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  • C12N2510/00—Genetically modified cells

Definitions

• the present invention relates to a method of culturing/proliferating cells in a serum-free or low-serum medium, a medium additive and a medium composition for the method.
• the present invention relates to a method for producing a cell population in which a desired number of cells is expanded in a serum-free or low-serum medium, and a medicament containing the cell population obtained by the method.
• serum-containing medium fetal bovine serum
• FBS fetal bovine serum
• the xenogeneic components may become a source of infection with blood-borne pathogens or xenoantigens.
• differences in serum between lots may cause variations in culture results. Therefore, in recent years, it has become mainstream to reduce the amount of serum used and to culture cells using a chemically defined medium. development is underway.
• CAR-T cells chimeric antigen receptor T cells
• CAR-T cell therapy such as Kymriah (trade name) and Yescarta (trade name), which have been approved in the United States, involves introducing the CAR gene into T cells collected from patients in vitro.
• Autologous CAR-T cell therapy in which the CAR-T cells are manufactured and administered to a patient is common.
• this method requires multiple steps over a long period of time, including activation/proliferation of T cells, preparation of viral vectors, and gene transfer into T cells, resulting in high costs associated with cell culture and viral vector preparation.
• manufacturing costs increase due to such factors. Therefore, iPS cell-derived CAR-T cells that enable allogeneic CAR-T therapy have been developed.
• a serum-containing medium is used in the step of producing iPS cell-derived CAR-T cells, particularly in the step of expanding culture, in order to obtain a sufficient cell proliferation effect.
• GMP Good Manufacturing Practice
• iPS cell-derived T cells are more susceptible to washing stress than primary T cells, there is a strong desire to use a serum-free or low-serum medium that does not require strong washing.
• Non-Patent Document 1 the cell proliferation effect of boric acid via its transporter NaBC1 has been reported.
• the medium used here is a serum-containing medium, and it is unclear whether a serum-free medium or a low-serum medium would have the same cell growth effect.
• Patent Document 1 describes a serum-free medium containing borate and suitable for culturing diploid cells.
• Non-Patent Document 2 describes that Taut, a taurine transporter, is important for the recall response of T cells.
• An object of the present invention is to provide a method for culturing and sufficiently proliferating cells, particularly immunocompetent cells, in a serum-free or low-serum medium, and a medium additive and a medium composition used in the method. Furthermore, the present invention provides 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 medicament containing the cell population obtained by the method.
• the challenge is to provide the following.
• the present inventors prepared various factors to be added to a serum-free medium or a low-serum medium, and investigated their effects on cell culture and proliferation. As a result, it was confirmed that by adding boric acid or its salt, even when a serum-free medium or a low-serum medium was used, cell proliferation maintenance and promotion effects comparable to culture in a serum-containing medium were confirmed. Based on this knowledge, more suitable culture conditions were found and the present invention was completed. That is, the present invention is as follows. [1] A method for culturing cells, which includes the step of culturing cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.
• the immunocompetent cells are T cells.
• the cells are derived from pluripotent stem cells.
• the pluripotent stem cells are iPS cells.
• the medium further contains taurine.
• a method for growing cells which includes the step of culturing cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.
• the serum-free medium or low serum medium is a serum-free medium.
• the medium further contains taurine.
• a method for preventing and/or treating cancer which comprises administering a cell population obtained by the method according to any one of [14] to [16] above to a subject in need thereof.
• cells that are susceptible to stress due to operations such as cell washing are preferably cultured in serum-free or low-serum media that do not require strong washing, and are suitable for culture under such conditions.
• FIG. 1 is a graph showing the results of a cell proliferation test of iPS cell-derived CAR-T cells to verify the effect of adding 0.3 mM boric acid in a 1% FBS-containing medium.
• the upper row represents the cell proliferation rate from day 0 of expansion culture (Day 0), and the lower row represents the results of measuring the cell survival rate.
• FIG. 2 is a graph showing the results of a cell proliferation test of iPS cell-derived CAR-T cells to verify the effect of adding 0.3 mM boric acid in a serum-free expansion culture medium.
• the upper row represents the cell proliferation rate from day 0 of expansion culture (Day 0), and the lower row represents the results of measuring the cell survival rate.
• FIG. 3 is a graph showing the results of a proliferation test of iPS cell-derived CAR-T cells when boric acid was added at different concentrations to a serum-free expansion culture medium.
• the upper row represents the cell proliferation rate from day 0 of expansion culture (Day 0), and the lower row represents the results of measuring the cell survival rate.
• the upper row represents the cell proliferation rate from day 0 of expansion culture (Day 0), and the lower row represents the results of measuring the cell survival rate.
• w/o;without, w/;with It is a graph showing the results of a cell proliferation test of human primary T cell-derived CAR-T cells to verify the effect of adding taurine alone in a serum-free medium containing 0.3 mM boric acid.
• the upper row represents the cell proliferation rate from day 0 of expansion culture (Day 0), and the lower row represents the results of measuring the cell survival rate. w/o;without, w/;with
• the culture method or proliferation method of the present invention includes the step of culturing cells in a serum-free medium or a low-serum medium containing boric acid or a salt thereof.
• Cells The cell type to be cultured in the culture method and proliferation method of the present invention is not particularly limited. Examples of such cell types include reproductive cells such as sperm and eggs, somatic cells constituting living organisms, stem cells (pluripotent stem cells, etc.), cells induced to differentiate from stem cells, progenitor cells, and cancer cells isolated from living organisms. , cells that have been isolated from a living body and have acquired immortality and are stably maintained outside the body (cell line), cells that have been isolated from a living body and have undergone artificial genetic modification, and cells that have been isolated from a living body and whose nucleus has been artificially modified. Includes replaced cells, etc.
• Examples of cells induced to differentiate from somatic cells and stem cells that make up living bodies include, but are not limited to, immunocompetent cells (natural killer (NK) cells, macrophages, monocytes, mast cells, dendritic cells, Cells, Langerhans cells, neutrophils, eosinophils, basophils, B cells, T cells, etc.), fibroblasts, bone marrow cells, red blood cells, platelets, osteocytes, pericytes, keratinocytes, adipocytes, mesenchyme cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, Included are esophageal cells, muscle cells (eg, smooth muscle cells or skeletal muscle cells), pancreatic beta cells, me
• Somatic cells include, for example, 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, and vascular tissue. , blood (including umbilical cord blood), bone marrow, heart, eye, brain or neurological tissue.
