Method for producing target cells, method for producing product by target cells, and serum-free medium

Abstract

To provide a cell culture technology using a serum substitute, which can maintain the cell culture efficiency when serum is used, and can solve the problems of cost when serum is used and the problems of quality stability of cultured cells when serum is used. [Solution] A method for producing target cells, comprising culturing starting cells in a serum-free medium containing at least one culture factor selected from a growth factor and a differentiation-inducing factor, human serum albumin, and at least one additive selected from the group consisting of a Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin, to produce target cells.

Description

[Technical field]

[0001] The present invention relates to a cell culture technique using a serum substitute. [Background technology]

[0002] Conventionally, animal cells, including human cells, have been cultured by adding serum to a synthetic medium. Serum contains various components necessary for maintaining the functions of the living body in addition to proteins such as albumin and globin. For example, fetal bovine serum is used for culturing animal cells, including human cells. Since serum is derived from animals, there are problems such as cost and lot differences. To solve these problems, serum-free media containing serum substitutes instead of serum have been developed (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2014 / 144198 Summary of the Invention [Problem to be solved by the invention]

[0004] As described above, since serum is derived from animals, there are problems with costs and quality stability of cultured cells due to lot differences, especially when culturing a large amount of cells. On the other hand, there is a concern that serum substitutes may reduce cell culture efficiency compared to serum. Therefore, the present invention aims to provide a cell culture technology using a serum substitute that can maintain the cell culture efficiency when serum is used and can solve the problems of costs and quality stability of cultured cells when serum is used. [Means for solving the problem]

[0005] According to one aspect, At least one culture factor selected from a growth factor and a differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. and culturing source cells in a serum-free medium comprising the above-mentioned component to produce the target cells.

[0006] In another aspect, there is provided a method for producing a product by a target cell, comprising culturing a source cell according to the above-described method to produce the target cell and to cause the target cell to produce the product.

[0007] According to yet another aspect, At least one culture factor selected from a growth factor and a differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. A serum-free medium for use in the production of a cell of interest is provided, comprising:

[0008] At least one culture factor selected from a growth factor and a differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. A serum-free medium for use in the production of a product by a cell of interest is provided, comprising: Effect of the Invention

[0009] According to the present invention, there is provided a cell culture technology using a serum substitute, which can maintain the cell culture efficiency when serum is used, and can solve the problems of cost and quality stability of cultured cells that occur when serum is used. [Brief description of the drawings]

[0010] [Figure 1] Graph showing the number of megakaryocytes differentiated in each medium. [Diagram 2] Graph showing the number of megakaryocytes differentiated in each medium. [Diagram 3] Graph showing the number of megakaryocytes differentiated in each medium. [Figure 4A] Photograph showing megakaryocytes stained with a nuclear staining reagent. [Figure 4B] Photograph showing megakaryocytes stained with a nuclear staining reagent. [Figure 4C] Photograph showing megakaryocytes stained with a nuclear staining reagent. [Figure 4D] Photograph showing megakaryocytes stained with a nuclear staining reagent. [Figure 4E] Photograph showing megakaryocytes stained with a nuclear staining reagent. [Diagram 5] Graph showing the positive cell rate for platelet markers (CD41 and CD42b). [Figure 6] Graph showing the positive cell rate for the platelet marker (PAC1). [Figure 7] Graph showing the number of iPS cells grown in each medium. [Figure 8] Graph showing the number of mesenchymal stem cells proliferated in each medium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below. However, the following description is for the purpose of explaining the present invention in detail and is not intended to limit the present invention.

[0012] The present inventors have screened various substances for serum substitutes. As a result, the present inventors have found that the culture efficiency of cells when serum is used can be maintained by using a combination of (a) human serum albumin and (b) at least one additive selected from the group consisting of Src inhibitors, PKC activators, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin as a serum substitute, and have completed the present invention. In this specification, the term "culture efficiency" refers to the differentiation efficiency in the case of differentiation culture, and refers to the proliferation efficiency in the case of proliferation culture.

[0013] <1. Method for producing target cells> According to one aspect, At least one culture factor selected from a growth factor and a differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. and culturing source cells in a serum-free medium comprising the above-mentioned component to produce the target cells.

[0014] In one embodiment, the culture factor is at least one differentiation-inducing factor, and the target cell is a differentiated cell. The differentiation-inducing factor has the effect of promoting differentiation of the starting cell. In this embodiment, the above method is a "method for producing differentiated cells". That is, the "method for producing differentiated cells" is At least one differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. The method includes culturing the source cells in a serum-free medium containing the above-mentioned components to produce differentiated cells.

