, more preferably T cells or NK cells, particularly preferably T cells into which CAR genes have been introduced, i.e., CAR-T cells, and NK cells into which CAR genes have been introduced, i.e., CAR-NK cells. CAR-T cells and CAR-NK cells are preferably derived from pluripotent stem cells, preferably iPS cells, and even more preferably CAR-T cells derived from pluripotent stem cells (particularly iPS cells).

[0112] The source of the "cell group containing the desired cells" is not particularly limited, as long as it contains the desired cells, and the cell group may be of natural origin or artificially prepared. For example, when immunocompetent cells (such as T cells) are desired, the cell group may be exemplified as a cell group containing peripheral blood mononuclear cells (PBMC), blood cells, hematopoietic stem cells, cord blood mononuclear cells, etc., collected, separated, purified or induced from body fluids such as blood (peripheral blood, umbilical cord blood, etc.) and bone marrow fluid. These cells may be collected from a living body, or obtained by in vitro culture, and for example, a cell group obtained by the production method of the present invention may be mentioned, whether original or frozen. In addition, the cell group is not limited to a cell group of a single cell, and may be a tissue block section obtained by biopsy, preferably a cell group of a single cell. With regard to a body fluid cell group, it is not limited to a cell group derived from a single tissue, and may be a mixed system of cell groups derived from different tissues. "A cell group containing desired cells" is preferably a cell group containing immunocompetent cells, more preferably a cell group containing T cells or NK cells, and particularly preferably a cell group containing T cells introduced with a CAR gene, i.e., a cell group containing CAR-T cells, or a cell group containing NK cells introduced with a CAR gene, i.e., a cell group containing CAR-NK cells. Even more preferably, a cell group containing CAR-T cells derived from pluripotent stem cells (preferably iPS cells), or a cell group containing CAR-NK cells derived from pluripotent stem cells (preferably iPS cells).

[0113] "Expanding the number of desired cells" means that the number of desired cells in a cell population is increased compared to a control, such as the number of cells obtained before expansion or without practicing the present invention, and means an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% compared to a control. In a specific embodiment of the invention, the number of desired cells in a cell population produced by the invention is expanded to achieve an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% compared to the number of cells in a control.

[0114] When the desired cells are CAR-T cells or CAR-NK cells, the cells are increased in number by activation culture and expansion culture (and recovery culture when necessary) as described above, i.e., amplified. After the amplification culture, the cells are washed and collected. When the culture method and proliferation method of the present invention are used for culture or proliferation, since the culture medium used is a serum-free culture medium or a low-serum culture medium, the washing operation is simpler than when a generally used serum-containing culture medium (serum concentration: 5% to 20%) is used for culture or proliferation, and the time required for washing can be shortened.

[0115] For example, when washing and collecting cells using (i) pipetting and centrifugation or (ii) a closed system automated cell processing device, continuous automated processing using techniques such as (i) pipetting and centrifugation or (ii) a closed system centrifugation / spin membrane filtration / hollow fiber membrane filtration needs to be repeated multiple times until the BSA contained in the serum reaches an acceptable amount for administration to humans. When culturing or proliferation is performed using the culture method and proliferation method of the present invention, multiple repetitions are not required (the amount of serum-derived BSA introduced into the culture system is already small). Alternatively, the desired cell population can be collected with fewer repetitions than when using a serum-containing culture medium (serum concentration: 5% to 20%).

[0116] In a preferred embodiment, a step of culturing cells in the presence of a stimulating substance is performed before the collection operation (after amplification culture). This step allows efficient amplification culture, and also has the advantage of improving cell viability. When desired, the CAR-T cell group or CAR-NK cell group after amplification culture can be subjected to a cell separation step such as washing operation and separation using beads. This embodiment enables the purity of the CAR-T cell group or CAR-NK cell group to be increased with a higher effect. At the same time in this embodiment, when the culture method and proliferation method of the present invention are used for culture or amplification, the washing operation can be simplified and / or the time required for washing can be shortened.

[0117] In this way, the number of desired cells is expanded in the cell population obtained by the production method of the present invention.