• Stem cells are cells that have the ability to both reproduce themselves and differentiate into multiple lineages of cells, including, but not limited to, embryonic tumor cells, pluripotent cells, These include sexual stem cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, reproductive stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, etc.
• Pluripotent stem cell means a cell that has self-renewal ability and differentiation/proliferation ability, and has the ability to differentiate into all tissues and cells that make up a living body.
• 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. I can list them.
• iPS cells are preferred as pluripotent stem cells. Confirmation that the cells are iPS cells can be performed using an undifferentiated marker, which is due to the undifferentiated nature of iPS cells, as an indicator.
• undifferentiated markers examples include alkaline phosphatase, Oct3/4, Sox2, Nanog, ERas, and Esgl.
• Methods for detecting these undifferentiated markers include methods for detecting mRNA (using primers and probes), immunological detection methods (using antibodies and labels), and the like.
• a cell induced to differentiate from a stem cell is any cell that has been subjected to differentiation induction treatment so that the stem cell differentiates into a specific type of cell.
• a cell line is a cell that has acquired unlimited proliferative ability through artificial manipulation in vitro, 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 (product name), Vero, etc.
• the cells to be cultured are immunocompetent cells.
• Immune-competent cells refer to cells that are involved in various immune reactions in vivo. Immune-competent cells include natural killer (NK) cells, macrophages, monocytes, mast cells, dendritic cells, Langerhans cells, neutrophils, eosinophils, basophils, B cells, T cells, etc. , preferably selected from dendritic cells, B cells, T cells, and natural killer (NK) cells. More preferably, the immunocompetent cell is a T cell.
• T cells include CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, ⁇ -T cells, ⁇ -T cells, regulatory T cells, NKT cells, and the like.
• T cells can be subsets such as naive T cells, effector T cells, or memory T cells.
• immunocompetent cells are cells isolated from humans (primary cells), they can be obtained by differentiation from stem cells such as pluripotent stem cells (e.g. iPS cells, ES cells), hematopoietic stem cells, mesenchymal stem cells, etc.
• the cells may be primary cells or cells obtained by differentiation from pluripotent stem cells (particularly iPS cells).
• the primary cells human-derived primary T cells are preferred.
• Induction of differentiation from stem cells into immunocompetent cells can be carried out by appropriate methods known in the art depending on the type of stem cells used and the target immunocompetent cells. As an example, induction of differentiation of T cells from iPS cells and induction of differentiation of NK cells from iPS cells can be performed by the method described in the Examples.
• the target cells may be either autologous cells or allogeneic cells.
• autologous cells refer to cells obtained from a subject receiving the culture method of the present invention or a cell population (described below) produced by the method of the present invention, or cells derived from the obtained cells.
• Allogeneic cells means cells that are not the above-mentioned “autologous cells.”
• the target cell may be a cell that has been artificially genetically modified.
• examples include immunocompetent cells into which a CAR gene has been introduced (T cells into which a CAR gene has been introduced, natural killer cells into which a CAR gene has been introduced), T cells into which an extrinsic T cell receptor (TCR) has been introduced, and cytokines. and/or immunocompetent cells into which genes for expressing chemokines have been introduced.
• CAR is a structure that contains, from the N-terminal side to the C-terminal side of the protein, a target-specific extracellular domain, a transmembrane domain, and an intracellular signaling domain for effector functions of immune cells.
• the gene is the one that codes for this receptor.
• the extracellular domain contains an antigen recognition site that exhibits target-specific binding.
• a transmembrane domain exists between the extracellular domain and the intracellular signaling domain.
• Intracellular signaling domains transmit signals necessary for immune cells to exert their effector functions. That is, when the extracellular domain binds to a target antigen, an intracellular signal domain is used that is capable of transmitting a signal necessary for activating immune cells.
• Preferred examples of cells to which the culture method and proliferation method of the present invention are applied include T cells into which a CAR gene has been introduced (CAR-T cells), NK cells into which a CAR gene has been introduced (CAR-NK cells), and more.
• CAR-T cells T cells into which a CAR gene has been introduced
• CAR-NK cells NK cells into which a CAR gene has been introduced
• pluripotent stem cell-derived CAR-T cells and CAR-NK cells more preferably iPS cell-derived CAR-T cells and iPS cell-derived CAR-NK cells, even more preferably iPS cell-derived CAR-T cells.
• Examples include cells.
• CAR-T cells and CAR-NK cells can be obtained by introducing a CAR gene into T cells and NK cells or their progenitor cells such as pluripotent stem cells, respectively.
• CAR-T cells include cells obtained by differentiating cells such as iPS cells, ES cells, hematopoietic stem cells, and mesenchymal stem cells into T cells into which a CAR gene has been introduced, and iPS cells into which a CAR gene has been introduced. They may also be cells obtained by differentiating into T cells from.
• iPS cell-derived CAR-T cells obtained by differentiating iPS cells into which a CAR gene has been introduced into T cells and CAR-T cells obtained by introducing a CAR gene into T cells obtained by differentiating iPS cells.
• CAR-T cells derived from iPS cells are obtained by introducing a CAR gene into T cells obtained by differentiating iPS cells.
• CAR-NK cells cells obtained by differentiating iPS cells, ES cells, hematopoietic stem cells, mesenchymal stem cells, etc. into NK cells into which the CAR gene has been introduced, or cells into which the CAR gene has been introduced.
• the cells may be obtained by differentiating iPS cells into NK cells.
• iPS cell-derived CAR-NK cells obtained by differentiating iPS cells into which a CAR gene has been introduced into NK cells, and CAR-NK cells obtained by introducing a CAR gene into NK cells obtained by differentiating iPS cells.
• CAR-NK cells derived from iPS cells and these may be collectively referred to as "iPS cell-derived CAR-NK cells.” More preferably, CAR-NK cells derived from iPS cells are obtained by introducing a CAR gene into NK cells obtained by differentiating iPS cells.
• Induction of differentiation from iPS cells (in which a CAR gene may be introduced, if desired) into T cells, or from iPS cells (into which a CAR gene may be introduced, if desired) into NK cells, can be carried out using known methods. Specifically, the method described in Examples can be used.
• the CAR gene is usually introduced into cells using a CAR expression vector.
