Cell culture method

Abstract

The present invention addresses the problem of providing: a method that enables sufficient proliferation of cells, in particular immune competent cells, even in a serum-free medium or a low-serum medium; and a medium composition for use in this method. By incorporating imidazole dipeptide or a salt thereof, taurine or a precursor thereof, or a salt of these, and lysophosphatidic acid or a derivative thereof, a cell proliferation maintenance / promoting effect comparable to that of culture in a serum-containing medium can be obtained even in a serum-free medium or a low-serum medium.

Description

Cell culture method

[0001] The present invention relates to a method for culturing / growing cells in a serum-free or low-serum medium, a medium composition for the method, 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 pharmaceutical comprising the cell population obtained by the method.

[0002] (Background of the Invention) Traditionally, cell culture has been carried out using media containing serum (hereinafter also referred to as "serum-containing media"). For example, fetal bovine serum (FBS) is widely used in cell culture as an additive important for cell growth. However, when cultured cells or their derivatives are used for medical purposes, heterologous components may become a source of infection for blood-borne pathogens or xenoantigens. Furthermore, differences between serum lots may cause variations in culture results. For this reason, in recent years, it has become common to minimize the amount of serum used and to culture cells using chemically defined media, and the development of serum-free and low-serum media has been progressing.

[0003] Recently, a new immunotherapy for cancer patients has been developed using chimeric antigen receptor (CAR) T cells (hereinafter simply referred to as CAR-T cells). This therapy involves genetically modifying the T cell receptors (TCRs) of cytotoxic T cells (CTLs) to allow the CTLs to directly and selectively recognize tumor cells, thereby exerting an antitumor effect. Because CAR-T cell cancer therapy kills cancer cells using a mechanism different from that of conventional anticancer drugs or radiation therapy, it is expected to be effective even against intractable or treatment-resistant cancers. Current CAR-T cell therapies, such as Kymriah (trade name) and Yescarta (trade name), approved in the United States, generally involve autologous CAR-T cell therapy, in which CAR-T cells are produced by ex vivo transfection of T cells collected from the patient with a CAR gene, and then the CAR-T cells are administered to the patient. However, this method requires multiple steps over a long period of time, such as T cell activation / proliferation, viral vector preparation, and gene transfer into T cells, resulting in high production costs due to the costs associated with cell culture and viral vector preparation. Therefore, iPS cell-derived CAR-T cells have been developed to enable allogeneic CAR-T therapy. This method also uses serum-containing media in the process of producing iPS cell-derived CAR-T cells, particularly in the expansion culture process, to achieve sufficient cell proliferation effects. However, from the standpoint of GMP (Good Manufacturing Practice), xeno-free media containing no xenogeneic components are preferred, and serum-free or low-serum media are desirable. Furthermore, considering that iPS cell-derived T cells are particularly susceptible to washing stress compared to primary T cells, there has been a strong demand for serum-free or low-serum media that do not require extensive washing.

[0004] There have been many reports on methods for maintaining or promoting desired cell proliferation effects in cell culture. Non-Patent Document 1 describes that lysophosphatidylcholine (LPC) and α-tocopherol are useful for mouse spermatogenesis in a synthetic medium containing no albumin. Non-Patent Document 2 describes that human peripheral blood-derived CD4 ＋It has been reported that carnosine is effective in extending cell survival in in vitro culture of T cells. Non-Patent Document 3 describes that LPC exhibits a cell proliferation effect in CHO cell culture in a protein-free medium. Non-Patent Document 4 describes that α-tocopherol, together with selenium, is involved in protecting cells against oxidative stress. Patent Document 1 describes that applying carnosine to cells can increase the Hayflick limit of cells during culture. Patent Document 2 describes that serum-free media containing taurine, carnosine, and vitamin E (α-tocopherol) can be used to improve the metabolic maturity state of cells. Non-Patent Document 5 describes that the addition of lysophosphatidic acid (LPA) is effective in promoting CHO cell proliferation in serum-free media. Furthermore, there is a report on the cell proliferation effect of boric acid via its transporter, NaBC1 (Non-Patent Document 6). Patent Document 3 describes a serum-free medium containing borate suitable for culturing diploid cells. Non-Patent Document 7 describes that Taut, a taurine transporter, is important for the recall response of T cells.

[0005] European Patent No. 0571390 Patent Publication No. 2014-521337 Chinese Patent No. 105462912

[0006] Sanjo, Hiroyuki et al., FASEB J. 2020 Jul;34(7):9480-9497.Hyland, P et al., Mech Ageing Dev. 2000 Dec 20;121(1-3):203-15.In-Kyoung Kim et al., Biotechnol Bioprocess Eng. 2008;13:396-400.Saito, Yoshiro et al., J Biol Chem. 2003 Oct 10;278(41):39428-34.Miki, Hideo et al., Cytotechnology. 2015 Aug;67(4):689-97.Park, Meeyoung et al., Mol Cell. 2004 Nov 5;16(3):331-41.Kaesler, Susanne et al., Eur. J. Immunol. 2012 42:831-841.

[0007] The present invention addresses the following objectives: to provide a method for culturing and sufficiently growing cells, particularly immunocompetent cells, in a serum-free or low-serum medium, as well as a medium additive and medium composition for use in the method. Furthermore, the present invention addresses the following objectives: to provide a method for producing a cell population in which the number of desired cells, particularly immunocompetent cells, is expanded in a serum-free or low-serum medium, and a pharmaceutical comprising the cell population obtained by the method.

[0008] In view of the above-mentioned problems, the present inventors prepared various factors to be added to serum-free or low-serum medium and investigated their effects on cell culture or proliferation. As a result, it was confirmed that the addition of imidazole dipeptide or a salt thereof (e.g., carnosine), taurine or a precursor thereof or a salt thereof (e.g., taurine), and lysophosphatidic acid or a derivative thereof (e.g., lysophosphatidylcholine (LPC)) maintained and promoted cell proliferation comparable to that achieved in serum-containing medium, even when serum-free or low-serum medium was used. Furthermore, it was found that vitamin E (e.g., α-tocopherol) and / or boric acid could be further added, if desired. Based on these findings, more suitable culture conditions were established, leading to the completion of the present invention. Specifically, the present invention is as follows: [1] A method for culturing cells, comprising the step of culturing cells in a serum-free or low-serum medium containing (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof. [2] The method of [1], comprising the step of culturing cells in a serum-free or low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof. [3] The method of [1] or [2], wherein the imidazole dipeptide or a salt thereof is carnosine or anserine, or a salt thereof. [4] The method of any of [1] to [3], wherein the imidazole dipeptide or a salt thereof is carnosine or a salt thereof. [5] The method of any of [2] to [4], wherein the medium further contains vitamin E. [6] The method of [1], comprising a step of culturing cells in a serum-free medium or a low-serum medium containing (ib) vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof. [7] The method of any of [1] to [6], wherein the lysophosphatidic acid or a derivative thereof is lysophosphatidylcholine (LPC). [8] The method of any of [1] to [7], wherein the medium further contains boric acid or a salt thereof. [9] The method of any of [1] to [8], wherein the serum-free medium or low-serum medium is a serum-free medium.

[10] The method of any one of [1] to [9], wherein the cell is an immunocompetent cell.

[11] The method of

[10] , wherein the immunocompetent cell is selected from dendritic cells, B cells, T cells, and natural killer cells.

[12] The method of

[10] or

[11] , wherein the immunocompetent cell is a T cell.

[13] The method of any one of [1] to

[12] , wherein the cell is derived from a pluripotent stem cell.

[14] The method of

[13] , wherein the pluripotent stem cell is an iPS cell. [14a] The method of any one of [1] to

[14] , wherein the cell is a cell into which a chimeric antigen receptor (CAR) has been introduced. [14b] The method of [14a], wherein the cell is a T cell into which a chimeric antigen receptor has been introduced (CAR-T cell) or a natural killer cell into which a chimeric antigen receptor has been introduced (CAR-NK cell).

[15] A method for growing cells, comprising the step of culturing cells in a serum-free or low-serum medium containing (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[16] The method of

[15] , comprising the step of culturing cells in a serum-free or low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[17] The method of

[15] or

[16] , wherein the imidazole dipeptide or a salt thereof is carnosine or anserine, or a salt thereof.

[18] The method of any of

[15] to

[17] , wherein the imidazole dipeptide or a salt thereof is carnosine or a salt thereof.

[19] The method of any of

[16] to

[18] , wherein the medium further contains vitamin E.

[20] The method of

[15] , comprising a step of culturing cells in a serum-free medium or a low-serum medium containing (ib) vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[21] The method of any of

[15] to

[20] , wherein the lysophosphatidic acid or a derivative thereof is lysophosphatidylcholine (LPC).

[22] The method of any of

[15] to

[21] , wherein the medium further contains boric acid or a salt thereof.

[23] The method according to any one of

[15] to

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

[24] A method for producing a cell population in which the number of desired cells has been expanded, comprising a step of culturing a cell population containing desired cells in a serum-free or low-serum medium containing (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[25] The method according to

[24] , wherein the number of desired cells has been expanded, comprising a step of culturing a cell population containing desired cells in a serum-free or low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[26] The method according to

[24] or

[25] , wherein the imidazole dipeptide or a salt thereof is carnosine or anserine, or a salt thereof.

[27] The method according to any one of

[24] to

[26] , wherein the imidazole dipeptide or a salt thereof is carnosine or a salt thereof.

[28] The method according to any one of

[25] to

[27] , wherein the medium further contains vitamin E.