[0118] The "cell population in which the number of desired cells is expanded" produced by the production method of the present invention can appropriately produce drugs according to the function of the cells. When the cells are CAR-T cells or CAR-NK cells, the cell population containing CAR-T cells or CAR-NK cells produced by the culture method and proliferation method of the present invention can be used to treat cancer, especially cancer expressing the target antigen of CAR-expressing immune cells. The cancer may be a solid tumor or a blood tumor. Specific examples of cancer include, but are not limited to, various B-cell lymphomas (follicular malignant lymphoma, diffuse large B-cell malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative neoplasms, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndrome, acute myeloid leukemia, neuroblastoma, brain tumors, Ewing's sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, bone and soft tissue sarcoma, renal cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, and colorectal cancer. In a preferred embodiment, the cancer is a solid tumor. Examples of solid tumors include neuroblastoma, brain tumors, Ewing's sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, ovarian cancer, rhabdomyosarcoma, bone and soft tissue sarcomas, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, and colorectal cancer.

[0119] The drug of the present invention is administered in a therapeutically effective amount appropriately determined according to the age, weight, body surface area, symptoms, etc. of the subject. The subject of the present disclosure is a mammal, specifically, generally a human, and preferably a cancer patient. The drug of the present invention can be administered, for example, 1x104 cells to 1x10 10 The administration route is not particularly limited, and can be intratumoral, peritumoral, intraventricular, intravenous, intraarterial, intraportal, intradermal, subcutaneous, intramuscular or intraperitoneal. The cell population disclosed herein can be administered systemically or locally. Local administration includes direct injection into the target tissue, internal organs or organs. The administration schedule is appropriately determined according to the age, weight, body surface area, symptoms, etc. of the subject, and can be a single administration or continuous or regular multiple administrations. The drug of the present invention can be used for autologous transplantation or allogeneic transplantation. It can also be used in combination with other drugs.

[0120] Specifically, the drug of the present invention may contain the following components in addition to the cell population to be administered to the subject, such as dimethyl sulfoxide (DMSO) or serum albumin for the purpose of protecting the cells, antibiotics for the purpose of preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) for the purpose of activating, proliferating or inducing differentiation of cells. The composition can be prepared by conventional methods (e.g., methods described in the Japanese Pharmacopoeia, etc.).

[0121] (3) Culture medium additive (hereinafter also referred to as "culture medium additive of the present invention") and culture medium composition containing the culture medium additive (hereinafter also referred to as "culture medium composition of the present invention")

[0122] The culture medium additive of the present invention contains boric acid or a salt thereof. The culture medium additive of the present invention is used to be added to a serum-free medium or a low-serum medium. "Boric acid or a salt thereof" is the same as described in the culture method of the present invention and the proliferation method of the present invention in (1) above. The amount of boric acid or a salt thereof to be added to the culture medium additive is not particularly limited as long as the desired effect is obtained. It can generally be 0.01% to 100% by weight, preferably 0.1% to 100% by weight, more preferably 1% to 100% by weight, further preferably 5% to 100% by weight, and particularly preferably 10% to 100% by weight.

[0123] The culture medium composition of the present invention is a composition containing culture medium components obtained by adding the culture medium additive of the present invention to a serum-free culture medium or a low-serum culture medium. As used herein, "serum-free culture medium or low-serum culture medium" has the same meaning as the "serum-free culture medium or low-serum culture medium" described in (1) above in the culture method of the present invention and the proliferation method of the present invention, except that boric acid or its salt has been removed. The culture medium additive of the present invention is added to the "serum-free culture medium or low-serum culture medium" so that the concentration of boric acid or its salt in the culture medium composition is in the range of 0.1mM to 0.9mM, preferably 0.2mM to 0.8mM, further preferably 0.2mM to 0.7mM, and particularly preferably 0.3mM to 0.6mM.

[0124] The present invention is described in detail below using examples, but the present invention is not limited in any way. Unless otherwise stated, the reagents and materials to be used are all commercially available or can be prepared according to known literature, etc. In addition, it should be understood by those skilled in the art that any substance with similar effects or functions can be used alternatively.

[0125] [Example]

[0126] (List of abbreviations)