• CAR expression vector means a nucleic acid molecule capable of transporting a nucleic acid molecule encoding a CAR gene into cells, regardless of whether it is DNA or RNA, and there are no particular restrictions on its form or origin.
• vectors can be viral or non-viral vectors. Examples of viral vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, Sendai virus vectors, vaccinia virus vectors, and the like.
• the target gene inserted into the vector is integrated into the host chromosome, and stable and long-term expression can be expected.
• Each viral vector can be produced according to a conventional method or using a commercially available dedicated kit.
• Non-viral vectors include plasmid vectors, liposome vectors, and 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.
• the CAR gene can be introduced into cells by the method described in the Examples.
• the medium used in the culture method and propagation method of the present invention is a serum-free medium or a low-serum medium containing boric acid or a salt thereof (hereinafter may be collectively referred to as "the medium of the present invention").
• boric acid is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include orthoboric acid, metaboric acid, tetraboric acid, and the like. Among these boric acids, orthoboric acid is preferably used.
• Salts of boric acid are not particularly limited as long as they are pharmaceutically acceptable; alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts; triethylamine and triethanol. Examples include organic amine salts such as amine, morpholine, piperazine, and pyrrolidine.
• boric acid or a salt thereof may be in the form of a hydrate, such as borax.
• the concentration of boric acid or its salt in the medium is not particularly limited as long as the desired effect is obtained, but it is usually 0.1-0.9mM, preferably 0.2-0.8mM, more preferably 0.2-0.8mM. 0.7mM, particularly preferably 0.3 to 0.6mM.
• the concentration of boric acid or a salt thereof is a concentration converted to boric acid unless otherwise specified. If the concentration is too high, it will affect the cell survival rate, and if the concentration is too low, the cell proliferation promoting effect will be weak.
• serum-free medium means a medium that does not substantially contain, preferably does not contain, unadjusted or unpurified serum, and does not contain purified blood-derived components or animal tissue-derived components (e.g., A medium containing a factor) is considered to be a serum-free medium.
• a medium used for culturing animal cells hereinafter also referred to as basal medium for convenience
• basal medium for perfusion culture is used.
• substantially free means that it is not contained at all, or even if it is contained, it is below the detection limit.
• basal medium examples include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, Ham's medium, and RPMI 1640.
• medium Fischer's medium, F12 medium, mixed medium thereof (eg, Advanced DMEM/F12 medium, etc.), but are not particularly limited.
• Perfusion culture is a culture method in which a culture medium is continuously supplied to a culture system containing cells, and the same amount of culture supernatant that does not contain cells is continuously removed from the culture system to maintain the culture system in a steady state.
• perfusion culture medium generally has a higher concentration of nutrient components than basal medium.
• basal medium can be purchased from companies such as Invitrogen, SIGMA, Fujifilm Wako Pure Chemical Industries, and Sumitomo Pharma, and as long as they have the same name or brand name, the composition of the culture medium is the same regardless of the manufacturer.
• the term "low serum medium” refers to a medium in which serum is added to a basal medium, and the concentration thereof is set to a commonly used serum-containing medium (serum concentration: 5 to 20% (in this specification, serum concentration: 5% to 20%). (expressed in v/v%)).
• the serum concentration of the low serum medium can be less than 5%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, but usually 0.1% or more, It can be 1% or more. Specifically, the serum concentration is preferably 0.1% or more and less than 5%, more preferably 1% or more and less than 4%.
• the medium used in this step may also contain a serum substitute.
• Serum substitutes include, for example, albumin (e.g. lipid-rich albumin), transferrin, fatty acids, collagen precursors, insulin, trace elements (e.g. zinc, selenium, etc.), ITS supplements (mixtures of insulin, transferrin, selenite). , B-27 supplement, N2 supplement, knockout serum replacement, 2-mercaptoethanol or 3' thiol glycerol, or equivalents thereof. These serum replacements are also commercially available. Knockout Serum Replacement is available from Invitrogen.
• serum substitutes can be purchased from companies such as Invitrogen, SIGMA, Fujifilm Wako Pure Chemicals, and Sumitomo Pharma, and the composition may vary depending on the manufacturer if they are reagents or additives with the same name or brand name. are equivalent.
• components preferable for cell proliferation can be added to the medium of the present invention.
• Such components include, for example, sugars such as glucose, fructose, sucrose, and maltose; glycine, serine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, proline, threonine, cysteine, asparagine, glutamine, and aspartic acid.
• N N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethane Sulfonic acid (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES)), N-[tris(hydroxymethyl)methyl]glycine (Tris(hydroxymethyl)methyl) buffering agents such as ne))
• Antibiotics such as amphotericin B, kanamycin, gentamicin, and streptomycin
• Cell adhesion factors and extracellular matrix components such as Type I collagen, Type II collagen, fibronectin, laminin,
• Cytokines and growth factors include hormones such as dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin, and appropriate components can be selected depending on the type of cells to be cultured and used at appropriate concentrations.
• taurine an amino acid derivative, can be added and typically used at a final concentration of 0.5-10 mM, preferably 2-5 mM.
• the growth factor may be from an animal species other than humans, but is preferably derived from humans (and may be recombinant).
• the medium may further contain additional factors to support cell survival and proliferation. Additional 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- ⁇ ), specifically IL-1 to IL- Preferred examples include IL-1, IL-2, IL-7, IL-15, IL-18, and IL-21.
• the additive factor can be prepared according to a conventional method, or a commercially available product can also be used. The additive factor may be from an animal species other than humans, but is preferably derived from humans (and may be recombinant).
• xeno-free refers to conditions in which components derived from a biological species different from that of the cells to be cultured are excluded.
• the present invention also provides a method for culturing cells (hereinafter sometimes referred to as "the culture method of the present invention"), which is characterized by culturing in the medium of the present invention (described above).
• the culture vessels used for cell culture are not particularly limited as long as they are capable of culturing cells, but include flasks, tissue culture flasks, dishes, petri dishes, tissue culture dishes, multi-dishes, microplates, and microwells. Mention may be made of plates, multiplates, multiwell plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, and bioreactors. It is selected as appropriate depending on the target cells.
• the culture vessel may be cell-adhesive or non-cell-adhesive, and is appropriately selected depending on the purpose.
• a cell-adhesive culture vessel may be coated with any cell-supporting substrate such as extracellular matrix (ECM) for the purpose of improving adhesion with cells on the surface of the culture vessel.