[29] The method according to

[24] , wherein the culture medium is a method for producing a cell population in which the number of desired cells is expanded, comprising culturing a cell population containing desired cells in a serum-free or low-serum medium containing (ib) vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[30] The method according to any one of

[24] to

[29] , wherein the lysophosphatidic acid or a derivative thereof is lysophosphatidylcholine (LPC).

[31] The method according to any one of

[24] to

[30] , wherein the medium further contains boric acid or a salt thereof.

[32] The method according to any one of

[24] to

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

[33] A serum-free or low-serum medium composition containing (ia) imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[34] The composition according to

[33] , containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[35] The composition according to

[33] , containing (ib) vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[36] A medium additive comprising (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, wherein the medium is a serum-free medium or a low-serum medium.

[37] A medium additive containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, wherein the medium is a serum-free medium or a low-serum medium.

[38] The agent according to

[36] or

[37] , wherein the imidazole dipeptide or a salt thereof is carnosine or anserine, or a salt thereof.

[39] The agent according to any of

[36] to

[38] , wherein the imidazole dipeptide or a salt thereof is carnosine or a salt thereof.

[40] The agent according to any of

[37] to

[39] , wherein the medium further contains vitamin E.

[41] A medium additive containing (ib) vitamin E, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, wherein the medium is a serum-free medium or a low-serum medium.

[42] The agent according to any one of

[36] to

[41] , wherein lysophosphatidic acid or a derivative thereof is lysophosphatidylcholine (LPC).

[43] The agent according to any one of

[36] to

[42] , wherein the medium further contains boric acid or a salt thereof.

[44] The agent according to any one of

[36] to

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

[45] A pharmaceutical comprising a cell population obtained by the method according to any one of

[24] to

[32] .

[46] The pharmaceutical according to

[45] , which is for use in the prevention and / or treatment of cancer.

[47] A method for preventing and / or treating cancer, comprising administering to a subject in need thereof a cell population obtained by the method of any of

[24] to

[32] .

[48] A cell population obtained by the method of any of

[24] to

[32] for use in the prevention and / or treatment of cancer.

[49] Use of a cell population obtained by the method of any of

[24] to

[32] in the manufacture of a medicament for preventing and / or treating cancer.

[0009] According to the present invention, even when a serum-free or low-serum medium is used, it is possible to obtain cell growth maintenance and promotion effects comparable to those obtained by culturing in a serum-containing medium. In particular, for cells that are sensitive to stress caused by operations such as cell washing, it is preferable to culture them in a serum-free or low-serum medium that does not require extensive washing, and they are suitable for culture under such conditions.

[0010] (Detailed Description of the Invention) The present invention will be described below. Terms used in this specification have the meanings commonly used in the art unless otherwise specified. (1) Cell Culture Method (hereinafter also referred to as "Culture Method of the Present Invention") and Cell Proliferation Method (hereinafter also referred to as "Proliferation Method of the Present Invention") The culture method or proliferation method of the present invention comprises the step of culturing cells in a serum-free or low-serum medium containing (i) imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof. If desired, the serum-free or low-serum medium may further contain (iv) vitamin E and / or (v) boric acid or a salt thereof. If desired, the culture method or proliferation method of the present invention may use vitamin E in addition to or instead of (i) imidazole dipeptide or a salt thereof. In this case, component (iv) is not necessary. Therefore, the culture method or proliferation method of the present invention may be a method comprising the step of culturing cells in a serum-free medium or a low-serum medium containing (ia) imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[0011] 1-1. Cells The cell types to be cultured using the culture method and proliferation method of the present invention are not particularly limited. Examples of such cell types include germ cells such as sperm and eggs, somatic cells that constitute an organism, stem cells (e.g., pluripotent stem cells) and cells induced to differentiate from stem cells, progenitor cells, cancer cells isolated from an organism, cells isolated from an organism that have acquired immortalization and are stably maintained ex vivo (cell lines), cells isolated from an organism that have been artificially genetically modified, and cells isolated from an organism that have undergone artificial nucleus exchange. Examples of cells induced to differentiate from somatic cells and stem cells that constitute a living organism 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 beta cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and mononuclear cells. 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.

[0012] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple 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.

[0013] "Pluripotent stem cells" refer to cells that have the ability to self-replicate and differentiate / proliferate, and have the ability to differentiate into all tissues and cells that make up 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 stress or cell stimulation. Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred 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 preferred as pluripotent stem cells. Identification of iPS cells can be performed using undifferentiated markers, which are due to the undifferentiated nature of iPS cells. Examples of undifferentiation markers include alkaline phosphatase, Oct3 / 4, Sox2, Nanog, ERas, Esgl, etc. Methods for detecting these undifferentiation markers include methods for detecting mRNA (using primers or probes), immunological detection methods (using antibodies or labels), etc.

[0014] A cell induced to differentiate from a stem cell (differentiation-induced cell) is any cell that has been subjected to a differentiation-inducing treatment to differentiate from a stem cell into a specific type of cell.

[0015] A cell line is a cell that has acquired the ability to proliferate indefinitely through artificial manipulation outside of a living body, and examples thereof include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human embryonic kidney cells), 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, and the like.

[0016] In a preferred embodiment, the cells to be cultured are immunocompetent cells. Immunocompetent cells refer to cells involved 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, the immunocompetent cells are T cells.

[0017] T cells include CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, αβ-T cells, γδ-T cells, regulatory T cells, NKT cells, etc. T cells may be subsets such as naive T cells, effector T cells, or memory T cells.

[0018] The immunocompetent cells may be cells isolated from humans (primary cells) or cells obtained by differentiation from stem cells such as pluripotent stem cells (e.g., iPS cells, ES cells), hematopoietic stem cells, or mesenchymal stem cells, and are preferably primary cells or cells obtained by differentiation from pluripotent stem cells (particularly iPS cells). Primary cells are preferably human-derived primary T cells. Stem cells can be induced to differentiate into immunocompetent cells by methods known in the art, depending on the stem cells used and the type of immunocompetent cells of interest. As an example, iPS cells can be induced to differentiate into T cells and NK cells by the methods described in the Examples.

[0019] Furthermore, the target cells may be either autologous cells or allogeneic cells. In the present invention, "autologous cells" refers to cells obtained from a subject receiving a cell population (described below) produced by the culture method or proliferation method of the present invention, or cells induced from the obtained cells, and "allogeneic cells" refers to cells that are not the above-mentioned "autologous cells."

[0020] In the present invention, the target cells may be artificially genetically modified cells, examples of which include immunocompetent cells into which a CAR gene has been introduced (T cells into which a CAR gene has been introduced or natural killer cells into which a CAR gene has been introduced), T cells into which an exogenous T cell receptor (TCR) has been introduced, and immunocompetent cells into which a gene for expressing a cytokine and / or a chemokine has been introduced.

[0021] A CAR is a structure comprising, from the N-terminus to the C-terminus of a protein, a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain for the effector function of immune cells, and a CAR gene is a gene encoding this receptor. The extracellular domain comprises an antigen recognition site that exhibits specific binding to the target. The transmembrane domain is located between the extracellular domain and the intracellular signaling domain. The intracellular signaling domain transmits a signal required for the immune cell to exert its effector function. That is, an intracellular signaling domain is used that can transmit a signal required for activating immune cells when the extracellular domain binds to a target antigen. There have been several reports of experiments and clinical studies 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), the CAR of the present invention can be constructed with reference to these reports.

[0022] Preferred examples of cells to which the culture method and proliferation method of the present invention can be applied include T cells into which a CAR gene has been introduced (CAR-T cells) and NK cells into which a CAR gene has been introduced (CAR-NK cells), more preferably CAR-T cells and CAR-NK cells derived from pluripotent stem cells, even more preferably CAR-T cells and CAR-NK cells derived from iPS cells, and even more preferably CAR-T cells derived from iPS cells. CAR-T cells and CAR-NK cells can be obtained by introducing a CAR gene into T cells and NK cells, respectively, or their precursor cells, such as pluripotent stem cells. For example, CAR-T cells may be cells obtained by introducing a CAR gene into cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, or mesenchymal stem cells into T cells, or cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, or mesenchymal stem cells into T cells. Preferably, the CAR-T cells are CAR-T cells derived from iPS cells obtained by differentiating iPS cells introduced with a CAR gene into T cells, and CAR-T cells derived from iPS cells obtained by introducing a CAR gene into T cells obtained by differentiating iPS cells; these may be collectively referred to as "iPS cell-derived CAR-T cells." More preferably, the CAR-T cells are CAR-T cells derived from iPS cells obtained by introducing a CAR gene into T cells obtained by differentiating iPS cells. For example, CAR-NK cells may be cells obtained by introducing a CAR gene into cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, or mesenchymal stem cells into NK cells, or cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, or mesenchymal stem cells introduced with a CAR gene into NK cells. Preferably, the CAR-NK cells are iPS cell-derived CAR-NK cells obtained by differentiating iPS cells introduced with a CAR gene into NK cells, and iPS cell-derived CAR-NK cells obtained by introducing a CAR gene into NK cells obtained by differentiating iPS cells, and these may be collectively referred to as "iPS cell-derived CAR-NK cells." More preferably, the CAR-NK cells are iPS cell-derived CAR-NK cells obtained by introducing a CAR gene into NK cells obtained by differentiating iPS cells.The differentiation of iPS cells (which may optionally be introduced with a CAR gene) into T cells, or the differentiation of iPS cells (which may optionally be introduced with a CAR gene) into NK cells can be induced using a known method, specifically, by the method described in the Examples.