[0015] In another embodiment, the culture factor is at least one growth factor, and the target cells are proliferating cells. The growth factor has the effect of promoting the proliferation of the starting cells. In this embodiment, the above method is a "method for producing proliferating cells". That is, the "method for producing proliferating cells" is At least one growth factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. The method includes culturing the source cells in a serum-free medium comprising:

[0016] The "method for producing differentiated cells" and the "method for producing proliferating cells" are specifically explained below.

[0017] <1-1. Method for producing differentiated cells> (cell) In the method for producing differentiated cells, starting cells are cultured in the presence of a differentiation-inducing factor to produce differentiated cells.

[0018] The source cells may be selected from the group consisting of megakaryocyte progenitor cells, iPS cells, mesenchymal stem cells, and fibroblasts, for example. The source cells may be selected from the group consisting of human megakaryocyte progenitor cells, human iPS cells, human mesenchymal stem cells, and human fibroblasts, for example.

[0019] The differentiated cells may be selected from the group consisting of, for example, megakaryocytes, mesenchymal stem cells, fibroblasts, and cardiomyocytes. The differentiated cells may be preferably selected from the group consisting of human megakaryocytes, human mesenchymal stem cells, human fibroblasts, and human cardiomyocytes.

[0020] According to one embodiment, the source cells are megakaryocyte progenitor cells and the differentiated cells are megakaryocytes. Preferably, the source cells are human megakaryocyte progenitor cells and the differentiated cells are human megakaryocytes. According to another embodiment, the source cells are iPS cells and the differentiated cells are mesenchymal stem cells. Preferably, the source cells are human iPS cells and the differentiated cells are human mesenchymal stem cells.

[0021] According to yet another embodiment, the source cells are iPS cells and the differentiated cells are fibroblasts. Preferably, the source cells are human iPS cells and the differentiated cells are human fibroblasts. According to yet another embodiment, the source cells are iPS cells and the differentiated cells are cardiomyocytes. Preferably, the source cells are human iPS cells and the differentiated cells are human cardiomyocytes.

[0022] According to yet another embodiment, the source cells are mesenchymal stem cells and the differentiated cells are cardiomyocytes. Preferably, the source cells are human mesenchymal stem cells and the differentiated cells are human cardiomyocytes. According to yet another embodiment, the source cells are fibroblasts and the differentiated cells are cardiomyocytes. Preferably, the source cells are human fibroblasts and the differentiated cells are human cardiomyocytes.

[0023] (serum substitute) The method for producing differentiated cells includes, as a serum substitute, (a) human serum albumin; (b) at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and a non-human animal serum albumin; The present invention is characterized in that a combination of the above is used.

[0024] Human serum albumin is prepared from human plasma or serum and is not recombinant. Human serum albumin is commercially available, for example from Sigma-Aldrich.

[0025] The additive is preferably a Src inhibitor. That is, the serum replacement is preferably a combination of human serum albumin and a Src inhibitor. "Src inhibitor" refers to a substance that inhibits Src, which is a tyrosine kinase, or the signal transduction pathway downstream thereof. The Src inhibitor can be a known Src inhibitor. Examples of Src inhibitors include A419259, SU6656, PP1, 1-naphthyl PP1, PP2, indirubin-3'-(2,3-dihydroxypropyl)-oximether, TX-1123, Src Kinase Inhibitor I (CAS 179248-59-0), AZM475271, bosutinib, herbimycin A, KB SRC 4, MNS, PD166285, and TC-S7003.

[0026] The Src inhibitor is preferably A419259, which has the following structure, and the compound name of A419259 is 7-[4-(4-methylpiperazin-1-yl)cyclohexyl]-5-(4-phenoxyphenyl)pyrrolo[2,3-d]pyrimidin-4-amine. [ka]

[0027] A419259 may be added in the form of a salt. A419259 is commercially available, for example, from FUJIFILM Wako Pure Chemical Industries, Ltd.

[0028] Alternatively, the additive is preferably a PKC activator. That is, the serum replacement is preferably a combination of human serum albumin and a PKC activator. The term "PKC activator" refers to a substance that activates protein kinase C (PKC) or its downstream signal transduction pathway. The PKC activator may be a known PKC activator. Examples of the PKC activator include phorbol 12-myristate 13-acetate (PMA), prostratin, bryostatin 1, bryostatin 2, FR236924, (-)-indolactam V, PEP005, phorbol 12,13-dibutyrate, and the like. 12,13-dibutyrate, SC-9, SC-10, 1-Oleoyl-2-acetyl-sn-glycerol, 1-O-Hexadecyl-2-O-arachidonyl-sn-glycerol, 1,2-Dioctanoyl-sn-glycerol, PIP2, Resiniferatoxin, Phorbol 12,13-Dihexanoate, Mezerein, Ingenol 3-Angelate 3-Angelate, RHC-80267, DCP-LA, Lipoxin A4, etc. are examples.