• ECM extracellular matrix
• the cell support substrate can be any material intended for cell attachment.
• the culture temperature is not particularly limited, but may be about 30 to 40°C, preferably about 37°C.
• the CO 2 concentration may be about 1-10%, preferably about 2-5%.
• the oxygen partial pressure can be between 1 and 21%.
• the frequency and culture period of medium exchange in cell culture generally depend on cell density, culture method (adherent culture/suspension culture), cell type to be cultured, medium composition, culture conditions (temperature, gas concentration), It is determined by comprehensively considering various conditions such as the amount of culture medium (total amount/partial amount), the cost of the culture medium, and the lifestyle of the worker.
• the culture medium is usually replaced once every 2 to 3 days, once a day, or multiple times a day (e.g., twice), and the culture method and propagation method of the present invention also require such a frequency.
• the medium can be exchanged by Moreover, continuous medium exchange can be performed by perfusion culture.
• the culture period is usually about 4 days to 4 weeks, preferably about 4 days to 3 weeks, and more preferably about 1 to 2 weeks.
• the cells cultured in a serum-free medium or low serum medium containing boric acid or a salt thereof are immunocompetent cells, preferably dendritic cells. , B cells, T cells, and natural killer (NK) cells, more preferably T cells and NK cells, and still more preferably T cells.
• the cells cultured in the medium of the present invention are T cells derived from primary T cells and pluripotent stem cells (especially iPS cells), more preferably derived from primary T cells and pluripotent stem cells.
• CAR-T cells derived from stem cells are cultured in the medium of the present invention.
• the cells are primary NK cells and NK cells derived from pluripotent stem cells (especially iPS cells), more preferably CAR-NK cells derived from primary NK cells and pluripotent stem cells (especially iPS cells), and even more preferably CAR-NK cells derived from pluripotent stem cells (especially iPS cells).
• culturing includes an activation step and an expansion step.
• a recovery step may be included.
• the cells can be cultured by any one of recovery culture consisting of a recovery step, activation culture consisting of an activation step, expansion culture consisting of an expansion step, or 2. It is a concept that includes one or all three.
• the target is expanded culture.
• Another embodiment of the invention provides CAR-T cells and CAR-NK cells, preferably CAR-T cells derived from pluripotent stem cells (especially iPS cells) and CAR-NK cells derived from pluripotent stem cells (especially iPS cells). It is a cell culture, which includes an activation step and an expansion step, and if necessary (such as when using cryopreserved cells) a recovery step.
• This step is performed after thawing the cryopreserved cells. Remove cryoprotectant from cells immediately after thawing, as prolonged exposure of cells to cryoprotectant can cause damage. Thereafter, cell damage and cell dysfunction caused by freezing and thawing are restored.
• the process/cultivation method varies depending on the target cells, the cryoprotectant used, the thawing method, etc., but is usually cultured in a medium containing no cryoprotectant for several days, preferably 1 to 5 days, and more preferably. Most of the cells will recover to normal by culturing them for about 3 days.
• the method of this step is not particularly limited as long as the desired effect on the cells is obtained, but it can be carried out, for example, by contacting with a stimulating substance.
• the culture is carried out in the presence of a stimulating substance, specifically in a medium containing a stimulating substance, for several days, preferably for 1 to 5 days, and more preferably for about 3 days.
• the stimulant is a signal molecule that controls cell activity by autocrine, paracrine, endocrine, etc. methods, and may be a substance that can be secreted from all cells in the culture system or a substance that can be secreted from the outside. It may be added. Specifically, this is carried out by contacting with a substrate bound to a ligand, culturing in a medium containing a ligand, or the like.
• the ligand used in the activation culture of CAR-T cells preferably CAR-T cells derived from pluripotent stem cells (particularly iPS cells), interacts with surface molecules of CAR-T cells and activates them.
• CAR-T cells preferably CAR-T cells derived from pluripotent stem cells (particularly iPS cells)
• CAR-T cells derived from pluripotent stem cells (particularly iPS cells)
• CD3 which is coupled to TCR and plays a role in TCR-mediated signal transduction
• surface molecules CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, and CD40L which are known as costimulatory factors for T cell activation.
• Examples include molecules that have the function of specifically binding to T cells and transmitting activation signals into T cells.
• Such a molecule may be a physiological ligand (or receptor) for the T cell surface molecule, or a non-physiological ligand (or receptor) having
• the T cell activation ligand used in the present invention includes an antibody against CD3.
• Antibodies against CD3 are complete antibodies as long as they have the ability to specifically bind to CD3 expressed on target T cells that induce activation, stimulate surface molecules of these T cells, and transmit signals into T cells. or a fragment thereof (eg, Fab, F(ab') 2 , Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc.).
• CAR-NK cells preferably CAR-NK cells derived from pluripotent stem cells (particularly iPS cells), interact with surface molecules of CAR-NK cells, and activate the CAR-NK cells.
• CAR-NK cells preferably CAR-NK cells derived from pluripotent stem cells (particularly iPS cells)
• iPS cells pluripotent stem cells
• the non-physiological ligand includes an antibody against CD3.
• Antibodies may be complete antibodies or fragments thereof (e.g., Fab, F(ab') 2 , Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc.). It's okay.
• cytokines include IFN- ⁇ / ⁇ , 1FN- ⁇ , 1L-2, 1L-4, IL-7, 1L-12, 1L-15, IL-18, IL-21, and the like.
• This step is a step of proliferating 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 can be obtained, and those skilled in the art can appropriately adjust the method while monitoring the number of cells and the like.
• the cells can be cultured in a medium containing the following: for example, for 7 days or more, 9 days or more, or 11 days or more, with appropriate adjustment while monitoring the number of cells and the like.
• the culture can be continued and the number of cells can be increased exponentially.
• the upper limit of the culture period is not particularly limited, and is, for example, 28 days or less, preferably 21 days or less.
• activated CAR-T cells preferably CAR-T cells derived from activated pluripotent stem cells (particularly iPS cells)
• contain cytokines such as IL-7, IL-15 and IL-2.
• This can be carried out by expanding culture in a medium for, for example, 2 days or more, preferably 3 days or more, more preferably 4 days or more, even more preferably 5 days or more, even more preferably 6 days or more.