[0023] The CAR gene is usually introduced into cells using a CAR expression vector. A CAR expression vector refers to a nucleic acid molecule capable of transporting a nucleic acid molecule encoding a CAR gene into cells. Various types of vectors can be used, regardless of whether they are DNA or RNA, and are not particularly limited in terms of form or origin. The vector can be a viral vector or a non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and vaccinia viral vectors. Among these, retroviral vectors, lentiviral vectors, and adeno-associated viral vectors integrate the target gene into the host chromosome, allowing for stable and long-term expression. Each viral vector can be prepared according to standard methods or using a commercially available dedicated kit. 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, artificial chromosome vectors, etc. As an example, the CAR gene can be introduced into cells by the method described in the Examples.

[0024] 1-2. Culture Medium / Culture Composition The culture medium used in the culture method and proliferation method of the present invention is a serum-free or low-serum medium containing (i) imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, and optionally further containing (iv) vitamin E and / or (v) boric acid or a salt thereof (hereinafter, these may be collectively referred to as "the culture medium of the present invention"). If desired, the culture medium of the present invention may contain vitamin E in addition to or instead of (i) imidazole dipeptide or a salt thereof. When vitamin E is used, the further inclusion of component (iv) is unnecessary. Therefore, the culture medium used in the culture method and proliferation method of the present invention may be a serum-free or low-serum medium containing (ia) imidazole dipeptide or a salt thereof or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof. The present invention can provide a serum-free or low-serum medium composition comprising a basal medium (described below) to which the above components (ia) to (iii) are added. The present invention can provide a serum-free or low-serum medium composition comprising a basal medium (described below) to which the above components (i) to (iii) are added, and optionally, further components (iv) and / or (v).

[0025] (i) Imidazole dipeptide or its salt (hereinafter also collectively referred to as imidazole dipeptides) Imidazole dipeptides are a type of peptide in which two amino acids are bonded via an imidazole ring. Examples of imidazole dipeptides include carnosine, which is composed of β-alanine and L-histidine, anserine, which is composed of β-alanine and 1-methyl-L-histidine, balenine, which is composed of valine and L-histidine, and homocarnosine, which has a structure similar to carnosine. Other examples include dipeptides composed of alanine and L-histidine, dipeptides composed of glycine and L-histidine, and dipeptides composed of lysine and L-histidine. Carnosine or anserine, or a salt thereof, is preferred. Salts of imidazole dipeptides are not particularly limited as long as they are pharmaceutically acceptable, and include inorganic acid salts such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate; and organic acid salts such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, salicylate, oxalate, stearate, ascorbate, malate, adipate, and gluconate. For example, salts of anserine include hydrochloride, nitrate, and acetate, and salts of carnosine include hydrochloride, nitrate, and acetate. In the present invention, preferred imidazole dipeptides are carnosine, anserine, and salts thereof, and more preferably carnosine or a salt thereof. Imidazole dipeptides are contained in the meat of seafood, livestock, poultry, whale, and the like, and can be prepared from these as raw materials according to conventionally known extraction and purification methods. Alternatively, commercially available imidazole dipeptides can be used for convenience. In the present invention, the origin of the imidazole dipeptide used does not matter, and the imidazole dipeptide and / or salt thereof may be used alone or in combination of two or more.The concentration of imidazole dipeptides in the culture medium is not particularly limited as long as the desired effect is obtained, and may be increased or decreased as appropriate depending on the type of imidazole dipeptide or salt thereof used. However, it is usually 1 to 50 mM, preferably 3 to 40 mM, more preferably 5 to 30 mM, and particularly preferably 10 to 25 mM. When two or more types are used in combination, the concentration refers to the total concentration. In this specification, the concentration of imidazole dipeptide or its salt is the concentration converted into carnosine, a representative imidazole dipeptide, unless otherwise specified. If the concentration is too high, it will affect cell viability, and if it is too low, the cell proliferation-promoting effect will be weak.

[0026] (ii) Taurine or its precursor or a salt thereof (hereinafter collectively referred to as taurines). Taurine is an organic sulfur compound and a type of non-essential amino acid synthesized from cysteine ​​or methionine. Taurine precursors are substances that are directly or indirectly converted to taurine and are not particularly limited as long as the desired effect is obtained when cells are cultured in their presence. For example, taurine precursors can include one or more selected from the group consisting of cysteine ​​sulfinic acid, cysteamine, cysteic acid, and hypotaurine. Salts of taurine or its precursors are not particularly limited as long as they are pharmaceutically acceptable, and include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; aluminum salt; and salts with organic amines such as triethylamine, triethanolamine, morpholine, piperazine, and pyrrolidine. In the present invention, preferred taurines are taurine, hypotaurine, and their salts, and more preferably taurine or its salt. Taurines are found in seafood and meat from livestock, and can be obtained using these raw materials in accordance with conventional extraction and purification methods, such as livestock-derived taurine and marine-derived taurine. Alternatively, commercially available synthetic taurine of reagent / pharmaceutical grade can be used for convenience. The concentration of taurines in the medium is not particularly limited as long as the desired effect is obtained, and can be adjusted depending on the type of taurine used. However, the concentration is typically 0.1 to 20 mM, preferably 0.5 to 15 mM, more preferably 1 to 10 mM, and particularly preferably 2 to 5 mM. Unless otherwise specified, the concentration of taurines herein is expressed in terms of taurine. A too high concentration can affect cell viability, while a too low concentration can weaken the cell proliferation-promoting effect.

[0027] (iii) Lysophosphatidic acid or its derivatives (hereinafter collectively referred to as lysophosphatidic acids) Lysophosphatidic acid (LPA) is a phospholipid derivative that has a structure in which the acyl group has been removed from phosphatidic acid and can transmit various signals into cells. LPA is known to exhibit a variety of pharmacological actions via several G protein-coupled receptors on the cell surface. There are currently eight LPA receptor subtypes, each with a different role, called LPA1, LPA2, LPA3, LPA4, LPA5, LPA6, GPR87, and GPR35. Derivatives of lysophosphatidic acid include lysophosphatidylserine (LPS), a serine-based lysophosphatidic acid; lysophosphatidylethanolamine (LPE), an ethanolamine-based lysophosphatidic acid; lysophosphatidylcholine (LPC), a choline-based lysophosphatidic acid; and lysophosphatidylinositol, an inositol-based lysophosphatidic acid. In the present invention, a preferred lysophosphatidic acid is lysophosphatidylcholine. Lysophosphatidic acid or its derivatives may exist as their salts. The salts are not particularly limited as long as they are pharmaceutically acceptable, and 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. There is no particular limitation on the method for producing lysophosphatidic acids, and those produced by known methods can be used. A specific production method involves enzymatically lyso-converting lecithin containing phosphatidic acid. Alternatively, commercially available lysophosphatidic acids can be used for convenience.The concentration of lysophosphatidic acids in the medium is not particularly limited as long as the desired effect is obtained, and may be increased or decreased as appropriate depending on the type of compound used, but is typically 0.1 to 50 mg / L, preferably 0.5 to 25 mg / L, more preferably 1 to 15 mg / L, and particularly preferably 2 to 10 mg / L. In this specification, the concentration of lysophosphatidic acid or a derivative thereof is the concentration converted into lysophosphatidylcholine, unless otherwise specified. If the concentration is too high, it will affect cell viability, and if it is too low, the cell proliferation-promoting effect will be weak.

[0028] (iv) Vitamin E Vitamin E is a fat-soluble antioxidant vitamin, and is mainly classified into two major groups: tocopherols and tocotrienols. These compounds are characterized by having a phenol ring and a long fatty chain bonded to it. Tocopherols have four isomers (α, β, γ, δ) that differ depending on the position of the methyl group, and tocotrienols also have similar isomers. These may be used alone or in combination of two or more. In the present invention, vitamin E is preferably tocopherol or a salt thereof, and more preferably α-tocopherol or a salt thereof. Vitamin E may be in the form of a salt. There are no particular limitations on the salt of vitamin E, as long as it is pharmaceutically acceptable. Examples of vitamin E include salts with organic bases (e.g., salts with organic amines such as methylamine, triethylamine, triethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, and picoline), and salts with inorganic bases (e.g., ammonium salts; alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and metal salts such as aluminum salts). Vitamin E may also be in the form of a derivative. Examples of vitamin E derivatives include vitamin E acetate (tocopherol acetate), vitamin E nicotinate, vitamin E succinate, and vitamin E linoleate. Commercially available vitamin E (including salts and derivatives) can be used as appropriate. The concentration of vitamin E in the medium is not particularly limited as long as the desired effect is obtained, and may be increased or decreased depending on the type of compound used. However, the concentration is typically 10 to 1,000 μM, preferably 20 to 500 μM, more preferably 40 to 300 μM, and particularly preferably 100 to 250 μM. In this specification, unless otherwise specified, the concentration of vitamin E is the concentration converted to α-tocopherol. If the concentration is too high, it will affect cell viability, and if it is too low, the effect of promoting cell proliferation will be weak.

[0029] (v) Boric Acid or Salts Thereof In this specification, boric acid is not particularly limited as long as it is pharmaceutically acceptable, but examples include orthoboric acid, metaboric acid, tetraboric acid, etc. Among these boric acids, orthoboric acid is preferably used. Salts of boric acid are not particularly limited as long as they are pharmaceutically acceptable, but examples 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. Boric acid or its salts may also be in the form of a hydrate, such as borax. Commercially available boric acid (including salts) can be used as appropriate. The concentration of boric acid or its salts in the medium is not particularly limited as long as the desired effect is obtained, but is typically 0.1 to 0.9 mM, preferably 0.2 to 0.8 mM, more preferably 0.2 to 0.7 mM, and particularly preferably 0.3 to 0.6 mM. In this specification, the concentration of boric acid or a salt thereof is the concentration converted to boric acid unless otherwise specified. If the concentration is too high, it will affect cell viability, and if it is too low, the effect of promoting cell proliferation will be weak.