[0029] The PKC activator is preferably prostratin. Prostratin has the following structure, and the compound name of prostratin is (1aR,1bS,4aR,7aS,7bR,8R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1,2-e]azulen-9a-yl acetate. Prostratin is commercially available, for example, from Fujifilm Wako Pure Chemical Industries, Ltd. [ka]

[0030] Alternatively, the additive is preferably methylcellulose. That is, the serum replacement is preferably a combination of human serum albumin and methylcellulose. Methylcellulose is commercially available, for example, from R&D Systems.

[0031] Alternatively, the additive is preferably Essential 8 medium. That is, the serum replacement is preferably a combination of human serum albumin and Essential 8 medium. According to the paper 1 (Chen G et al, Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011 May;8(5):424-9. doi: 10.1038 / nmeth.1593. Epub 2011 Apr 10.), Essential 8 medium has the following composition: DMEM / F12, L-ascorbic acid-2-phosphate magnesium (64 mg / l), sodium selenium (14 μg / l), FGF2 (100 μg / l), insulin (19.4 mg / l), NaHCO3 (543 mg / l), transferrin (10.7 mg / l), and either TGFβ1 (2 μg / l) or NODAL (100 μg / l). Essential 8 medium is commercially available, for example from Invitrogen.

[0032] Alternatively, the additive is preferably recombinant human serum albumin or non-human animal serum albumin. That is, the serum replacement is preferably a combination of human serum albumin and either recombinant human serum albumin or non-human animal serum albumin. The recombinant human serum albumin is, for example, human serum albumin expressed in rice or human serum albumin expressed in yeast. The non-human animal serum albumin is, for example, bovine serum albumin. Recombinant human serum albumin and non-human animal serum albumin are commercially available, for example, from Sigma-Aldrich or Fujifilm Wako Pure Chemical Corporation.

[0033] Serum replacements can be used at the concentrations listed below, which represent the final concentrations in the medium. The concentration of human serum albumin in the serum-free medium is, for example, 0.01 to 2.0% by mass, preferably 0.05 to 1.5% by mass, and more preferably 0.075 to 0.75% by mass.

[0034] When an Src inhibitor such as A419259 is used as an additive, the concentration of the Src inhibitor in the serum-free medium is, for example, 0.001 to 10 μM, preferably 0.01 to 1.0 μM, and more preferably 0.03 to 0.2 μM. When a PKC activator such as prostratin is used as an additive, the concentration of the PKC activator in the serum-free medium is, for example, 0.001 to 10 μM, preferably 0.01 to 1.0 μM, and more preferably 0.015 to 0.2 μM.

[0035] When methylcellulose is used as an additive, the concentration of methylcellulose in the serum-free medium is, for example, 0.001 to 3.0% by mass, preferably 0.01 to 1.0% by mass, and more preferably 0.018 to 0.3% by mass. When Essential 8 medium is used as an additive, the concentration of Essential 8 medium in the serum-free medium is, for example, 0.5 to 100% by mass, preferably 0.5 to 50% by mass, and more preferably 0.625 to 30% by mass.

[0036] When recombinant human serum albumin is used as an additive, the concentration of recombinant human serum albumin in the serum-free medium is, for example, 0.001 to 1.0% by mass, preferably 0.01 to 0.5% by mass, and more preferably 0.015 to 0.3% by mass. When non-human animal serum albumin is used as an additive, the concentration of non-human animal serum albumin in the serum-free medium is, for example, 0.01 to 2.0 mass%, preferably 0.05 to 1.5 mass%, and more preferably 0.125 to 0.75 mass%.

[0037] (culture) The method for producing differentiated cells can be carried out in the same manner as conventional differentiation culture using serum, except that a combination of (a) human serum albumin and (b) at least one additive selected from the group consisting of Src inhibitor, PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin is used as a serum substitute. More specifically, the method for producing differentiated cells can be carried out by culturing raw material cells in a serum-free medium containing a basal medium (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.), a differentiation inducer, and the above-mentioned serum substitute.