[0127] BMP-4: bone morphogenetic protein-4

[0128] bFGF: basic fibroblast growth factor

[0129] VEGF: vascular endothelial growth factor

[0130] SCF: Stem Cell Factor

[0131] TPO: thrombopoietin

[0132] FLT3L: Fms-related tyrosine kinase 3 ligand

[0133] Fc-DLL4: recombinant human DLL4 Fc chimeric protein

[0134] DLL4: Delta-like protein 4

[0135] IL7: interleukin-7

[0136] SDF1α: stromal cell-derived factor 1α

[0137] αMEM: α modified Eagle's minimum essential medium

[0138] TCR: T cell receptor

[0139] Example 1

[0140] 1. Preparation of iPS cell-derived CAR-T cells

[0141] The TCR gene was introduced into the iPS cell line QHJI01S04 provided by the iPS Cell Research and Application Center (CiRA) of Kyoto University using a lentiviral vector. The iPS cells into which the TCR gene was introduced were differentiated into hematopoietic progenitor cells with reference to the known method described in Nature Communication 2021; 12: 430. Specifically, iPS cells were cultured for 4 days in the presence of CHIR99021, SB431542, BMP-4, bFGF, and VEGF to induce differentiation into mesoderm. In addition, hematopoietic cytokines SCF, TPO, and FLT3L were used to differentiate into hematopoietic progenitor cells. The obtained hematopoietic progenitor cells were differentiated into cytotoxic T lymphocytes (CTLs) according to known patent methods (WO2017 / 221975) and literature information (Nature Communication 2021; 12: 430). Specifically, from the obtained hematopoietic progenitor cells, CD34-positive cells were purified using magnetic beads (Myltenyi Biotec) and cultured in α-MEM medium containing SCF, TPO, FLT3L, IL7, SDF1α and SB203580 on a plate fixed with Fc-DLL4 and RetroNectin (recombinant human fibronectin fragment, TakaraBio) for 3 weeks. The CAR gene that recognizes a specific antigen is introduced into the obtained TCR-positive CTL by using a retroviral vector. Next, the cells were cryopreserved in liquid nitrogen and used as iPS cell-derived CAR-T cells.

[0142] 2. Recovery culture of iPS cell-derived CAR-T cells

[0143] iPS cell-derived CAR-T cells were suspended at 500,000 cells / mL in IMDM medium containing 15% fetal bovine serum (FBS) to which the additives shown in the "Recovery medium" column in Table 1 were added, seeded on G-Rex (registered trademark) 10M (Wilson Wolf), and incubated at 5% CO 2 / Cultivate at 37°C for 3 days.

[0144] [Table 1]

[0145]

[0146] The additives added in each group are indicated by +.

[0147] 3. Reagents and Antibodies

[0148] RetroNectin (trade name) was purchased from Takara Bio Inc. The anti-CD3 agonist antibody used was anti-CD3 mAb GMP grade, anti-CD3 monoclonal antibody (clone: ​​OKT3) purchased from Takara Bio Inc.

[0149] 4. Immobilization of anti-CD3 agonist antibody and RetroNectin (trade name) on culture plates

[0150] Anti-CD3 agonist antibody (OKT3, final concentration 3 μg / mL) and RetroNectin (trade name) (final concentration 150 μg / mL) dissolved in PBS at the desired concentration were added to a T225 flask and left overnight at 4° C. After washing with PBS, the cells were tested.

[0151] 5. Activation and culture of iPS cell-derived CAR-T cells

[0152] The iPS cell-derived CAR-T cells after recovery culture were suspended at 133,333 cells / mL in IMDM medium containing 15% FBS to which the additives shown in the "Activation medium" column in Table 1 were added, seeded in a T225 flask immobilized with an anti-CD3 agonist antibody (OKT3) and RetroNectin (trade name), and incubated at 5% CO. 2 / Cultivate at 37°C for 3 days.

[0153] 6. Expansion of iPS cell-derived CAR-T cells in two types of culture medium containing 1% FBS

[0154] Two types of expansion medium (a medium supplemented with 0 mM boric acid and a medium supplemented with 0.3 mM boric acid) were prepared: a modified DMEM / F12 medium (Thermo Fisher) containing 1% FBS and further containing the additives shown in the "Expansion Medium" column in Table 1, and the medium further supplemented with 0.3 mM boric acid (Sigma-Aldrich). After 3 days of activation culture, cells were collected from the T225 flask and suspended at 40,000 cells / mL in each of the two types of expansion medium, and incubated with G-Rex (registered trademark) 10 M (Wilson Wolf) at 5% CO. 2 / 37 ℃ and culture. Then, on the fifth day of culture, some cells were recovered from G-Rex (registered trademark) 10M and cell counts were performed, and the culture medium was replaced with the corresponding culture medium. On the sixth day of culture, some cells were recovered from G-Rex (registered trademark) 10M and cell counts were performed. On the seventh day of culture, some cells were recovered from the plate, counted, and inoculated at 1,375,000 cells / mL on a G-Rex (registered trademark) 24-well plate (Wilson Wolf) containing each culture medium. Thereafter, on the 10th day of culture, some cells were collected from a G-Rex (registered trademark) 24-well plate, counted, and inoculated at 50,000 cells / mL on a G-Rex (registered trademark) 24-well plate (Wilson Wolf) containing the corresponding culture medium. On the 12th and 14th days of culture, some cells were similarly collected from a G-Rex (registered trademark) 24-well plate, counted, and the culture medium was replaced with each culture medium.