[0030] As used herein, "serum-free medium" refers to a medium that is substantially free of, and preferably free of, unconditioned or unpurified serum, and includes media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors). As the medium, a medium typically used for culturing animal cells (hereinafter also referred to as basal medium for convenience) or a medium for perfusion culture is used. Here, "substantially free" means that the medium is completely free of, or if present, is below the detection limit. Examples of basal media include, but are not limited to, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved 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 mixtures thereof (e.g., Advanced DMEM / F12 medium, etc.). Perfusion culture is a culture method in which a medium is continuously supplied to a cell-containing culture system while an equal amount of cell-free culture supernatant is continuously removed from the culture system, thereby maintaining the culture system in a steady state. Because perfusion culture requires culture at a higher cell density than conventional culture methods, media for perfusion culture generally have a higher concentration of nutrients than basal media. These media are commercially available from Invitrogen, SIGMA, Fujifilm Wako Pure Chemical Industries, Sumitomo Pharma, and other companies. Media with the same name or trade name have equivalent composition regardless of manufacturer. As used herein, "low-serum medium" refers to a medium in which serum is added to a basal medium, characterized by a reduced serum concentration compared to commonly used serum-containing media (serum concentration: 5-20% (herein, serum concentration is expressed as v / v%)). The serum concentration of a low-serum medium can be less than 5%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, or less than 0.001%, but is usually 0.1% or greater, 1% or greater. Specifically, the serum concentration is preferably 0.1% or greater but less than 5%, more preferably 1% or greater but less than 4%.

[0031] The medium used in the present culture and expansion methods may also contain a serum substitute. Examples of serum substitutes include albumin (e.g., lipid-rich albumin), transferrin, fatty acids, collagen precursors, insulin, trace elements (e.g., zinc, selenium, etc.), ITS supplement (a mixture of insulin, transferrin, and selenite), B-27 supplement, N2 supplement, knockout serum replacement, 2-mercaptoethanol, or 3'-thiolglycerol, or equivalents thereof. These serum substitutes are also commercially available. Knockout serum replacement is available from Invitrogen. Other serum substitutes are available from Invitrogen, SIGMA, Fujifilm Wako Pure Chemical Industries, Ltd., Sumitomo Pharma, etc. Reagents or additives with the same name or trade name have similar compositions regardless of manufacturer.

[0032] The medium of the present invention may further contain components favorable for cell growth in addition to the basal medium. Examples of such components include sugars 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, betaine, carnitine, creatine, and pantothenic acid; proteins such as albumin and transferrin; peptides such as glycylglycylglycine and soybean peptide; serum; choline, B vitamins (thiamine, riboflavin, pyridoxine, cyanocobalamin, biotin, folic acid, pantothenic acid, nicotinamide, etc.), vitamin C (ascorbic acid, niacin ... vitamins such as rubidium acid), vitamin C derivatives; fatty acids such as oleic acid, arachidonic acid, linoleic acid; lipids such as cholesterol; nucleosides such as adenosine, thymidine, guanosine, cytidine, uridine, 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; N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffering agents 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, and streptomycin;Examples of such components include cell adhesion factors and extracellular matrix components such as Type I collagen, Type II 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, and appropriate components can be selected and used at appropriate concentrations depending on the type of cells to be cultured.

[0033] The medium may further contain supplemental factors to support cell survival and proliferation. Examples of supplemental factors include type 1 cytokine family members, type 2 cytokine family members, TNF superfamily cytokines, IL-1 family cytokines, and other cytokines (such as TNF-β), specifically IL-1 to IL-41, and preferably IL-1, IL-2, IL-7, IL-15, IL-18, and IL-21. Supplemental factors can be prepared according to standard methods, or commercially available products can be used. Supplemental factors may be derived from animals other than humans, but are preferably derived from humans (which may be recombinant).

[0034] As used herein, "xeno-free" refers to conditions under which components derived from organisms different from the organism species of the cells to be cultured are excluded.

[0035] 1-3. Cell Culture and Cell Proliferation The present invention also provides a method for culturing cells (the culture method of the present invention) and a method for proliferating cells (the proliferation method of the present invention), characterized by culturing cells in the medium of the present invention (described above). The culture vessel used for cell culture is not particularly limited as long as it is capable of culturing cells, and examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, Petri dishes, tubes, trays, culture bags, roller bottles, and bioreactors. The type of vessel is appropriately selected depending on the target cells.

[0036] The culture vessel may be cell-adhesive or cell-non-adhesive, and is selected appropriately depending on the purpose. A cell-adhesive culture vessel may be coated with any cell-supporting substrate such as an extracellular matrix (ECM) for the purpose of improving adhesion of cells to the surface of the culture vessel. The cell-supporting substrate may be any substance intended for cell adhesion.

[0037] Other culture conditions can be set appropriately. For example, the culture temperature is not particularly limited, but can be about 30 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 about 1 to 21%.

[0038] The frequency of medium exchange and culture period in cell culture are generally determined by comprehensively considering various conditions, such as cell density, culture method (adherent culture / suspension culture), cell type to be cultured, medium composition, culture conditions (temperature, gas concentration), amount of medium to be exchanged (total amount / partial amount), cost of medium, and lifestyle of the operator. Medium exchange is usually performed once every 2 to 3 days, once a day, or multiple times a day (e.g., twice a day). Medium exchange can also be performed at such frequencies in the culture method and proliferation method of the present invention. Continuous medium exchange can also be performed by perfusion culture. The culture period is usually about 2 days to 4 weeks, preferably about 3 days to 3 weeks, and more preferably about 3 days to 2 weeks.

[0039] As used herein, "cell proliferation" refers to the phenomenon in which cells increase in number through repeated cell division. This phenomenon includes the process in which cells go through the cell cycle to form new cells, and is achieved by cell division in an appropriate culture environment. Indicators of proliferation include an increase in cell number, an increase in DNA content, or the expression of specific proliferation-related markers. Specifically, this refers to an increase in cell number compared to a control, such as before proliferation (i.e., before the start of culture), 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 the control. In certain embodiments of the invention, the increase is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% increase compared to the number of cells in a control.

[0040] In one embodiment of the present invention, the cells cultured in a serum-free or low-serum medium containing (i) imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, and optionally further containing (iv) vitamin E and / or (v) boric acid or a salt thereof (i.e., the medium of the present invention) 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 even more preferably T cells. In another embodiment of the present invention, the cells cultured in the medium of the present invention are primary T cells and T cells derived from pluripotent stem cells (particularly iPS cells), more preferably CAR-T cells derived from primary T cells and pluripotent stem cells (particularly iPS cells), and even more preferably CAR-T cells derived from pluripotent stem cells (particularly iPS cells). In yet 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 (particularly iPS cells), more preferably CAR-NK cells derived from primary NK cells and pluripotent stem cells (particularly iPS cells), and even more preferably CAR-NK cells derived from pluripotent stem cells (particularly iPS cells). In one embodiment of the present invention, the culture comprises an activation step and an expansion step. When cryopreserved cells are used, the culture may comprise a recovery step. In the present invention, when the cells to be cultured are cryopreserved cells, the culture of the cells is a concept that includes any one, two, or all three of recovery culture comprising a recovery step, activation culture comprising an activation step, and expansion culture comprising an expansion step. Preferably, the culture is expansion culture. Another embodiment of the present invention is a culture of CAR-T cells and CAR-NK cells, preferably pluripotent stem cell (particularly iPS cell)-derived CAR-T cells and pluripotent stem cell (particularly iPS cell)-derived CAR-NK cells, which comprises an activation step and an expansion step, and optionally comprises a recovery step (when cryopreserved cells are used, for example).

[0041] (Recovery step / recovery culture) This step is carried out after thawing cryopreserved cells. Since prolonged exposure of cells to a cryoprotectant can cause damage, the cryoprotectant is quickly removed from the cells after thawing. Then, cell damage and dysfunction caused by freezing and thawing are recovered. The method of this step / culture varies depending on the target cells, the cryoprotectant used, the thawing method, etc., but most cells usually recover to normal by culturing them in a medium not containing a cryoprotectant for several days, preferably 1 to 5 days, and more preferably about 3 days.

[0042] (Activation Step / Activation Culture) The method for this step is not particularly limited as long as the desired effect on the cells is obtained, and can be carried out, for example, by contact with a stimulating substance. Typically, the cells are cultured in the presence of a stimulating substance, specifically in a medium containing the stimulating substance, for several days, preferably 1 to 5 days, and more preferably about 3 days. The stimulating substance is a signal molecule that controls cellular 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 one that is added exogenously. Specifically, the step is carried out by contact with a substrate to which a ligand is bound, or by culture in a medium containing the ligand, or the like.

[0043] For example, the ligand used in the activation culture of CAR-T cells, preferably CAR-T cells derived from pluripotent stem cells (particularly iPS cells), is not particularly limited as long as it is a molecule that interacts with a surface molecule of CAR-T cells and promotes their activation. Examples of such a ligand include CD3, which conjugates with TCR and is responsible for TCR-mediated signal transduction, and molecules that specifically bind to surface molecules known as costimulators for T cell activation, such as CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, and CD40L, and thus function to transmit an activation signal into T cells. Such molecules may be physiological ligands (or receptors) for 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.