• Target cells are primary T cells (especially CAR-T cells) and NK cells (preferably pluripotent stem cell (especially iPS cell-derived)-derived NK cells (especially CAR-NK cells)), and the number of cells is increased. For example, for 2 days or more, preferably 4 days or more, more preferably 6 days or more in a medium containing cytokines such as IL-7, IL-15, IL-21, and IL-2. , more preferably for 8 days or more, even more preferably for 10 days or more, even more preferably for 11 days or more.
• cytokines such as IL-7, IL-15, IL-21, and IL-2.
• the production method of the present invention is a method for producing a cell population in which the number of desired cells is expanded. the step of culturing a cell population containing the cell population.
• the term "serum-free medium or low-serum medium containing boric acid or a salt thereof” has the same meaning as described in (1) above for the culture method of the present invention and the propagation method of the present invention.
• Desired cells are cells whose proliferation is aimed at, and include those listed in the section "1-1.
• Cells in (1) above, preferably immunocompetent cells, more preferably. are T cells and NK cells, and particularly preferably T cells into which a CAR gene has been introduced, ie, CAR-T cells, and NK cells into which a CAR gene has been introduced, ie, CAR-NK cells.
• CAR-T cells and CAR-NK cells derived from pluripotent stem cells preferably iPS cells
• CAR-T cells derived from pluripotent stem cells especially iPS cells.
• the "cell population containing desired cells” is not particularly limited in its origin as long as it contains desired cells, and may be naturally derived or artificially prepared.
• the cell population may be collected, isolated, purified, or derived from body fluids such as blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, etc.
• body fluids such as blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, etc.
• Examples include cell populations including peripheral blood mononuclear cells (PBMC), blood cells, hematopoietic stem cells, and cord blood mononuclear cells.
• PBMC peripheral blood mononuclear cells
• These cells may be collected from a living body or obtained through in vitro culture, for example, a cell population obtained by the production method of the present invention may be preserved as is or frozen.
• Cell population containing desired cells is preferably a cell population containing immunocompetent cells, more preferably a cell population containing T cells or NK cells, particularly preferably T cells into which a CAR gene has been introduced, That is, it is a cell population containing CAR-T cells, or a cell population containing NK cells into which a CAR gene has been introduced, that is, CAR-NK cells. More preferably, it is a cell population comprising pluripotent stem cells, preferably iPS cell-derived CAR-T cells, or a cell population comprising pluripotent stem cells, preferably iPS cell-derived CAR-NK cells.
• the desired number of cells has been expanded means that the number of desired cells in the cell population is greater than the number of cells obtained prior to expansion or without carrying out the present invention.
• the number of desired cells in the cell population produced according to the invention is at least 10%, 20%, 30%, 40%, 50% compared to the number of cells in the control. , 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%. Expanded to increase by 1000%.
• the desired cells are CAR-T cells or CAR-NK cells
• the cells are increased in number, that is, expanded, through activation culture and expansion culture (recovery culture if necessary), as described above. .
• After expansion culture cells are washed and collected.
• the medium used is a serum-free medium or a low-serum medium
• a commonly used serum-containing medium (serum concentration: 5 to 20%) is used for culturing. Compared to the case of propagation, the washing operation is simpler and the time required for washing can be shortened.
• the BSA contained in the serum reaches an acceptable amount for human administration.
• Petting and centrifugation processes or (ii) continuous automated processes using techniques such as closed system centrifugation/spinning membrane filtration/hollow fiber membrane filtration need to be repeated multiple times.
• the desired cell population can be recovered by repeating the procedure fewer times than when using a serum-containing medium (serum concentration: 5-20%).
• a step of culturing the expanded cells in the presence of a stimulating substance is performed before the recovery operation.
• This step enables efficient expansion culture and also has the advantage of increasing cell survival rate.
• the expanded cultured CAR-T cell population or CAR-NK cell population may be subjected to a washing operation and cell separation step using bead separation or the like. According to this aspect, it is possible to increase the purity of a CAR-T cell population or a CAR-NK cell population that has higher efficacy.
• the washing operation can be simplified and/or the time required for washing can be shortened. In this manner, the desired number of cells is expanded in the cell population obtained by the production method of the present invention.
• the "cell population in which the number of desired cells is expanded" produced by the production method of the present invention can be used to appropriately produce medicines depending on the functions of the cells.
• the cells are CAR-T cells or CAR-NK cells
• the cell population containing CAR-T cells or CAR-NK cells produced by the culture method and proliferation method of the present invention can be used for cancer treatment, especially the It can be used to treat cancers that express target antigens of CAR-expressing immune cells.
• the cancer may be a solid tumor or a hematologic tumor.
• Specific cancers include various B-cell lymphomas (follicular malignant lymphoma, diffuse large B-cell malignant lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.), bone marrow Proliferative tumors, myelodysplastic/myeloproliferative tumors (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, neuroblastoma, brain tumors, Ewing's sarcoma, osteosarcoma, retinal buds Cytoma, 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, endometrial cancer, cervical cancer, ovarian cancer , colon cancer, etc., but are not limited to these.
• the cancer is a solid tumor.
• 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, and kidney cancer.
• pancreatic cancer malignant mesothelioma, prostate cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, and colorectal cancer.
• the medicament of the present invention is administered in a therapeutically effective amount that is appropriately determined depending on the age, weight, body surface area, symptoms, etc. of the subject.
• the subject in this disclosure is a mammal, particularly usually a human, preferably a cancer patient.
• the medicament of the present invention can be administered, for example, at a dose of 1 ⁇ 10 4 to 1 ⁇ 10 10 at a time.
• the route of administration is not particularly limited, and administration can be intratumoral, peritumoral, intraventricular, intravenous, intraarterial, intraportal, intradermal, subcutaneous, intramuscular, or intraperitoneal.
• the cell populations of the present disclosure may be administered systemically or locally, and local administration includes direct injection into the target tissue, organ, or organ.
• the administration schedule is appropriately determined depending on the subject's age, body weight, body surface area, symptoms, etc., and may be a single administration or continuous or periodic multiple administrations.
• the medicament of the present invention may be used for autologous or allogeneic transplantation. It may also be used in combination with other medicines.
• the medicament of the present invention can be administered with dimethyl sulfoxide (DMSO), serum albumin, etc. for the purpose of cell protection, and antibiotics, etc. for the purpose of preventing bacterial contamination. It may contain components such as various components (vitamins, cytokines, growth factors, steroids, etc.) for the purpose of activating, proliferating, or inducing differentiation.