[0155] Experimental Example 1

[0156] Proliferation assay to verify the effect of adding 0.3 mM boric acid to medium containing 1% FBS

[0157] In the expansion culture of Item 6 in Example 1, the cell number and viability of iPS cell-derived CAR-T cells were measured on days 3, 5, 6, 7, 12, and 14 of culture. Figure 1 The cell proliferation folds from day 0 of the expansion culture are shown. The proliferation of iPS cell-derived CAR-T cells was promoted by adding 0.3 mM boric acid to the culture medium containing 1% FBS ( Figure 1 ).

[0158] Example 2

[0159] 1. Recovery culture of iPS cell-derived CAR-T cells

[0160] iPS cell-derived CAR-T cells were suspended at 500,000 cells / mL in IMDM medium containing 15% FBS to which the additives shown in the "Recovery Medium" column in Table 2 were added, seeded on G-Rex (registered trademark) 10M (Wilson Wolf), and incubated at 5% CO 2 / Cultivate at 37°C for 3 days.

[0161] [Table 2]

[0162]

[0163]

[0164] The additives added in each group are indicated by +.

[0165] 2. Reagents and Antibodies

[0166] RetroNectin (trade name) was purchased from Takara Bio Inc. The anti-CD3 agonist antibody used was anti-CD3 mAb GMP grade, anti-CD3 monoclonal antibody (clone: ​​OKT3) purchased from Takara Bio Inc.

[0167] 3. Immobilization of anti-CD3 agonist antibody and RetroNectin (trade name) on culture plates

[0168] Anti-CD3 agonist antibody (OKT3, final concentration 3 μg / mL) and RetroNectin (trade name) (final concentration 150 μg / mL) dissolved in PBS at the desired concentration were added to a T225 flask and left overnight at 4° C. After washing with PBS, the cells were tested.

[0169] 4. Activation and culture of iPS cell-derived CAR-T cells

[0170] The iPS cell-derived CAR-T cells after recovery culture were suspended at 133,333 cells / mL in IMDM medium containing 15% FBS to which the additives shown in the "Activation medium" column in Table 2 were added, seeded in a T225 flask immobilized with an anti-CD3 agonist antibody (OKT3) and RetroNectin (trade name), and incubated at 5% CO. 2 / Cultivate at 37°C for 3 days.

[0171] 5. Expansion of iPS cell-derived CAR-T cells in two types of serum-free culture media

[0172] Two types of expansion medium were prepared: an expansion medium (without addition of boric acid) which was a modified DMEM / F12 medium containing the additives shown in the "Expansion Medium" column in Table 2, and the medium further supplemented with 0.3 mM boric acid. After 3 days of activation culture, cells were collected from the T225 flask and suspended at 40,000 cells / mL in each of the two types of expansion medium, and incubated with G-Rex (registered trademark) 10 M (Wilson Wolf) at 5% CO. 2 / 37 ° C. Then, on the fifth day of culture, some were collected from G-Rex (registered trademark) 10M and counted, and the culture medium was replaced with each culture medium. On the 7th and 10th days of culture, cells were counted in a similar manner, inoculated in a G-Rex (registered trademark) 24-well plate at 50,000 cells / mL, and cultured in each culture medium. On the 12th and 14th days of culture, some cells were similarly collected from the plate, counted, and the culture medium was replaced with each culture medium.

[0173] Experimental Example 2

[0174] Proliferation assay to verify the effect of adding boric acid to serum-free expansion medium

[0175] In order to verify the effect of the addition of boric acid on the serum-free expansion medium, in the expansion culture of item 5 in Example 2, the cell number and viability of iPS cell-derived CAR-T cells were measured on days 3, 5, 6, 7, 10, 12 and 14 of culture. Figure 2 The cell proliferation folds from day 0 of the expansion culture are shown. The proliferation of iPS cell-derived CAR-T cells was promoted by adding 0.3 mM boric acid to the serum-free medium ( Figure 2 ).

[0176] Example 3

[0177] 1. Recovery culture of iPS cell-derived CAR-T cells

[0178] iPS cell-derived CAR-T cells were suspended at 500,000 cells / mL in IMDM medium containing 15% FBS to which the additives shown in the "Recovery Medium" column in Table 3 were added, seeded on G-Rex (registered trademark) 10M (Wilson Wolf), and incubated at 5% CO 2 / Cultivate at 37°C for 3 days.

[0179] [Table 3]

[0180]

[0181] The additives added in each group are indicated by +.