[0038] The differentiation induction factor may be a known factor depending on the type of starting cell and differentiated cell. The differentiation induction factor may have the effect of promoting cell proliferation in addition to the effect of promoting differentiation of the starting cell. The differentiation culture may be composed of a single culture step using one or more specific differentiation induction factors, or may be composed of multiple culture steps using one or more different differentiation induction factors for each culture step.

[0039] By culturing, the starting cells are differentiated to produce differentiated cells, and during the culture, in addition to cell differentiation, cell proliferation may also occur.

[0040] For example, the differentiation culture of megakaryocyte progenitor cells into megakaryocytes can be carried out by adding megakaryocyte differentiation inducer and the above-mentioned serum substitute to basic medium to prepare serum-free medium, and culturing megakaryocyte progenitor cells in this serum-free medium.As the basic medium, for example, IMDM can be used, and as the megakaryocyte differentiation inducer, for example, SCF, TA-316, KP-457, GNF-351 and Y39983 can be used.

[0041] SCF is a known protein called stem cell factor, and is available, for example, from R&D Systems. TA-316 has the following structure and is available, for example, from Nissan Chemical Industries, Ltd. KP-457 has the following structure and is available, for example, from GLPBIO (https: / / www.glpbio.com / kp-457.html). GNF-351 has the following structure and is available, for example, from Santa Cruz Biotechnology (https: / / www.scbt.com / ja / p / ahr-antagonist-iii-gnf351). Y39983 has the following structure and is available, for example, from Selleck Biotech Co., Ltd. (https: / / www.selleck.co.jp / products / y-39983-hcl.html).

[0042] [ka]

[0043] [ka]

[0044] It is known that KP-457 functions as a substance that maintains the function of platelets produced by megakaryocytes, i.e., as a platelet function maintaining agent (see WO2012 / 157586). Also, GNF-351 and Y-39983 are known to function as substances that promote platelet production by megakaryocytes, i.e., as platelet production promoters (see WO2016 / 204256).

[0045] In the above explanation, a megakaryocyte differentiation-inducing factor is given as an example, but when cell differentiation occurs by carrying out the method for producing differentiated cells, it is clear that the serum-free medium contains a differentiation-inducing factor, and it is not necessarily necessary to specify which component in the serum-free medium is the differentiation-inducing factor.

[0046] (effect) The above-mentioned method uses a serum substitute, and is advantageous in that it can maintain the cell differentiation efficiency when serum is used. Furthermore, the above-mentioned method can solve the problems of cost and quality stability of cultured cells when serum is used by using a serum substitute.

[0047] <1-2. Method for producing proliferating cells> In the method for producing proliferating cells, starting cells are cultured in the presence of a growth factor to produce proliferating cells. As used herein, the term "growth factor" refers to any substance that contributes to cell proliferation, such as a substance that serves as a nutrient source for cell proliferation or a substance that promotes cell proliferation.

[0048] The source cells and the target cells may be selected from the group consisting of, for example, iPS cells and mesenchymal stem cells. The source cells and the target cells may be preferably selected from the group consisting of human iPS cells and human mesenchymal stem cells.

[0049] The method for producing proliferating cells is characterized by using, as a serum replacement, a combination of (a) human serum albumin and (b) at least one additive selected from the group consisting of a Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and a non-human animal serum albumin.

[0050] The serum substitute is as described above in the section <1-1. Method for producing differentiated cells>. The serum substitute can be used at the concentration described above in the section <1-1. Method for producing differentiated cells>.

[0051] The method for producing proliferating cells can be carried out in the same manner as conventional proliferating culture using serum, except that a combination of (a) human serum albumin and (b) at least one additive selected from the group consisting of Src inhibitor, PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin is used as a serum substitute. More specifically, the method for producing proliferating cells can be carried out by culturing raw material cells in a serum-free medium containing a basal medium (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.) containing growth factors and the above-mentioned serum substitute.

[0052] The basal medium usually contains growth factors such as amino acids, vitamins, and inorganic salts, and in addition to the growth factors contained in the basal medium, additional growth factors may be added to the basal medium.

[0053] For example, the proliferation and culture of iPS cells can be carried out by preparing a serum-free medium by adding the above-mentioned serum substitute to a commercially available medium for proliferation of iPS cells, and culturing iPS cells in this serum-free medium. As the medium for proliferation of iPS cells, for example, DMEM / F12 can be used, and DMEM / F12 contains, for example, L-glutamine, vitamin B12, etc. as an iPS cell growth factor. As the serum substitute, a combination of human serum albumin and prostratin, or a combination of human serum albumin and recombinant human serum albumin can be preferably used.