• DMSO dimethyl sulfoxide
• serum albumin etc.
• antibiotics, etc. for the purpose of preventing bacterial contamination.
• the composition can be prepared by a conventional method (for example, the method described in the Japanese Pharmacopoeia).
• a culture medium additive (hereinafter also referred to as “the culture medium additive of the present invention") and a culture medium composition formed by adding the culture medium additive (hereinafter also referred to as “the culture medium composition of the present invention”)
• the medium additive of the present invention contains boric acid or a salt thereof.
• the medium additive of the present invention is used to add to a serum-free medium or a low-serum medium.
• Boric acid or its salt has the same meaning as described in (1) above for the culture method of the present invention and the propagation method of the present invention.
• the amount of boric acid or its salt added to the medium additive is not particularly limited as long as the desired effect is obtained, but it is usually 0.01 to 100% by weight, preferably 0.1 to 100% by weight, more preferably It may be 1 to 100% by weight, more preferably 5 to 100% by weight, particularly preferably 10 to 100% by weight.
• the medium composition of the present invention is a composition containing a medium component obtained by adding the medium additive of the present invention to a serum-free medium or a low-serum medium.
• serum-free medium or low-serum medium refers to the "serum-free medium or low-serum medium” described in the above (1) culture method of the present invention and growth method of the present invention, excluding boric acid or its salt. It is synonymous with
• the concentration of boric acid or its salt is 0.1 to 0.9mM, preferably 0.2 to 0.8mM, more preferably 0.2 to 0.7mM, particularly preferably 0.3 to 0.
• the medium additive of the present invention is added to a "serum-free medium or low serum medium” so that the concentration is in the range of .6mM.
• 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
• DLL4 Delta-like protein 4
• IL7 Interleukin-7
• SDF1 ⁇ Stromal cell-derived factor 1 ⁇ ⁇ MEM:alpha Modified Eagle Minimum Essential Medium
• TCR T-cell receptor
• Example 1 Preparation of iPS Cell-Derived CAR-T Cells
• the TCR gene was introduced into iPS cell line QHJI01S04 provided by Kyoto University iPS Cell Research Institute (CiRA) using a lentivirus vector.
• the iPS cells into which the TCR gene was introduced were differentiated into hematopoietic progenitor cells with reference to the known method described in Nature Communication 2021; 12: 430. Specifically, the iPS cells were cultured for 4 days in the presence of CHIR99021, SB431542, BMP-4, bFGF, and VEGF to induce differentiation into mesoderm.
• the cells were differentiated into hematopoietic progenitor cells using hematopoietic cytokines SCF, TPO, and FLT3L.
• Differentiation of the obtained hematopoietic progenitor cells into cytotoxic T lymphocytes (CTLs) was performed according to the known patent method (WO2017/221975) and literature information (Nature Communication 2021; 12: 430).
• CD34-positive cells were purified using magnetic beads (Myltenyi Biotec), and then placed on a plate immobilized with Fc-DLL4 and RetroNectin (Recombinant Human Fibronectin Fragment, TakaraBio).
• the cells were cultured for 3 weeks in ⁇ MEM medium containing SCF, TPO, FLT3L, IL7, SDF1 ⁇ , and SB203580.
• a CAR gene that recognizes a specific antigen was introduced into the obtained TCR-positive CTL using a retrovirus vector.
• cells cryopreserved in liquid nitrogen were used as iPS cell-derived CAR-T cells.
• iPS cell-derived CAR-T cells were cultured in an IMDM medium containing 15% Fetal Bovine Serum (FBS) with the additives shown in the "Recovery culture medium" column of Table 1 added. The cells were suspended at 500,000 cells/mL, seeded on G-Rex (registered trademark) 10M (Wilson Wolf), and cultured for 3 days at 5% CO 2 /37°C.
• FBS Fetal Bovine Serum
• RetroNectin (trade name) was purchased from Takara Bio.
• Anti-CD3 mAb GMP grade Anti-CD3 monoclonal antibody (Clone: OKT3) purchased from Takara Bio Inc. was used.
• Activation culture of iPS cell-derived CAR-T cells After recovery culture, iPS cell-derived CAR-T cells were added to IMDM medium containing 15% FBS with the additives shown in the "Activation culture medium" column of Table 1. Suspend cells at 133,333 cells/mL in culture medium, seed in a T225 flask immobilized with anti-CD3 agonist antibody (OKT3) and RetroNectin (trade name), and store at 37°C in 5% CO2 . The cells were cultured for 3 days.
• the cells were collected from the T225 flask, suspended in two types of expansion culture media at 40,000 cells/mL, and treated with G-Rex (registered trademark) 10M (Wilson Wolf). The cells were cultured under 5% CO 2 /37°C. Thereafter, on the fifth day of culture, some cells were collected from G-Rex (registered trademark) 10M, the number of cells was counted, and the medium was replaced with the respective medium. On the 6th day of culture, some cells were collected from G-Rex (registered trademark) 10M and the number of cells was counted.
• G-Rex registered trademark
• Test example 1 Proliferation test to verify the effect of adding 0.3mM boric acid in a 1% FBS-containing medium Item 6 of Example 1.
• the cell number and survival rate of iPS cell-derived CAR-T cells were measured on days 3, 5, 6, 7, 12, and 14 of culture.
• Figure 1 shows the cell proliferation rate from day 0 of expansion culture.
• Proliferation of iPS cell-derived CAR-T cells was enhanced by adding 0.3 mM boric acid to the medium containing 1% FBS ( Figure 1).
• Example 2 Recovery culture of iPS cell-derived CAR-T cells 500,000 iPS cell-derived CAR-T cells were cultured in a medium containing IMDM medium containing 15% FBS with the additives shown in the "Recovery culture medium" column of Table 2. The cells were suspended at a concentration of 100 mL/mL, seeded on G-Rex (registered trademark) 10M (Wilson Wolf), and cultured for 3 days at 37° C. in 5% CO 2 .
• G-Rex registered trademark
• 10M Woodson Wolf
• RetroNectin (trade name) was purchased from Takara Bio.
• Anti-CD3 mAb GMP grade Anti-CD3 monoclonal antibody (Clone: OKT3) purchased from Takara Bio Inc. was used.