[0182] 2. Reagents and Antibodies

[0183] RetroNectin (trade name) was purchased from Takara Bio Inc. The anti-CD3 agonist antibody used was anti-CD3 mAb GMP grade, anti-CD3 monoclonal antibody (clone: ​​OKT3) purchased from Takara Bio Inc.

[0184] 3. Immobilization of anti-CD3 agonist antibody and RetroNectin (trade name) on culture plates

[0185] Anti-CD3 agonist antibody (OKT3, final concentration 3 μg / mL) and RetroNectin (trade name) (final concentration 150 μg / mL) dissolved in PBS at the desired concentration were added to a T225 flask and left overnight at 4° C. After washing with PBS, the cells were tested.

[0186] 4. Activation and culture of iPS cell-derived CAR-T cells

[0187] The iPS cell-derived CAR-T cells after recovery culture were suspended at 133,333 cells / mL in IMDM medium containing 15% FBS to which the additives shown in the "Activation medium" column in Table 3 were added, seeded in a T225 flask immobilized with an anti-CD3 agonist antibody (OKT3) and RetroNectin (trade name), and incubated at 5% CO. 2 / Cultivate at 37°C for 3 days.

[0188] 5. Expansion and culture of iPS cell-derived CAR-T cells in five types of serum-free culture media

[0189] Five types of expansion medium were prepared: modified DMEM / F12 medium containing the additives shown in the "Expansion Medium" column in Table 3, and each further containing 0.2 mM, 0.3 mM, 0.4 mM, 0.6 mM, or 0.9 mM boric acid. After 3 days of activation culture, cells were collected from T225 flasks and suspended in each of the five types of expansion medium at 50,000 cells / mL, and incubated using a G-Rex (registered trademark) 24-well plate (Wilson Wolf) at 5% CO. 2 / 37 ° C. Then, on the fifth day of culture, some cells were recovered from the G-Rex (registered trademark) 24-well plate and the cell count was performed, and the culture medium was replaced with the corresponding culture medium. On the seventh day of culture, the cells were counted in a similar manner, inoculated into a fresh G-Rex (registered trademark) 24-well plate at 50,000 cells / mL, and cultured in each culture medium. On the 10th and 12th days of culture, some cells were similarly collected from the plate, counted, and the culture medium was replaced with each culture medium.

[0190] Experimental Example 3

[0191] Proliferation test of iPS cell-derived CAR-T cells when different concentrations of boric acid were added to serum-free expansion medium

[0192] In order to verify the optimal concentration of boric acid to be added to the serum-free expansion medium, the number of iPS cell-derived CAR-T cells was counted over time in the expansion culture of item 5 in Example 3. Figure 3 The cell proliferation folds on day 0 of the self-expansion culture are shown. The proliferation folds were maximized when the final concentration of boric acid was in the range of 0.3 mM to 0.6 mM. It was also confirmed that cell viability was not affected in the boric acid concentration range of 0.2 mM to 0.9 mM ( Figure 3 ).

[0193] Example 4

[0194] 1. Recovery culture of iPS cell-derived CAR-T cells

[0195] iPS cell-derived CAR-T cells were suspended at 500,000 cells / mL in IMDM medium containing 15% FBS to which the additives shown in the "Recovery Medium" column in Table 4 were added, seeded on G-Rex (registered trademark) 10M (Wilson Wolf), and incubated at 5% CO 2 / Cultivate at 37°C for 3 days.

[0196] [Table 4]

[0197]

[0198] The additives added in each group are indicated by +.

[0199] 2. Reagents and Antibodies

[0200] RetroNectin (trade name) was purchased from Takara Bio Inc. The anti-CD3 agonist antibody used was anti-CD3 mAb GMP grade, anti-CD3 monoclonal antibody (clone: ​​OKT3) purchased from Takara Bio Inc.

[0201] 3. Immobilization of anti-CD3 agonist antibody and RetroNectin (trade name) on culture plates

[0202] Anti-CD3 agonist antibody (OKT3, final concentration 3 μg / mL) and RetroNectin (trade name) (final concentration 150 μg / mL) dissolved in PBS at the desired concentration were added to a T225 flask and left overnight at 4° C. After washing with PBS, the cells were tested.

[0203] 4. Activation and culture of iPS cell-derived CAR-T cells

[0204] The iPS cell-derived CAR-T cells after recovery culture were suspended at 133,333 cells / mL in IMDM medium containing 15% FBS to which the additives shown in the "Activation medium" column in Table 4 were added, seeded in a T225 flask immobilized with an anti-CD3 agonist antibody (OKT3) and RetroNectin (trade name), and incubated at 5% CO. 2 / Cultivate at 37°C for 3 days.