[0054] For example, the proliferation and culture of mesenchymal stem cells can be carried out by preparing a serum-free medium by adding the above-mentioned serum substitute to a commercially available mesenchymal stem cell proliferation medium, and culturing mesenchymal stem cells in this serum-free medium. As the mesenchymal stem cell proliferation medium, for example, DMEM / F12 can be used, and DMEM / F12 contains, for example, L-glutamine, vitamin B12, etc. as mesenchymal stem cell growth factors. As the serum substitute, a combination of human serum albumin and A419259, or a combination of human serum albumin and recombinant human serum albumin can be preferably used.

[0055] Alternatively, the above-mentioned method can be applied to megakaryocyte progenitor cells to proliferate and culture the megakaryocyte progenitor cells, or the above-mentioned method can be applied to cardiomyocytes to proliferate and culture the cardiomyocytes.

[0056] In the above explanation, iPS cell growth factor and mesenchymal stem cell growth factor are given as examples, but when cell proliferation occurs by carrying out the method for producing proliferative cells, it is clear that the serum-free medium contains a growth factor, and it is not necessarily necessary to specify which component in the serum-free medium is the growth factor.

[0057] The above-mentioned method uses a serum substitute, and is advantageous in that it can maintain the cell growth efficiency when serum is used. Furthermore, the above-mentioned method can solve the problems of cost and quality stability of cultured cells when serum is used by using a serum substitute.

[0058] <2. Method for producing a product using target cells> According to another aspect, there is provided a method for producing a product by a target cell, comprising culturing a source cell according to the above-mentioned method to produce the target cell and to allow the target cell to produce the product. At least one culture factor selected from a growth factor and a differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. The present invention provides a method for producing a product from a target cell, comprising culturing a source cell in a serum-free medium containing the above compound to produce the target cell and to allow the target cell to produce the product.

[0059] In this method, the starting cells can be cultured according to the same method as described above in the section <1-1. Method for producing differentiated cells>. This method may further include a step of recovering the product produced by the target cells.

[0060] According to one embodiment, in a method for producing a product from target cells, the source cells are megakaryocyte progenitor cells, the target cells are megakaryocytes, and the product is platelets. At least one megakaryocyte differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. The present invention provides a method for producing platelets by megakaryocytes, comprising culturing megakaryocyte precursor cells in a serum-free medium containing the above compound to produce megakaryocytes and to allow the megakaryocytes to produce platelets.

[0061] For example, the production of platelets by megakaryocytes can be carried out by preparing a serum-free medium by adding megakaryocyte differentiation inducer and the above-mentioned serum substitute to a basic medium, and culturing megakaryocyte progenitor cells in this serum-free medium.As the basic medium, for example, IMDM can be used, and as the megakaryocyte differentiation inducer, for example, SCF, TA-316, KP-457, GNF-351, and Y39983 can be used.By culturing, megakaryocyte progenitor cells differentiate to produce megakaryocytes, and platelets are produced by the produced megakaryocytes.

[0062] As described above, KP-457 is known to function as a substance that maintains the function of platelets produced by megakaryocytes, i.e., as a platelet function maintaining agent (see WO2012 / 157586). In addition, GNF-351 and Y-39983 are known to function as substances that promote platelet production by megakaryocytes, i.e., as platelet production promoting agents (see WO2016 / 204256).

[0063] Alternatively, according to another embodiment, iPS cells or mesenchymal stem cells can be expanded in culture to cause these cells to produce cytokines.

[0064] That is, according to another aspect, At least one iPS cell growth factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. The present invention provides a method for producing cytokines by iPS cells, comprising culturing iPS cells in a serum-free medium containing the above compound to cause the iPS cells to produce cytokines.

[0065] In this method, the culture of iPS cells can be performed according to the same method as that described for the proliferation and culture of iPS cells in the above section <1-2. Method for producing proliferated cells>. The iPS cells proliferate during the culture and produce cytokines.

[0066] According to yet another aspect, at least one mesenchymal stem cell growth factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. The present invention provides a method for producing cytokines by mesenchymal stem cells, comprising culturing the mesenchymal stem cells in a serum-free medium containing the above compound to cause the mesenchymal stem cells to produce cytokines.

[0067] In this method, the mesenchymal stem cells can be cultured according to the same method as that described for the proliferation and culture of mesenchymal stem cells in the above section <1-2. Method for producing proliferated cells>. Through the culture, the mesenchymal stem cells proliferate and produce cytokines.