• Activation culture of iPS cell-derived CAR-T cells After recovery culture, iPS cell-derived CAR-T cells were added to IMDM medium containing 15% FBS with the additives shown in the "Activation culture medium” column of Table 2. Suspend cells at 133,333 cells/mL in culture medium, seed in T225 flasks immobilized with anti-CD3 agonist antibody (OKT3) and RetroNectin (trade name), and store at 37°C in 5 % CO2. It was cultured for 3 days.
• Expansion culture of iPS cell-derived CAR-T cells in two types of serum-free media Expansion culture medium prepared by adding the additives shown in the "Expansion culture medium" column of Table 2 to Advanced DMEM/F12 medium (no boric acid) ) and a medium to which 0.3mM boric acid was added, a total of two types of expansion culture medium were prepared. After 3 days of activation culture, cells were collected from the T225 flask, suspended in two types of expansion culture media at 40,000 cells/mL, and cultured using G-Rex (registered trademark) 10M (Wilson Wolf). , and cultured under 5% CO 2 /37°C.
• G-Rex registered trademark
• Test example 2 Proliferation test to verify the effect of addition of boric acid in serum-free expansion culture medium
• item 5. of Example 2 was conducted.
• the cell number and survival rate of iPS cell-derived CAR-T cells were measured on days 3, 5, 6, 7, 10, 12, and 14 of culture.
• Figure 2 shows the cell proliferation rate from day 0 of expansion culture.
• Proliferation of iPS cell-derived CAR-T cells was enhanced by adding 0.3 mM boric acid to the serum-free medium (FIG. 2).
• Example 3 Recovery culture of iPS cell-derived CAR-T cells 500,000 iPS cell-derived CAR-T cells were cultured in a medium containing IMDM medium containing 15% FBS and the additives shown in the "Recovery culture medium" column of Table 3. The cells were suspended at a concentration of 100ml/ml, seeded on G-Rex (registered trademark) 10M (Wilson Wolf), and cultured for 3 days at 37°C in 5% CO 2 .
• G-Rex registered trademark
• 10M Woodson Wolf
• RetroNectin (trade name) was purchased from Takara Bio.
• Anti-CD3 mAb GMP grade Anti-CD3 monoclonal antibody (Clone: OKT3) purchased from Takara Bio Inc. was used.
• Activation culture of iPS cell-derived CAR-T cells After recovery culture, iPS cell-derived CAR-T cells were added to IMDM medium containing 15% FBS with the additives shown in the "Activation culture medium” column of Table 3. Suspend cells at 133,333 cells/mL in culture medium, seed in T225 flasks immobilized with anti-CD3 agonist antibody (OKT3) and RetroNectin (trade name), and store at 37°C in 5 % CO2. It was cultured for 3 days.
• Expansion culture of iPS cell-derived CAR-T cells in five types of serum-free media The additives shown in the "Expansion culture medium” column of Table 3 were added to Advanced DMEM/F12 medium, and boric acid was added at 0. Five types of expansion culture media containing 2mM, 0.3mM, 0.4mM, 0.6mM, and 0.9mM were prepared. After 3 days of activation culture, cells were collected from the T225 flask, suspended in each expansion culture medium at 50,000 cells/mL, and cultured using a G-Rex (registered trademark) 24-well plate (Wilson Wolf). , and cultured under 5% CO 2 /37°C.
• G-Rex registered trademark
• Test example 3 Proliferation test of iPS cell-derived CAR-T cells when boric acid was added at different concentrations to a serum-free expansion culture medium. Item 5 of 3. The number of iPS cell-derived CAR-T cells was counted over time in the expanded culture. Figure 3 shows the cell proliferation rate from day 0 of expansion culture. Maximum fold growth was achieved at a final concentration of boric acid ranging from 0.3 to 0.6 mM. It was also confirmed that boric acid concentration in the range of 0.2mM to 0.9mM did not affect cell viability (FIG. 3).
• Example 4 Recovery culture of iPS cell-derived CAR-T cells 500,000 iPS cell-derived CAR-T cells were cultured in a medium containing IMDM medium containing 15% FBS and the additives shown in the "Recovery culture medium" column of Table 4. The cells were suspended at a concentration of 100 mL/mL, seeded on G-Rex (registered trademark) 10M (Wilson Wolf), and cultured for 3 days at 37° C. in 5% CO 2 .
• G-Rex registered trademark
• 10M Woodson Wolf
• RetroNectin (trade name) was purchased from Takara Bio.
• Anti-CD3 mAb GMP grade Anti-CD3 monoclonal antibody (Clone: OKT3) purchased from Takara Bio Inc. was used.
• Activation culture of iPS cell-derived CAR-T cells After recovery culture, iPS cell-derived CAR-T cells were added to IMDM medium containing 15% FBS with the additives shown in the "Activation culture medium” column of Table 4. Suspend cells at 133,333 cells/mL in culture medium, seed in a T225 flask immobilized with anti-CD3 agonist antibody (OKT3) and RetroNectin (trade name), and store at 37°C in 5% CO2 . The cells were cultured for 3 days.
• Expansion culture of iPS cell-derived CAR-T cells in five types of serum-free media Expansion culture medium prepared by adding the additives shown in the "Expansion culture medium" column of Table 4 to Advanced DMEM/F12 medium (Thermo Fisher), and 0.3mM boric acid (Sigma-Aldrich), 0.3mM boric acid and 1xITS-X ((Insulin-Transferrin-Selenium-Ethanolamine Supplements)Gibco), and 0.3mM boric acid and 1xITS-X, respectively.
• a total of 5 types of expansion culture media were added with 1mM taurine (Sigma-Aldrich), 0.3mM boric acid, 1xITS-X and 3mM taurine (additives in Table 4 only, additives in Table 4 + 0.3mM boric acid). Supplemented medium, Table 4 additives + 0.3mM boric acid + ITS-X supplemented medium, Table 4 additives + 0.3mM boric acid + ITS-X + 1mM taurine supplemented medium, Table 4 additives + 0.3mM boric acid + ITS-X + 3mM taurine supplemented medium) was prepared.
• Test example 4 Proliferation test to verify the additive effect of ITS-X and taurine in serum-free medium containing 0.3mM boric acid Item 5 of Example 4.
• the cell number and survival rate of iPS cell-derived CAR-T cells were measured on days 3, 5, 6, 7, 10, 12, and 14 after culture.
• Figure 4 shows the cell proliferation rate from day 0 of expansion culture.