[0205] 5. Expansion and culture of iPS cell-derived CAR-T cells in five types of serum-free culture media

[0206] Five types of expansion medium were prepared: modified DMEM / F12 medium (Thermo Fisher), and the medium further supplemented with 0.3 mM boric acid (Sigma-Aldrich), 0.3 mM boric acid and 1×ITS-X ((insulin-transferrin-seleno-ethanolamine supplement) Gibco), 0.3 mM boric acid, 1×ITS-X and 1 mM taurine (Sigma-Aldrich), or 0.3 mM boric acid, 1×ITS-X and 3 mM taurine (medium supplemented with the additives in Table 4 alone, medium supplemented with the additives in Table 4 + 0.3 mM boric acid, medium supplemented with the additives in Table 4 + 0.3 mM boric acid + ITS-X, medium supplemented with the additives in Table 4 + 0.3 mM boric acid + ITS-X + 1 mM taurine, medium supplemented with the additives in Table 4 + 0.3 mM boric acid + ITS-X + 3 mM taurine). After 3 days of activation culture, the cells were collected from the T225 flask and suspended in each of five types of expansion medium at 40,000 cells / mL, and incubated with G-Rex (registered trademark) 10 M (Wilson Wolf) at 5% CO. 2 / 37 ℃ culture. Then, on the fifth day of culture, some cells were recovered from G-Rex (registered trademark) 10M and cell counts were performed, and the culture medium was replaced with the corresponding culture medium. On the sixth day of culture, some cells were recovered from G-Rex (registered trademark) 10M and cell counts were performed. On the 7th and 10th days of culture, some cells were recovered from the plate, counted, and inoculated at 50,000 cells / mL on a G-Rex (registered trademark) 24-well plate (Wilson Wolf) containing each culture medium. On the 12th and 14th days of culture, some cells were similarly collected from the G-Rex (registered trademark) 24-well plate, counted, and the culture medium was replaced with each culture medium.

[0207] Experimental Example 4

[0208] Proliferation test to verify the effect of adding the additives ITS-X and taurine to serum-free medium containing 0.3 mM boric acid

[0209] In the expansion culture of item 5 in Example 4, the cell number and viability of iPS cell-derived CAR-T cells were measured on days 3, 5, 6, 7, 10, 12, and 14 of culture. Figure 4 The cell proliferation folds from day 0 of the expansion culture are shown. The proliferation of iPS cell-derived CAR-T cells was promoted by further adding 1×ITS-X and 1 mM or 3 mM taurine to the serum-free medium containing 0.3 mM boric acid ( Figure 4 ), and the viability was increased by 1% to 4% by adding 1 mM or 3 mM taurine to the culture medium (Table 5).

[0210] [Table 5]

[0211]

[0212]

[0213] Bold numbers indicate the maximum value for each measurement date.

[0214] Example 5

[0215] 1. Activation and culture of primary human T cells

[0216] Human primary T cells (Leukopak-SoloPak, Charles River Laboratories Cell Solutions) were suspended at 1,000,000 cells / mL in OpTmizer medium (Thermo Fisher) containing 2% CTS immune cell SR (Thermo Fisher) and the additives shown in the activation medium in Table 6, seeded in PL240 bags (OriGen), and incubated in 5% CO 2 / Cultivate at 37°C for 2 days.

[0217] [Table 6]

[0218]

[0219] The additives added in each group are indicated by +.

[0220] 2. Reagents

[0221] RetroNectin (trade name) was purchased from Takara Bio Inc.

[0222] 3. Fixation of RetroNectin (product name) and retrovirus on culture plates

[0223] RetroNectin dissolved in PBS at a necessary concentration (final concentration 150 μg / mL) was added to the PL120 bag and left at room temperature for 2 hours. After washing with PBS, the retrovirus solution was added and fixed overnight at 4°C while shaking at 50 rpm. After washing with PBS containing 1.5% HSA, the plate was tested.

[0224] 4. Transduction and culture of primary human T cells

[0225] The activated human primary T cells were suspended at 722,222 cells / mL in OpTmizer medium containing 2% CTS immune cell SR and the additives shown in the transduction medium in Table 6, inoculated into a PL120 bag fixed with RetroNectin (trade name) and a retroviral vector containing a CAR gene that recognizes a specific antigen, and incubated at 5% CO. 2 / Cultivate at 37°C for one day.