[0044] More preferably, the T cell activation ligand 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')), as long as it has the ability to specifically bind to CD3 expressed on target T cells that induce activation, stimulate the surface molecules of these T cells, and transmit a signal into the T cells. ２ , Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc.).

[0045] For example, the molecule used in the activation culture of CAR-NK cells, preferably CAR-NK cells derived from pluripotent stem cells (particularly iPS cells), is not particularly limited as long as it is a molecule that interacts with a surface molecule of CAR-NK cells and promotes their activation. For example, it may be a physiological ligand (or receptor) for the above-mentioned NK cell surface molecule, or a non-physiological ligand (or receptor) having agonist activity. It may also be a cytokine. A preferred example of the 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.) Cytokines include IFN-α / β, IFN-γ, IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, etc.

[0046] (Expansion Step / Expansion Culture) This step is a step of expanding the cells activated as described above. The method of expansion culture is not particularly limited as long as the desired effect on the activated cells is obtained, and those skilled in the art can adjust the method appropriately while monitoring the cell number, etc. When the goal is to increase the number of cells, the cells can be cultured in a medium containing the above-mentioned additives, for example, for 2 days or more, preferably 3 days or more, more preferably 4 days or more, even more preferably 5 days or more, and even more preferably 6 days or more. The culture can also be adjusted appropriately, for example, for 7 days or more, 9 days or more, or 11 days or more, while monitoring the cell number, etc. Furthermore, by alternately repeating activation culture and expansion culture, the culture can be continued and the cell number can be exponentially increased. The upper limit of the culture period is not particularly limited, and is, for example, 28 days or less, preferably 21 days or less. For example, in the case of activated CAR-T cells, preferably CAR-T cells derived from activated pluripotent stem cells (particularly iPS cells), expansion culture can be performed in a medium containing cytokines such as IL-7, IL-15, and IL-2, for 2 days or more, preferably 3 days or more. When the target cells are primary T cells (particularly CAR-T cells) or NK cells (preferably pluripotent stem cell (particularly iPS cell-derived)-derived NK cells (particularly CAR-NK cells)), and the purpose is to increase the number of cells, the cells can be expanded in a medium containing cytokines such as IL-7, IL-15, IL-21, and IL-2 for, for example, 2 days or more, preferably 4 days or more, more preferably 6 days or more, even more preferably 8 days or more, still more preferably 10 days or more, and even more preferably 11 days or more.

[0047] (2) Method for producing a cell population (hereinafter also referred to as "the production method of the present invention") and a pharmaceutical comprising the cell population (hereinafter also referred to as "the pharmaceutical of the present invention"). The production method of the present invention is a method for producing a cell population in which the number of desired cells has been expanded, and the method comprises the step of culturing a cell population comprising the desired cells in a serum-free or low-serum medium containing (i) imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, and optionally further containing (iv) vitamin E and / or (v) boric acid or a salt thereof. Therefore, the production method may be a method comprising the step of culturing a cell population comprising the desired cells in a serum-free or low-serum medium containing (ia) imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

[0048] The "serum-free or low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, and optionally further containing (iv) vitamin E and / or (v) boric acid or a salt thereof" and the "serum-free or low-serum medium containing (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof" have the same meanings as those described in the culture method and proliferation method of the present invention in (1) above. The desired cells are cells targeted for proliferation, and include those exemplified in the section "1-1. Cells" in (1) above, but are preferably immunocompetent cells, more preferably T cells and NK cells, and particularly preferably T cells into which a CAR gene has been introduced, i.e., CAR-T cells, and NK cells into which a CAR gene has been introduced, i.e., CAR-NK cells. More preferably, the CAR-T cells and CAR-NK cells are derived from pluripotent stem cells, preferably iPS cells, and even more preferably, the CAR-T cells are derived from pluripotent stem cells (particularly iPS cells).

[0049] The origin of a "cell population containing desired cells" is not particularly limited, as long as it contains the desired cells, and may be naturally derived or artificially prepared. For example, when immunocompetent cells such as T cells are desired, the cell population may be derived from body fluids such as blood (peripheral blood, umbilical cord blood, etc.) and bone marrow fluid, as well as cell populations containing peripheral blood mononuclear cells (PBMCs), blood cells, hematopoietic stem cells, umbilical cord blood mononuclear cells, etc., collected, isolated, purified, or induced from these. These cells may be collected from a living body or obtained through ex vivo culture, for example, a cell population obtained by the production method of the present invention, either directly or cryopreserved. Furthermore, the cell population is not limited to a single-cell cell population, and may also be a section of a tissue mass obtained by biopsy, although a single-cell cell population is preferred. In the case of a liquid cell population, it is not limited to a cell population derived from a single tissue, but may also be a mixed system of cell populations derived from different tissues. The "cell population containing desired cells" is preferably a cell population containing immunocompetent cells, more preferably a cell population containing T cells or NK cells, and particularly preferably a cell population containing T cells into which a CAR gene has been introduced, i.e., CAR-T cells, or a cell population containing NK cells into which a CAR gene has been introduced, i.e., CAR-NK cells. The cell population containing CAR-T cells derived from pluripotent stem cells, preferably iPS cells, or a cell population containing CAR-NK cells derived from pluripotent stem cells, preferably iPS cells, is even more preferred.

[0050] "Expanded number of desired cells" refers to an increase in the number of desired cells in a cell population compared to a control, such as the number of cells before expansion or obtained 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 the control. In certain embodiments of the invention, the number of desired cells in a cell population produced by the invention is expanded by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% increase compared to the number of cells in a control.

[0051] When the desired cells are CAR-T cells or CAR-NK cells, the cells are expanded, i.e., increased in number, through activation culture and expansion culture (and recovery culture, if necessary), as described above. After expansion culture, the cells are washed and recovered. When cells are cultured or expanded using the culture method and expansion method of the present invention, the medium used is serum-free or low-serum medium. This simplifies the washing procedure and shortens the time required for washing, compared to when cells are cultured or expanded using a commonly used serum-containing medium (serum concentration: 5-20%). For example, when cells are washed and recovered 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) closed-system centrifugation / spinning membrane filtration / hollow fiber membrane filtration must be repeated multiple times until the BSA contained in the serum reaches an acceptable amount for human administration. In this regard, when cells are cultured or expanded using the culture method and expansion method of the present invention, multiple repetitions are not necessary (the amount of serum-derived BSA introduced into the culture system is originally small). Alternatively, the desired cell population can be recovered with fewer repetitions compared to when a serum-containing medium (serum concentration: 5 to 20%) is used. In a preferred embodiment, a step of culturing the cells after expansion in the presence of a stimulating substance is carried out prior to the recovery procedure. This step enables efficient expansion and also has the advantage of increasing the cell viability. If desired, the CAR-T cell population or CAR-NK cell population after expansion may be subjected to a washing procedure and cell separation step using beads or the like. This embodiment can increase the purity of a more effective CAR-T cell population or CAR-NK cell population. Even in this embodiment, when cells are cultured or expanded using the culture method and expansion method of the present invention, the washing procedure can be simplified and / or the time required for washing can be shortened. Thus, the number of desired cells is expanded in the cell population obtained by the production method of the present invention.

[0052] A "cell population in which the number of desired cells is expanded" produced by the production method of the present invention can be used to produce a pharmaceutical as appropriate depending on the function of the cells. When the cells are CAR-T cells or CAR-NK cells, a 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, particularly cancer that expresses the target antigen of the CAR-expressing immune cells. The cancer may be a solid tumor or a blood tumor. Specific cancers include, but are not limited to, various B-cell lymphomas (follicular lymphoma, diffuse large B-cell 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 syndromes, 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, kidney 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 tumor, Ewing's sarcoma, osteosarcoma, retinoblastoma, small cell lung cancer, non-small cell lung cancer, melanoma, ovarian cancer, rhabdomyosarcoma, bone and soft tissue sarcoma, kidney cancer, pancreatic cancer, malignant mesothelioma, prostate cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, and colorectal cancer.

[0053] The medicament of the present invention is administered in a therapeutically effective amount that is appropriately determined depending on the age, body weight, body surface area, symptoms, etc. of the subject. The subject in the present disclosure is a mammal, particularly, usually a human, and preferably a cancer patient. The medicament of the present invention is administered in a dose of, for example, 1 x 10 ４ pieces ~ 1x10 １０The cell population of the present disclosure may be administered in individual doses. The route of administration is not particularly limited, and may be administered intratumorally, peritumorally, intravenously, intraarterially, intraportally, intradermally, subcutaneously, intramuscularly, or intraperitoneally. The cell population of the present disclosure may be administered systemically or locally, and local administration may include direct injection into the target tissue, organ, or tissue. The administration schedule is appropriately determined depending on the subject's age, weight, body surface area, symptoms, etc., and may be a single administration or multiple continuous or regular administrations. The pharmaceutical agent of the present invention may be used for autotransplantation or allotransplantation. It may also be used in combination with other pharmaceutical agents.

[0054] In particular, the pharmaceutical of the present invention may contain, in addition to the cell population to be administered to a subject, components 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 the cells. The composition can be prepared by conventional methods (for example, methods described in the Japanese Pharmacopoeia, etc.).