[0068] The above-mentioned method uses a serum substitute, and is advantageous in that it can maintain the cell culture efficiency and cell product production efficiency when serum is used. Furthermore, the above-mentioned method can solve the problems of cost and quality stability of cultured cells when serum is used by using a serum substitute.

[0069] <3. Serum-free medium> <3-1. Serum-free medium for use in the production of target cells> In another aspect, At least one culture factor selected from a growth factor and a differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. A serum-free medium for use in the production of a cell of interest is provided, comprising:

[0070] This serum-free medium is used in the production of target cells, and specifically, can be used in the method described in the above section <1. Method for producing target cells>. That is, the serum-free medium can be used in the production of proliferating cells or differentiated cells. Therefore, for details of the serum-free medium, the description of the medium described in the above section <1. Method for producing target cells> can be referred to.

[0071] The serum-free medium is characterized by comprising a combination of (a) human serum albumin and (b) at least one additive selected from the group consisting of Src inhibitors, PKC activators, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin as serum substitutes. The serum substitute is as described above in the section <1-1. Method for producing differentiated cells>. The serum substitute may be contained in the serum-free medium at a concentration described above in the section <1-1. Method for producing differentiated cells>.

[0072] In one embodiment, the serum-free medium is a medium in which the serum contained in a known cell differentiation-inducing medium containing a differentiation-inducing factor is replaced with the above-mentioned serum substitute. In this embodiment, the serum-free medium contains a basal medium (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.), a differentiation-inducing factor, and the above-mentioned serum substitute.

[0073] In another embodiment, the serum-free medium is a medium in which the serum contained in a known cell growth medium containing a growth factor is replaced with the above-mentioned serum substitute. In this embodiment, the serum-free medium contains a basal medium containing a growth factor (e.g., MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.) and the above-mentioned serum substitute.

[0074] According to a specific embodiment, in the serum-free medium for use in producing a target cell, the target cell is a megakaryocyte, and the culture factor is a megakaryocyte differentiation-inducing factor.That is, in this specific embodiment, the serum-free medium comprises a basal medium (such as IMDM), a megakaryocyte differentiation-inducing factor, and the above-mentioned serum replacement.As mentioned above, the megakaryocyte differentiation-inducing factor is, for example, SCF, TA-316, KP-457, GNF-351, and Y39983.

[0075] The above-mentioned serum-free medium contains a serum substitute, but is superior in that it can maintain the cell culture efficiency (specifically, differentiation efficiency and proliferation efficiency) when the corresponding medium containing serum is used. Furthermore, the above-mentioned serum-free medium can solve the problems of cost and quality stability of cultured cells when serum is used by using a serum substitute.

[0076] <3-2. Serum-free medium for use in the production of products by target cells> In another aspect, At least one culture factor selected from a growth factor and a differentiation-inducing factor; Human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. A serum-free medium for use in the production of a product by a cell of interest is provided, comprising:

[0077] This serum-free medium is used in the production of a product by a target cell, and specifically, can be used in the method described above in the section <2. Method for producing a product by a target cell>. Therefore, for details of the serum-free medium, the description of the medium described in the section <2. Method for producing a product by a target cell> can be referred to.

[0078] The serum-free medium is characterized by comprising a combination of (a) human serum albumin and (b) at least one additive selected from the group consisting of Src inhibitors, PKC activators, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin as serum substitutes. The serum substitute is as described above in the section <1-1. Method for producing differentiated cells>. The serum substitute may be contained in the serum-free medium at a concentration described above in the section <1-1. Method for producing differentiated cells>.

[0079] According to a specific embodiment, in the serum-free medium for use in the production of a product by a target cell, the production of a product by a target cell is the production of platelets by megakaryocytes, and the culture factor is a megakaryocyte differentiation-inducing factor.That is, in this specific embodiment, the serum-free medium comprises a basal medium (e.g., IMDM, etc.), a megakaryocyte differentiation-inducing factor, and the above-mentioned serum replacement.As mentioned above, the megakaryocyte differentiation-inducing factor is, for example, SCF, TA-316, KP-457, GNF-351, and Y39983.

[0080] As described above, KP-457 is known to function as a substance that maintains the function of platelets produced by megakaryocytes, i.e., as a platelet function maintaining agent (see WO2012 / 157586). In addition, GNF-351 and Y-39983 are known to function as substances that promote platelet production by megakaryocytes, i.e., as platelet production promoting agents (see WO2016 / 204256).