[0226] 5. Expansion and culture of CAR-T cells derived from human primary T cells in four types of serum-free culture media

[0227] Four types of expansion medium were prepared: an expansion medium that was an OpTmizer medium containing 2% CTS immune cell SR and the additives shown in Expansion Medium 1 in Table 6, an expansion medium that was a medium containing the components shown in Table 7 (hereinafter referred to as BM220720) and the additives shown in Expansion Medium 2 in Table 6, the medium further supplemented with 0.3 mM boric acid (Sigma-Aldrich), and the medium further supplemented with 0.3 mM boric acid and 3 mM taurine (Sigma-Aldrich) (OpTmizer medium containing only the additives in Table 6, and BM220720 medium containing (i) only the additives in Table 6, (ii) the additives in Table 6 + 0.3 mM boric acid, or (iii) the additives in Table 6 + 0.3 mM boric acid and 3 mM taurine). One day after the transduction culture, the cells were collected from the PL120 bag and suspended in each of the expansion medium at 220,000 cells / mL and incubated with G-Rex (registered trademark) 6M (Wilson Wolf) at 5% CO. 2 / 37 ° C. Then, on the seventh day of culture, some cells were recovered from G-Rex (registered trademark) 6M and the cells were counted, and 40 IU of IL-2 was added to each culture medium. On the ninth day of culture, some cells were recovered from G-Rex (registered trademark) 6M and the cells were counted, and they were inoculated into fresh G-Rex (registered trademark) 6M containing the corresponding culture medium at 220,000 cells / mL. Similarly, on the thirteenth and fourteenth days, some cells were recovered from G-Rex (registered trademark) 6M and the cells were counted.

[0228] [Table 7]

[0229]

[0230]

[0231] Experimental Example 5

[0232] Proliferation test to verify the effect of adding boric acid and taurine to CAR-T cells derived from human primary T cells

[0233] To verify the effect of adding boric acid and taurine to serum-free expansion medium on different cell types (primary T cells of human origin), the cell number and viability of CAR-T cells derived from primary T cells of human origin were measured on days 4, 6, 10 and 11 in the expansion culture described in item 5 of Example 5. Figure 5 The cell proliferation fold from day 0 of the expansion culture is shown. The proliferation of human primary T cell-derived CAR-T cells was promoted by adding 0.3 mM boric acid or 0.3 mM and 3 mM taurine to the serum-free medium ( Figure 5 ).

[0234] Example 6

[0235] 1. Preparation of iPS cell-derived NK cells

[0236] The iPS cell line FfI01s04 provided by the Center for iPS Cell Research and Application (CiRA) of Kyoto University was differentiated into NK cells according to known methods (e.g., the method described in WO2021 / 174004). The obtained cells were frozen and stored in liquid nitrogen and used as iPS cell-derived NK cells.

[0237] 2. Recovery culture of iPS cell-derived NK cells

[0238] iPS cell-derived NK cells were cultured at 1.25×10 5The cells were suspended in a recovery medium containing 15% fetal bovine serum (FBS) and the additives shown in Table 8, which was IMDM medium (Thermo Fisher Scientific), seeded in a G-Rex (registered trademark) 6-well plate (Wilson Wolf), and incubated in 5% CO. 2 / cultured in a 37°C incubator for 3 days (culture day 0 to day 3).

[0239] [Table 8]

[0240]

[0241] 3. Activation and culture of iPS cell-derived NK cells

[0242] After recovery, iPS cell-derived NK cells were cultured at 1.84×10 5 The cells were suspended in activation medium containing 15% fetal bovine serum (FBS) and IMDM medium (Thermo Fisher Scientific) supplemented with the additives shown in Table 9, seeded in 6-well plates (TPP), and incubated in 5% CO 2 / Cultivate in a 37°C incubator for 3 days (from the 3rd to the 6th day of cultivation).

[0243] [Table 9]

[0244]

[0245] 4. Expansion and culture of iPS cell-derived NK cells in five types of serum-free culture media

[0246] After 3 days, a total of five types of expansion medium were produced by adding 2 g / L human serum albumin (Albuminer (trade name), CSL Behring) to the BM220720 medium in Table 7, further adding 0.3 mM boric acid (Sigma Aldrich), 0.3 mM boric acid + 1 mM taurine (Sigma Aldrich), and 0.3 mM boric acid + 3 mM taurine to the basal medium to obtain four types of basal medium, and one type of X-VIVO (trademark) 15 medium (Lonza). To these five types of basal medium, the additives shown in Table 9 were added to obtain five types of expansion medium. The cells that had been activated and cultured for three days were incubated at 1.25×10 5 10 cells / mL were suspended in each of five types of expansion medium, re-seeded in G-Rex (registered trademark) 24-well plates (Wilson Wolf), and incubated in 5% CO 2 / Cultivate in a 37°C incubator for two days (the 6th to 8th day of culture).