[0055] (3) A medium additive (hereinafter also referred to as "the medium additive of the present invention") and a medium composition containing the medium additive (hereinafter also referred to as "the medium composition of the present invention"). The medium additive of the present invention contains (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof, and optionally further contains (iv) vitamin E and / or (v) boric acid or a salt thereof. Therefore, the medium additive of the present invention may contain (ia) an imidazole dipeptide or a salt thereof, or vitamin E, (ii) taurine or a precursor thereof or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof. The medium additive of the present invention is used to add to a serum-free medium or a low-serum medium. The above components (i) to (iii), and optionally components (iv) and / or (v), have the same meanings as those described in the culture method of the present invention and the proliferation method of the present invention in (1) above. The above components (ia) to (iii) have the same meanings as those described in the culture method and proliferation method of the present invention in (1) above. The amount of each component to be incorporated into the medium additive is not particularly limited as long as the desired effect is obtained, but is usually 0.01 to 100% by weight, preferably 0.1 to 100% by weight, more preferably 1 to 100% by weight, even more preferably 5 to 100% by weight, and particularly preferably 10 to 100% by weight. If desired, the medium additive of the present invention may contain vitamin E in addition to or instead of (i) imidazole dipeptide or a salt thereof. When vitamin E is used, the addition of component (iv) is not necessary.

[0056] The medium composition of the present invention is a composition containing medium components obtained by adding the medium additive of the present invention to a serum-free medium or a low-serum medium. Here, the "serum-free medium or low-serum medium" has the same meaning as the "serum-free medium or low-serum medium" described in the culture method of the present invention and the proliferation method of the present invention above, excluding components (i) to (iii) and, optionally, component (iv) and / or (v). The medium additive of the present invention is added to the "serum-free medium or low-serum medium" so that the concentrations of components (i) to (iii) and, optionally, component (iv) and / or (v) in the medium composition are as follows: (i) Imidazole dipeptides: usually 1 to 50 mM, preferably 3 to 40 mM, more preferably 5 to 30 mM, and particularly preferably 10 to 25 mM. (ii) Taurines: usually 0.1 to 20 mM, preferably 0.5 to 15 mM, more preferably 1 to 10 mM, and particularly preferably 2 to 5 mM. (iii) Lysophosphatidic acids: usually 0.1 to 50 mg / L, preferably 0.5 to 25 mg / L, more preferably 1 to 15 mg / L, and particularly preferably 2 to 10 mg / L. (iv) Vitamin E: usually 10 to 1,000 μM, preferably 20 to 500 μM, more preferably 40 to 300 μM, and particularly preferably 100 to 250 μM. (v) Boric acid or a salt thereof: usually 0.1 to 0.9 mM, preferably 0.2 to 0.8 mM, more preferably 0.2 to 0.7 mM, and particularly preferably 0.3 to 0.6 mM. The medium composition of the present invention may be a composition containing medium components obtained by adding the medium additive of the present invention to a serum-free medium or a low-serum medium. Here, the term "serum-free medium or low-serum medium" has the same meaning as the "serum-free medium or low-serum medium" described in the above (1) culture method of the present invention and the growth method of the present invention, except that components (ia) to (iii) are removed. The medium additive of the present invention is added to a "serum-free medium or low-serum medium" so that the concentrations of components (ia) to (iii) in the medium composition are as follows: (i) Imidazole dipeptides: usually 1 to 50 mM, preferably 3 to 40 mM, more preferably 5 to 30 mM, and particularly preferably 10 to 25 mM. Vitamin E: usually 10 to 1,000 μM, preferably 20 to 500 μM, more preferably 40 to 300 μM, and particularly preferably 100 to 250 μM.(ii) Taurines: usually 0.1 to 20 mM, preferably 0.5 to 15 mM, more preferably 1 to 10 mM, particularly preferably 2 to 5 mM. (iii) Lysophosphatidic acids: usually 0.1 to 50 mg / L, preferably 0.5 to 25 mg / L, more preferably 1 to 15 mg / L, particularly preferably 2 to 10 mg / L.

[0057] As used in this specification and claims, singular terms include plurals and plural terms include the singular, unless the context otherwise requires. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept, unless otherwise specified.

[0058] The present invention will be described in detail below using examples, but the present invention is not limited in any way. Unless otherwise specified, the reagents and materials used are commercially available or can be prepared according to known literature. Furthermore, those skilled in the art will understand that any substance having the same effect or action can be substituted.

[0059] (List of Abbreviations) BMP-4: bone morphogenetic protein-4 bFGF: basic fibroblast growth factor VEGF: vascular endothelial growth factor SCF: stem cell factor TPO: thrombopoietin Flt3L: Fms-related tyrosine kinase 3 ligand Fc-DLL4: Recombinant Human DLL4 Fc Chimera Protein DLL4: Delta-like protein 4 IL-7: Interleukin-7 IL-2: Interleukin-2 IL-15: Interleukin-15 IL-18: Interleukin-18 IL-21: Interleukin-21 SDF1α: Stromal cell-derived factor 1α αMEM: alpha Modified Eagle Minimum Essential Medium IMDM: Iscove's Modified Dulbecco's Medium TCR: T-cell receptor FBS: Fetal Bovine Serum PBS: Phosphate-Buffered Saline AA2P: L-Ascorbic acid 2-phosphate

[0060] Example 11. Preparation of iPS cell-derived CAR-T cells. The TCR gene was introduced into the QHJI01S04 iPS cell line provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, using a lentiviral vector. The TCR gene-introduced iPS cells were differentiated into hematopoietic progenitor cells according to a known method described in Nature Communication 2021; 12: 430. Specifically, the iPS cells were cultured for four days in the presence of CHIR99021, SB431542, BMP-4, bFGF, and VEGF to induce differentiation into mesoderm. Furthermore, the iPS cells were differentiated into hematopoietic progenitor cells using the hematopoietic cytokines SCF, TPO, and FLT3L. The resulting hematopoietic progenitor cells were differentiated into cytotoxic T lymphocytes (CTLs) according to a known patented method (WO2017 / 221975) and literature information (Nature Communication 2021; 12: 430). Specifically, CD34+ cells were purified from the resulting hematopoietic progenitor cells using magnetic beads (Myltenyi Biotec). The cells were then cultured for three weeks in α-MEM medium containing SCF, TPO, FLT3L, IL-7, SDF1α, and SB203580 on plates coated with Fc-DLL4 and RetroNectin (Recombinant Human Fibronectin Fragment, TakaraBio). A CAR gene recognizing a specific antigen was introduced into the resulting TCR-positive CTLs using a retroviral vector. These cells were cryopreserved in liquid nitrogen and used as iPS cell-derived CAR-T cells.

[0061] 2. Recovery Culture of iPS Cell-Derived CAR-T Cells iPS cell-derived CAR-T cells were suspended at 500,000 cells / mL in IMDM medium containing 15% FBS and the additives shown in the "Recovery Culture Medium" column in Table 1, and seeded onto G-Rex (registered trademark) 6M (Wilson Wolf). The cells were incubated at 5% CO ２ The cells were cultured at 37°C for 3 days.

[0062]

[0063] 3. Reagents and Antibodies The anti-CD3 agonist antibody used was Anti-CD3 mAb GMP grade, Anti-CD3 monoclonal antibody (Clone: ​​OKT3) purchased from Takara Bio Inc.

[0064] 4. Immobilization of anti-CD3 agonist antibody on culture plate An anti-CD3 agonist antibody (OKT3, final concentration 3 μg / mL) dissolved in PBS at the required concentration was added to a T225 flask and then allowed to stand overnight at 4° C. After washing with PBS, the plate was subjected to the test.

[0065] 5. Activation Culture of iPS Cell-Derived CAR-T Cells After recovery culture, the iPS cell-derived CAR-T cells were suspended in IMDM medium containing 15% FBS to which the additives shown in the "Activation Culture Medium" column in Table 1 were added, at a concentration of 133,333 cells / mL, and seeded into a T225 flask onto which an anti-CD3 agonist antibody (OKT3) had been immobilized. The suspension was incubated at 5% CO ２ The cells were cultured at 37°C for 3 days.

[0066] 6. Expansion of iPS cell-derived CAR-T cells in three types of serum-free media A total of three types of expansion culture media were prepared: a medium containing the components listed in Table 2 (hereinafter referred to as BM220720) further supplemented with the additives listed in the "Expansion culture medium 2" column in Table 1, and a medium further supplemented with 20 mM carnosine (Sigma-Aldrich) or 100 μM α-tocopherol (Sigma-Aldrich). As a control for the experiment, a medium was prepared by adding the additives listed in the "Expansion culture medium 1" column in Table 1 to IMDM medium containing 15% FBS. After 3 days of activation culture, cells were collected from the T225 flasks and suspended in each of the four types of expansion culture media at a concentration of 40,000 cells / mL. The cells were then incubated in G-Rex® 6M (Wilson Wolf) at 5% CO ２ The cells were cultured at 37°C. On the second day of culture, some of the cells were recovered from G-Rex (registered trademark) 6M, the cell count was counted, and the medium was replaced with the respective medium. On the third day of culture, some of the cells were recovered from G-Rex (registered trademark) 6M, and the cell count was counted. On the fourth day of culture, some of the cells were recovered from G-Rex (registered trademark) 6M, and the cell count was counted.

[0067]

[0068] Test Example 1-1: Proliferation test verifying the effect of adding carnosine or α-tocopherol to serum-free medium In the expansion culture described in item 6 of Example 1, the cell number and viability of iPS cell-derived CAR-T cells were measured on days 2, 3, and 4 of expansion culture. Table 3 shows the cell proliferation fold from day 0 of expansion culture. The addition of 20 mM carnosine or 100 μM α-tocopherol to serum-free medium enhanced the proliferation of iPS cell-derived CAR-T cells (Table 3). The addition of 20 mM carnosine to serum-free medium resulted in a higher proliferation fold compared to medium containing 15% FBS. Table 4 shows the cell viability from day 0 of expansion culture. The addition of 20 mM carnosine or 100 μM α-tocopherol to serum-free medium improved the viability of iPS cell-derived CAR-T cells (Table 4).