[0081] The above-mentioned serum-free medium contains a serum substitute, but is advantageous in that it can maintain the cell culture efficiency and cell product production efficiency when the corresponding medium containing serum is used. Furthermore, the above-mentioned serum-free medium can solve the problems of cost and quality stability of cultured cells when serum is used by using the serum substitute.

[0082] <4. Preferred embodiment> Preferred embodiments of the present invention are summarized below.

[0083] [A1] At least one culture factor selected from a growth factor and a differentiation-inducing factor, human serum albumin, and and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. A method for producing a target cell, comprising culturing a source cell in a serum-free medium containing the above compound to produce the target cell. [A2] The method according to [A1], wherein the culture factor is at least one differentiation-inducing factor, and the target cell is a differentiated cell. [A3] The method according to [A2], wherein the source cells are selected from the group consisting of megakaryocyte progenitor cells, iPS cells, mesenchymal stem cells, and fibroblasts. [A4] The method according to [A2] or [A3], wherein the source cells are selected from the group consisting of human megakaryocyte progenitor cells, human iPS cells, human mesenchymal stem cells, and human fibroblasts. [A5] The method according to any one of [A2] to [A4], wherein the target cells are selected from the group consisting of megakaryocytes, mesenchymal stem cells, fibroblasts, and cardiomyocytes.

[0084] [A6] The method according to any one of [A2] to [A5], wherein the target cells are selected from the group consisting of human megakaryocytes, human mesenchymal stem cells, human fibroblasts, and human cardiomyocytes. [A7] The method according to any one of [A2], [A3], and [A5], wherein the source cells are megakaryocyte progenitor cells and the target cells are megakaryocytes. [A8] The method according to any one of [A2] to [A7], wherein the source cells are human megakaryocyte progenitor cells and the target cells are human megakaryocytes. [A9] The method according to any one of [A2], [A3] and [A5], wherein the source cells are iPS cells and the target cells are mesenchymal stem cells. [A10] The method according to any one of [A2] to [A6] and [A9], wherein the source cells are human iPS cells and the target cells are human mesenchymal stem cells.

[0085] [A11] The method according to any one of [A2], [A3] and [A5], wherein the source cells are iPS cells and the target cells are fibroblasts. [A12] The method according to any one of [A2] to [A6] and [A11], wherein the source cells are human iPS cells and the target cells are human fibroblasts. [A13] The method according to any one of [A2], [A3] and [A5], wherein the source cells are iPS cells and the target cells are cardiomyocytes. [A14] The method according to any one of [A2] to [A6] and [A13], wherein the source cells are human iPS cells and the target cells are human cardiomyocytes. [A15] The method according to any one of [A2], [A3] and [A5], wherein the source cells are mesenchymal stem cells and the target cells are cardiomyocytes.

[0086] [A16] The method according to any one of [A2] to [A6] and [A15], wherein the source cells are human mesenchymal stem cells and the target cells are human cardiomyocytes. [A17] The method according to any one of [A2], [A3] and [A5], wherein the source cells are fibroblasts and the target cells are cardiomyocytes. [A18] The method according to any one of [A2] to [A6] and [A17], wherein the source cells are human fibroblasts and the target cells are human cardiomyocytes.

[0087] [B1] The method according to [A1], wherein the culture factor is at least one growth factor, and the target cells are proliferating cells. [B2] The method according to [B1], wherein the source cells and the target cells are selected from the group consisting of iPS cells and mesenchymal stem cells. [B3] The method according to [B1] or [B2], wherein the source cells and the target cells are selected from the group consisting of human iPS cells and human mesenchymal stem cells. [B4] The method according to [B1] or [B2], wherein the source cells and the target cells are iPS cells. [B5] The method according to any one of [B1] to [B4], wherein the source cells and the target cells are human iPS cells.

[0088] [B6] The method according to [B1] or [B2], wherein the source cells and the target cells are mesenchymal stem cells. [B7] The method according to any one of [B1] to [B3] and [B6], wherein the source cells and the target cells are human mesenchymal stem cells. [B8] The method according to [B1], wherein the source cells and the target cells are megakaryocyte progenitor cells. [B9] The method according to [B1] or [B8], wherein the source cells and the target cells are human megakaryocyte progenitor cells. [B10] The method according to [B1], wherein the source cells and the target cells are cardiomyocytes. [B11] The method according to [B1] or [B10], wherein the source cells and the target cells are human cardiomyocytes.