[0247] Two days later, the culture medium was replaced with five types of basal culture medium supplemented with the additives shown in Table 10, and the cells were incubated at 5% CO. 2 / Cultivate in a 37°C incubator for two days (the 8th to 10th day of culture).

[0248] Thereafter, every 2-3 days, cells were plated at 1.25 × 10 5 The cells / mL were reseeded in the same culture medium on a G-Rex (registered trademark) 24-well plate (Wilson Wolf) and incubated in 5% CO. 2 The cells were cultured in a 37°C incubator until the 17th day.

[0249] [Table 10]

[0250]

[0251] Experimental Example 6

[0252] Proliferation test to verify the effect of boric acid and taurine additives on iPS cell-derived NK cells

[0253] In order to verify the effect of adding boric acid and taurine to the serum-free expansion medium on iPS cell-derived NK cells, the cell number and viability of iPS cell-derived NK cells were measured over time during the expansion culture in Item 4 of Example 6. The proliferation fold and cell viability on day 11 of the expansion culture are shown in Table 11. The proliferation of iPS cell-derived NK cells was promoted by adding 0.3 mM boric acid and 1 mM taurine or 3 mM taurine to the serum-free basal medium (Table 11).

[0254] [Table 11]

[0255]

[0256] [Industrial Applicability]

[0257] According to the present invention, even when a serum-free medium or a low-serum medium is used, a cell proliferation maintenance-promoting effect comparable to that in a serum-containing medium can be obtained. Specifically, for cells affected by stress caused by operations such as cell washing, it is preferred to culture in a serum-free medium or a low-serum medium that does not require a large amount of washing, and the present invention is suitable for culture under such conditions.

[0258] This application is based on patent application No. 2022-102986 filed in Japan (filing date: June 27, 2022), the contents of which are incorporated herein in their entirety.

Claims

1. A method for culturing cells, comprising the step of culturing cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.

2. The method according to claim 1, wherein the final concentration of boric acid or its salt in the culture medium is 0.1 mM to 0.9 mM.

3. The method according to claim 1, wherein the final concentration of boric acid or its salt in the culture medium is 0.3 mM to 0.6 mM. The method according to claim 1 , wherein the serum-free medium or low-serum medium is a serum-free medium.

5. The method of claim 1, wherein the cells are immunocompetent cells.

6. The method of claim 5, wherein the immunocompetent cells are selected from the group consisting of dendritic cells, B cells, T cells and natural killer cells.

7. The method of claim 6, wherein the immunocompetent cells are T cells.

8. The method of claim 1, wherein the cells are derived from pluripotent stem cells.

9. The method according to claim 8, wherein the pluripotent stem cells are iPS cells.

10. The method of claim 1, wherein the culture medium further comprises taurine.

11. A method for proliferating cells, comprising the step of culturing the cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof. The method according to claim 11 , wherein the serum-free medium or the low-serum medium is a serum-free medium.

13. The method of claim 11, wherein the culture medium further comprises taurine.

14. A method for producing a cell population in which the number of desired cells is expanded, comprising the step of culturing the cell population containing the desired cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof. The method according to claim 14 , wherein the serum-free medium or reduced serum medium is a serum-free medium.

16. The method of claim 14, wherein the culture medium further comprises taurine.

17. A medicament comprising a cell population obtained by the method according to claim 14.

18. The medicament according to claim 17, which is used for preventing and / or treating cancer.

19. A culture medium additive, characterized in that The medium additive comprises boric acid or a salt thereof, and the medium is a serum-free medium or a low-serum medium.

20. The additive according to claim 19, wherein the serum-free medium or reduced serum medium is a serum-free medium.

21. The additive according to claim 19, wherein the culture medium further comprises taurine.

22. A medium composition comprising boric acid or a salt thereof, wherein the composition is obtained by adding the medium additive according to claim 19 to a serum-free medium or a low-serum medium.

23. The culture medium composition according to claim 22, wherein the serum-free medium or low-serum medium is a serum-free medium.

24. The culture medium composition according to claim 22, further comprising taurine.

25. A method for preventing and / or treating cancer, comprising administering a cell population obtained by the method according to claim 14 to a subject in need thereof.

26. A cell population obtained by the method according to claim 14, wherein the cell population is used for preventing and / or treating cancer.

27. Use of the cell population obtained by the method according to claim 14 in producing a medicament for preventing and / or treating cancer.

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