[0069]

[0070]

[0071] Test Example 1-2: Test to confirm proliferation ability of cryopreserved cells in serum-containing medium In the expansion culture described in item 6 of Example 1, cells were collected on day 3 of expansion culture of iPS cell-derived CAR-T cells, and the cell number was counted. A suspension containing a predetermined number of cells was centrifuged at 300 x g for 5 minutes, and the supernatant was removed. The cell mass was suspended at 8,000,000 cells / mL in MEM medium to which Albumin 25% I.V. Injection 12.5 g / 50 mL (product name) had been added so that the final albumin concentration was 5%, and an equal volume of CryoStor (registered trademark) CS10 (STEMCELL Technologies) was added to this suspension and cryopreserved. After thawing the cryopreserved cells, they were suspended in IMDM medium containing 15% FBS, 2 mM glutamine, ITS (x1), and 50 μg / mL ascorbic acid 2-phosphate sesquimagnesium salt hydrate at a density of 125,000 cells / mL, and then cultured in a G-Rex 24-well plate at 5% CO ２The cells were cultured at 37°C. The viable cell count and cell viability were then measured on days 0, 3, and 7 of culture. The results are shown in Table 5. Cells cultured in an expansion culture medium containing 20 mM carnosine or 100 μM α-tocopherol and then cryopreserved showed improved viability and proliferation rates from day 0 to day 7 after thawing in a proliferation ability confirmation test (Table 5). However, the proliferation rates were lower than those of cells expanded in a medium containing 15% FBS.

[0072]

[0073] Example 21 Recovery Culture of iPS Cell-Derived CAR-T Cells iPS cell-derived CAR-T cells were suspended in IMDM medium containing 15% FBS to which the additives shown in the "Recovery Culture Medium" column in Table 6 were added at a concentration of 500,000 cells / mL, and seeded onto G-Rex (registered trademark) 6M (Wilson Wolf). The cells were then incubated at 5% CO ２ The cells were cultured at 37°C for 3 days.

[0074]

[0075] 2. Reagents and Antibodies The anti-CD3 agonist antibody used was Anti-CD3 mAb GMP grade, Anti-CD3 monoclonal antibody (Clone: ​​OKT3) purchased from Takara Bio Inc.

[0076] 3. Immobilization of anti-CD3 agonist antibody on culture plate An anti-CD3 agonist antibody (OKT3, final concentration 3 μg / mL) dissolved in PBS at the required concentration was added to a T225 flask and then allowed to stand overnight at 4° C. After washing with PBS, the plate was subjected to the test.

[0077] 4. Activation Culture of iPS Cell-Derived CAR-T Cells After recovery culture, the iPS cell-derived CAR-T cells were suspended in IMDM medium containing 15% FBS to which the additives shown in the "Activation Culture Medium" column in Table 6 were added, at a concentration of 133,333 cells / mL, and seeded into a T225 flask onto which an anti-CD3 agonist antibody (OKT3) had been immobilized. The suspension was incubated at 5% CO ２ The cells were cultured at 37°C for 3 days.

[0078] 5. Expansion of iPS cell-derived CAR-T cells in two types of serum-free media Two types of serum-free expansion culture media were prepared: BM220720 medium supplemented with the additives shown in the "Expansion culture medium 2" column in Table 6 and further supplemented with 20 mM carnosine (Sigma-Aldrich), and a medium supplemented with 20 mM carnosine and 200 μM α-tocopherol (Sigma-Aldrich). As a control for the experiment, a medium was prepared by adding the additives shown in the "Expansion culture medium 1" column in Table 6 to IMDM medium containing 15% FBS. After 3 days of activation culture, cells were collected from the T225 flask and suspended in each of the three types of expansion culture media at a concentration of 40,000 cells / mL. The cells were then incubated in 5% CO using G-Rex® 100M (Wilson Wolf). ２ The cells were cultured at 37°C. On the second day of culture, some of the cells were recovered from G-Rex (registered trademark) 100M and the cell count was counted, and the medium was replaced with the respective medium. On the second day of culture, some of the cells were recovered from G-Rex (registered trademark) 100M and the cell count was counted. On the third day of culture, some of the cells were recovered from G-Rex (registered trademark) 100M and the cell count was counted.

[0079] Test Example 2-1 Proliferation test verifying the additive effect of carnosine and α-tocopherol in serum-free medium To verify the additive effect of 20 mM carnosine and 200 μM α-tocopherol in serum-free expansion culture medium, the cell number and viability of iPS cell-derived CAR-T cells were measured on days 2 and 3 of culture in the expansion culture described in item 5 of Example 2. The cell proliferation fold from day 0 of expansion culture is shown in Table 7. The iPS cell-derived CAR-T cells exhibited a higher proliferation fold in all serum-free medium conditions than in medium containing 15% FBS (Table 7). The cell viability from day 0 of expansion culture is shown in Table 8. The iPS cell-derived CAR-T cells exhibited a viability of 90% or higher in all serum-free medium conditions (Table 8).

[0080]

[0081]

[0082] Test Example 2-2: Test to confirm proliferation ability of cryopreserved cells in serum-containing medium In the expansion culture described in item 5 of Example 2, cells were collected on day 3 of expansion culture of iPS cell-derived CAR-T cells, and the cell number was counted. A suspension containing a predetermined number of cells was centrifuged at 300 x g for 5 minutes, and the supernatant was removed. The cell mass was suspended at 8,000,000 cells / mL in MEM medium to which Albumin 25% I.V. Injection 12.5 g / 50 mL (product name) had been added so that the final albumin concentration was 5%, and an equal volume of CS10 was added to this suspension and the suspension was cryopreserved. After thawing the cryopreserved cells, they were suspended in IMDM medium containing 15% FBS, 2 mM glutamine, ITS (x1), and 50 μg / mL ascorbic acid 2-phosphate sesquimagnesium salt hydrate at a density of 125,000 cells / mL, and then cultured in a G-Rex 24-well plate at 5% CO ２ The cells were cultured at 37°C. The viable cell count and cell viability were then measured on days 0, 3, and 7 of culture. The results are shown in Table 9. Cells cultured in an expansion culture medium containing 20 mM carnosine and 200 μM α-tocopherol and then cryopreserved showed an improved proliferation rate in a proliferation assay, and the proliferation rate was higher than that of cells cultured in an expansion culture medium containing 15% FBS and then cryopreserved. In this test, an additive effect of carnosine and α-tocopherol was observed in expansion culture (Table 9).

[0083]

[0084] Example 31 Activation Culture of Human-Derived Primary T Cells Human-derived 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) supplemented with the additives shown in Table 10 for the activation culture medium, and seeded in a PL240 bag (OriGen). The cells were cultured at 5% CO ２ The cells were cultured at 37°C for 2 days.

[0085]

[0086] 2. Reagents RetroNectin (trade name) was purchased from Takara Bio.

[0087] 3. Immobilization of Retronectin (trade name) and retrovirus on culture plates Retronectin (final concentration 20 μg / mL) dissolved in PBS at the required concentration was added to a PL120 bag and allowed to stand at room temperature for 2 hours. After washing with PBS, a retrovirus solution was added and the plate was immobilized overnight at 4°C while shaking at 50 rpm. After washing with PBS containing 1.5% HSA, the plate was subjected to testing.

[0088] 4. Transduction culture of human-derived primary T cells After activation culture, the human-derived primary T cells were suspended in OpTmizer medium containing 2% CTS Immune Cell SR to the additives shown in Table 10 for the transduction culture medium, at a concentration of 722,222 cells / mL. The cells were then seeded onto a PL120 bag onto which RetroNectin (trade name) and a retroviral vector containing a CAR gene that recognizes a specific antigen were immobilized, and the cells were incubated at 5% CO ２ The cells were cultured at 37°C for 1 day.

[0089] 5. Expansion of CAR-T Cells Derived from Human Primary T Cells in Three Types of Serum-Free Media A total of three types of expansion culture media were prepared: an expansion culture medium prepared by adding the additives shown in Expansion Culture Medium 1 in Table 10 to OpTmizer medium containing 2% CTS Immune Cell SR; an expansion culture medium prepared by adding the additives shown in Expansion Culture Medium 2 in Table 10 to BM220720 medium; and an expansion culture medium prepared by further adding 0.3 mM boric acid (Sigma-Aldrich), 3 mM taurine (Sigma-Aldrich), 5 mg / L lysophosphatidylcholine (Sigma-Aldrich), 20 mM carnosine (Sigma-Aldrich), and 200 μM α-tocopherol (Sigma-Aldrich). One day after transduction, cells were collected from the PL120 bag and suspended in the respective expansion culture medium at 220,000 cells / mL. The cells were then cultured in G-Rex® 6M (Wilson Wolf) under 5% CO ２The cells were then cultured for expansion at 37°C. On day 4 of the expansion culture, some of the cells were recovered from the G-Rex® 6M and counted, and 40 IU of IL-2 was added to each medium. On day 6 of the expansion culture, some of the cells were recovered from the G-Rex® 6M and counted, and the cells were seeded at 220,000 cells / mL into new G-Rex® 6M containing each medium. On day 10 of the expansion culture, some of the cells were recovered from the G-Rex® 6M and counted, and 40 IU of IL-2 was added to each medium. On day 11, some of the cells were recovered from the G-Rex® 6M and counted.