• Proliferation of iPS cell-derived CAR-T cells was enhanced by further adding 1xITS-X and 1mM or 3mM taurine to a serum-free medium containing 0.3mM boric acid ( Figure 4), and 1mM or 3mM taurine The survival rate improved by 1 to 4% by adding .
• Example 5 Activation culture of human-derived primary T cells
• Human-derived primary T cells (Leukopak-SoloPak, Charles River Laboratories Cell Solutions) were cultured in OpTmizer medium (Thermo Fisher) containing 2% CTS Immune Cell SR (Thermo Fisher) according to Table 6. Suspend at 1,000,000 cells/mL in a medium containing the additives shown in the activation culture medium, seed in PL240 bag (OriGen), and culture for 2 days under 5% CO 2 /37°C. did.
• OpTmizer medium Thermo Fisher
• CTS Immune Cell SR Thermo Fisher
• Reagent RetroNectin (trade name) was purchased from Takara Bio.
• RetroNectin (trade name) and retrovirus on a culture plate RetroNectin (final concentration 150 ⁇ g/mL) dissolved in PBS at the required concentration was added to a PL120 bag, and then left at room temperature for 2 hours. After washing with PBS, a retrovirus solution was added and solidified overnight at 4°C while shaking at 50 rpm. After washing with PBS containing 1.5% HSA, it was subjected to the test.
• human-derived primary T cells After activation culture, human-derived primary T cells were cultured in OpTmizer medium containing 2% CTS Immune Cell SR with the additives shown in the transduction culture medium in Table 6. The cells were suspended at 722,222 cells/mL and seeded in a PL120 bag immobilized with a retroviral vector containing RetroNectin (trade name) and a CAR gene that recognizes a specific antigen, and incubated at 5% CO 2 /mL. The cells were cultured at 37°C for 1 day.
• Expansion culture of CAR-T cells derived from human primary T cells in four types of serum-free media Expansion culture medium prepared by adding the additives shown in Expansion Culture Medium 1 in Table 6 to OpTmizer medium containing 2% CTS Immune Cell SR, An expansion culture medium containing the components listed in Table 7 (hereinafter referred to as BM220720) with the additives shown in Expansion Culture Medium 2 in Table 6, and an additional 0.3mM boric acid (Sigma-Aldrich) or A total of four types of expansion culture media containing 0.3mM boric acid and 3mM taurine (Sigma-Aldrich) were used (OpTmizer medium with only the additives in Table 6, BM220720 medium with (i) only the additives in Table 6, (ii) A medium to which either the additives shown in Table 6 + 0.3mM boric acid or (iii) the additives shown in Table 6 + 0.3mM boric acid + 3mM taurine were added was prepared.
• Test example 5 Proliferation test to verify the effect of addition of boric acid and taurine on CAR-T cells derived from human primary T cells. Item 5 of Example 5 for verification. In the expanded culture, the cell number and survival rate of CAR-T cells derived from human primary T cells were measured on days 4, 6, 10, and 11. Figure 5 shows the cell proliferation rate from day 0 of expansion culture. The proliferation of human primary T cell-derived CAR-T cells was enhanced by further adding 0.3 mM boric acid to the serum-free medium or 3 mM taurine to 0.3 mM boric acid (FIG. 5).
• Example 6 Preparation of iPS cell-derived NK cells
• the iPS cell FfI01s04 strain provided by Kyoto University iPS Cell Research Institute (CiRA) was used to differentiate into NK cells according to a known method (for example, the method described in WO2021/174004).
• the obtained cells were cryopreserved in liquid nitrogen and used as iPS cell-derived NK cells.
• iPS cell-derived NK cells were cultured 1.25 ⁇ in a recovery culture medium prepared by adding the additives shown in Table 8 to IMDM medium (Thermo Fisher Scientific) containing 15% Fetal Bovine Serum (FBS). They were suspended at 10 5 cells/mL, seeded on a G-Rex (registered trademark) 6-well plate (Wilson Wolf), and cultured in a 5% CO 2 /37°C incubator for 3 days (culture 0-3). day).
• IMDM medium Thermo Fisher Scientific
• FBS Fetal Bovine Serum
• iPS cell-derived NK cells were activated in IMDM medium (Thermo Fisher Scientific) containing 15% Fetal Bovine Serum (FBS) with the additives shown in Table 9 added. They were suspended in a medium to a density of 1.84 x 10 5 cells/mL, seeded in a 6-well plate (TPP), and cultured in a 5% CO 2 /37°C incubator for 3 days (days 3 to 6 of culture). ).
• IMDM medium Thermo Fisher Scientific
• FBS Fetal Bovine Serum
• Cells that had been activated and cultured for 3 days were suspended in 5 types of expansion culture media at a density of 1.25 x 10 5 cells/mL, and then seeded again on G-Rex (registered trademark) 24-well plates (Wilson Wolf).
• the cells were cultured in a 5% CO 2 /37°C incubator for 2 days (6th to 8th day of culture).
• the medium was changed to a medium containing the above five types of basal medium plus the additives shown in Table 10, and cultured in a 5% CO 2 /37°C incubator for 2 days (8th to 10th day of culture).
• the cells were replated into G-Rex (registered trademark) 24-well plates (Wilson Wolf) at 1.25 x 10 5 cells/mL in the same medium, and incubated with 5% CO 2 / Culture was continued in a 37°C incubator until the 17th day of culture.
• G-Rex registered trademark
• Test example 6 Proliferation test to verify the additive effects of boric acid and taurine on iPS cell-derived NK cells Item 4 of Example 6 was used to verify the effects of boric acid and taurine addition in a serum-free expansion culture medium on iPS cell-derived NK cells. In the expanded culture, the cell number and survival rate of iPS cell-derived NK cells were measured over time. Table 11 shows the proliferation rate and cell survival rate on the 11th day of expansion culture. Proliferation of iPS cell-derived NK cells was enhanced by further adding 0.3mM boric acid and 1mM taurine or 3mM taurine to the serum-free basal medium (Table 11).
• the present invention even when using a serum-free medium or a low-serum medium, it is possible to obtain cell proliferation maintenance and promotion effects comparable to those of culture in a serum-containing medium.
• cells that are susceptible to stress due to operations such as cell washing are preferably cultured in serum-free or low-serum media that do not require strong washing, and are suitable for culture under such conditions.

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