[0090] Test Example 3: Proliferation test to verify the effect of adding boric acid, taurine, lysophosphatidylcholine, carnosine, and α-tocopherol to human-derived primary T cell-derived CAR-T cells To verify the effect of adding boric acid, taurine, lysophosphatidylcholine, carnosine, and α-tocopherol to a serum-free expansion culture medium on human-derived primary T cells, the cell number and viability of human-derived primary T cell-derived CAR-T cells were measured on days 4, 6, 10, and 11 of the expansion culture described in item 5 of Example 3. The cell proliferation fold from day 0 of expansion culture is shown in Table 11. The proliferation of human-derived primary T cell-derived CAR-T cells was enhanced by adding 0.3 mM boric acid, 3 mM taurine, 5 mg / L lysophosphatidylcholine, 20 mM carnosine, and 200 μM α-tocopherol to the serum-free medium (Table 11). The cell viability from day 0 of expansion culture is shown in Table 12. The viability of human-derived primary T cell-derived CAR-T cells was improved from day 10 of expansion culture onwards by adding 0.3 mM boric acid, 3 mM taurine, 5 mg / L lysophosphatidylcholine, 20 mM carnosine, and 200 μM α-tocopherol to the serum-free medium (Table 12).

[0091]

[0092]

[0093] Example 41. Preparation of iPS cell-derived NK cells The iPS cell line FfI01s04, provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University, was differentiated into NK cells according to known methods (e.g., the method described in WO 2021 / 174004). The resulting cells were cryopreserved in liquid nitrogen and used as iPS cell-derived NK cells.

[0094] 2. Recovery culture of iPS cell-derived NK cells iPS cell-derived NK cells were cultured at 1.25 × 10 in a recovery culture medium containing 15% FBS in IMDM medium (Thermo Fisher Scientific) with the additives shown in Table 13. ５ The cells were suspended at 100 cells / mL and seeded onto a G-Rex (registered trademark) 6-well plate (Wilson Wolf). ２ The cells were cultured in an incubator at 37°C for 3 days (culture days 0 to 3).

[0095]

[0096] 3. Activation culture of iPS cell-derived NK cells After recovery culture, the iPS cell-derived NK cells were cultured at 1.84 × 10 in an activation culture medium containing 15% FBS in IMDM medium (Thermo Fisher Scientific) with the additives shown in Table 14. ５ The cells were suspended at 1000 cells / mL and seeded in a 6-well plate (TPP). ２ The cells were cultured in a 37°C incubator for 3 days (culture days 3 to 6).

[0097]

[0098] 4. Expansion culture of iPS cell-derived NK cells in three types of serum-free media and one type of serum-containing media After 3 days, the iPS cell-derived NK cells were cultured in three types of serum-free media and one type of serum-containing media. The first type of serum-free media was a basal medium in which human serum albumin (25% Albuminar I.V. Injection 12.5g / 50mL (product name)) was added to the BM220720 medium in Table 2 to a final concentration of 2g / L, and the second type of serum-free media was a basal medium in which human serum albumin (25% Albuminar I.V. Injection 12.5g / 50mL (product name)) was added to the BM220720 medium in Table 2 to a final concentration of 2g / L. The four types of basal media were supplemented with 12.5 g / 50 mL (product name) to a final concentration of 2 g / L, boric acid (Sigma-Aldrich) to a final concentration of 0.3 mM, taurine (Sigma-Aldrich) to a final concentration of 3 mM, lysophosphatidylcholine (Sigma-Aldrich) to a final concentration of 5 mg / L, carnosine (Sigma-Aldrich) to a final concentration of 20 mM, and α-tocopherol (Sigma-Aldrich) to a final concentration of 200 μM (second type of serum-free medium), a basal medium based on X-VIVO™ 15 medium (Lonza) (third type of serum-free medium), and a basal medium prepared by adding FBS to IMDM medium (Thermo Fisher Scientific) to a final concentration of 15%. The additives listed in Table 14 were added to these four types of basal media to prepare four types of expansion culture media. The cells were cultured for 3 days as an activation culture, and 1.25 × 10 cells were added to each of the four types of expansion culture media. ５ The cells were suspended at 1000 cells / mL and reseeded onto a G-Rex® 24-well plate (Wilson Wolf). ２ The cells were cultured in a 37°C incubator for 2 days (culture days 6-8). After 2 days, the medium was replaced with the four types of basal medium supplemented with the additives shown in Table 15, and the cells were incubated in a 5% CO ２ The cells were cultured in a 37°C incubator for 2 days (8th to 10th day of culture). After that, 1.25 × 10 cells were cultured in the same medium every 2 to 3 days. ５ The cells were reseeded into a G-Rex® 24-well plate (Wilson Wolf) at 200 cells / mL and incubated in 5% CO ２ The culture was continued in a 37°C incubator until the 17th day of culture.

[0099]

[0100] Test Example 4: Proliferation test to verify the effect of adding boric acid, taurine, lysophosphatidylcholine, carnosine, and α-tocopherol to iPS cell-derived NK cells To verify the effect of adding boric acid, taurine, lysophosphatidylcholine, carnosine, and α-tocopherol to a serum-free expansion culture medium on iPS cell-derived NK cells, the cell number and viability of iPS cell-derived NK cells were measured over time in the expansion culture described in Item 4 of Example 4. The proliferation fold and cell viability on day 11 of expansion culture are shown in Table 16. The proliferation of iPS cell-derived NK cells was enhanced by more than sevenfold by adding 0.3 mM boric acid (Sigma-Aldrich), 3 mM taurine (Sigma-Aldrich), 5 mg / L lysophosphatidylcholine (Sigma-Aldrich), 20 mM carnosine (Sigma-Aldrich), and 200 μM α-tocopherol (Sigma-Aldrich) to serum-free basal medium (Table 16).

[0101]

[0102] According to the present invention, even when a serum-free or low-serum medium is used, it is possible to obtain cell growth maintenance and promotion effects comparable to those obtained when cultured in a serum-containing medium. In particular, cells that are sensitive to stress from operations such as cell washing are preferably cultured in a serum-free or low-serum medium that does not require extensive washing, and are suitable for culture under such conditions. This application is based on Patent Application No. 2023-217677 filed in Japan (filing date: December 25, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for culturing cells, comprising the step of culturing cells in a serum-free medium or a low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

2. The method according to claim 1, wherein the imidazole dipeptide or a salt thereof is carnosine or anserine, or a salt thereof.

3. The method according to claim 1, wherein the imidazole dipeptide or a salt thereof is carnosine or a salt thereof.

4. The method according to claim 1, wherein the lysophosphatidic acid or a derivative thereof is lysophosphatidylcholine (LPC).

5. The method according to claim 1, wherein the medium further contains vitamin E.

6. The method according to claim 1, wherein the medium further contains boric acid or a salt thereof.

7. The method according to claim 1, wherein the serum-free medium or low-serum medium is a serum-free medium.

8. The method according to claim 1, wherein the cells are immune cells.

9. The method according to claim 8, wherein the immune cells are selected from dendritic cells, B cells, T cells, and natural killer cells.

10. The method according to claim 8, wherein the immune cells are T cells.

11. The method according to any one of claims 1 to 10, wherein the cells are derived from pluripotent stem cells.

12. The method according to claim 11, wherein the pluripotent stem cells are iPS cells.

13. A method for proliferating cells, comprising the step of culturing cells in a serum-free medium or a low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

14. The method according to claim 13, wherein the imidazole dipeptide or a salt thereof is carnosine or anserine, or a salt thereof.

15. The method according to claim 13, wherein the imidazole dipeptide or a salt thereof is carnosine or a salt thereof.

16. The method according to claim 13, wherein the lysophosphatidic acid or a derivative thereof is lysophosphatidylcholine (LPC).

17. The method according to claim 13, wherein the medium further contains vitamin E.

18. The method according to claim 13, wherein the medium further contains boric acid or a salt thereof.

19. The method according to claim 13, wherein the serum-free medium or low-serum medium is a serum-free medium.

20. A method for producing a cell population in which the number of desired cells has been expanded, the method comprising culturing a cell population containing the desired cells in a serum-free medium or a low-serum medium containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

21. The method according to claim 20, wherein the imidazole dipeptide or a salt thereof is carnosine or anserine, or a salt thereof.

22. The method according to claim 20, wherein the imidazole dipeptide or a salt thereof is carnosine or a salt thereof.

23. The method according to claim 20, wherein the lysophosphatidic acid or a derivative thereof is lysophosphatidylcholine (LPC).

24. The method according to claim 20, wherein the medium further contains vitamin E.

25. The method according to claim 20, wherein the medium further contains boric acid or a salt thereof.

26. The method according to claim 20, wherein the serum-free medium or the low-serum medium is a serum-free medium.

27. A serum-free or low-serum medium composition containing (i) an imidazole dipeptide or a salt thereof, (ii) taurine or a precursor thereof, or a salt thereof, and (iii) lysophosphatidic acid or a derivative thereof.

28. A medicament comprising a cell population obtained by the method according to any one of claims 20 to 26.

29. The medicament according to claim 28, for use in the prevention and / or treatment of cancer.

30. A method for the prevention and / or treatment of cancer, comprising administering to a subject in need thereof a cell population obtained by the method according to any one of claims 20 to 26.

31. A cell population obtained by the method according to any one of claims 20 to 26, for use in the prevention and / or treatment of cancer.

32. Use of a cell population obtained by the method according to any one of claims 20 to 26 in the manufacture of a medicament for the prevention and / or treatment of cancer.

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