[0089] [C1] The method according to any one of [A1] to [A18] and [B1] to [B11], wherein the additive is an Src inhibitor, preferably A419259. [C2] The method according to any one of [A1] to [A18] and [B1] to [B11], wherein the additive is a PKC activator, preferably prostratin. [C3] The method according to any one of [A1] to [A18] and [B1] to [B11], wherein the additive is methylcellulose. [C4] The method according to any one of [A1] to [A18] and [B1] to [B11], wherein the additive is Essential 8 medium. [C5] The method according to any one of [A1] to [A18] and [B1] to [B11], wherein the additive is recombinant human serum albumin or non-human animal serum albumin.

[0090] [C6] The method according to any one of [A1] to [A18], [B1] to [B11] and [C5], wherein the additive is recombinant human serum albumin, preferably recombinant human serum albumin expressed in rice or recombinant human serum albumin expressed in yeast. [C7] The method according to any one of [A1] to [A18], [B1] to [B11] and [C5], wherein the additive is a non-human animal serum albumin, preferably bovine serum albumin. [C8] The method according to any one of [A1] to [A18], [B1] to [B11] and [C1] to [C7], wherein the concentration (final concentration) of human serum albumin in the serum-free medium is 0.01 to 2.0% by mass, preferably 0.05 to 1.5% by mass, and more preferably 0.075 to 0.75% by mass. [C9] The method according to any one of [A1] to [A18], [B1] to [B11], [C1] and [C8], wherein the additive is a Src inhibitor, preferably A419259, and the concentration (final concentration) of the Src inhibitor in the serum-free medium is 0.001 to 10 μM, preferably 0.01 to 1.0 μM, and more preferably 0.03 to 0.2 μM. [C10] The method according to any one of [A1] to [A18], [B1] to [B11], [C2] and [C8], wherein the additive is a PKC activator, preferably prostratin, and the concentration (final concentration) of the PKC activator in the serum-free medium is 0.001 to 10 μM, preferably 0.01 to 1.0 μM, more preferably 0.015 to 0.2 μM.

[0091] [C11] The method according to any one of [A1] to [A18], [B1] to [B11], [C3] and [C8], wherein the additive is methylcellulose, and the concentration (final concentration) of methylcellulose in the serum-free medium is 0.001 to 3.0% by mass, preferably 0.01 to 1.0% by mass, and more preferably 0.018 to 0.3% by mass. [C12] The method according to any one of [A1] to [A18], [B1] to [B11], [C4] and [C8], wherein the additive is Essential 8 medium, and the concentration (final concentration) of Essential 8 medium in the serum-free medium is 0.5 to 100% by mass, preferably 0.5 to 50% by mass, and more preferably 0.625 to 30% by mass. [C13] The method according to any one of [A1] to [A18], [B1] to [B11], [C5], [C6] and [C8], wherein the additive is recombinant human serum albumin, and the concentration (final concentration) of the recombinant human serum albumin in the serum-free medium is 0.001 to 1.0% by mass, preferably 0.01 to 0.5% by mass, and more preferably 0.015 to 0.3% by mass. [C14] The method according to any one of [A1] to [A18], [B1] to [B11], [C5], [C7] and [C8], wherein the additive is non-human animal serum albumin, and the concentration (final concentration) of the non-human animal serum albumin in the serum-free medium is 0.01 to 2.0% by mass, preferably 0.05 to 1.5% by mass, and more preferably 0.125 to 0.75% by mass.

[0092] [D1] A method for producing a product from a target cell, comprising culturing a starting cell according to any one of the methods described in [A1] to [A18], [B1] to [B11] and [C1] to [C14], to produce the target cell and to cause the target cell to produce the product. [D2] The method according to [D1], wherein the source cells are megakaryocyte progenitor cells, the target cells are megakaryocytes, and the product is platelets. [D3] The method according to [D1] or [D2], wherein the source cells are human megakaryocyte progenitor cells, the target cells are human megakaryocytes, and the product is human platelets. [D4] The method according to [D1], wherein the source cells and the target cells are iPS cells, and the product is a cytokine. [D5] The method according to [D1] or [D4], wherein the source cells and the target cells are human iPS cells, and the product is a human cytokine. [D6] The method according to [D1], wherein the source cells and the target cells are mesenchymal stem cells, and the product is a cytokine. [D7] The method according to [D1] or [D6], wherein the source cells and the target cells are human mesenchymal stem cells, and the product is a human cytokine.

[0093] [E1] At least one culture factor selected from a growth factor and a differentiation-inducing factor, human serum albumin, and and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. 1. A serum-free medium for use in the production of a target cell, comprising: [E2] The serum-free medium according to [E1], wherein the target cells are megakaryocytes and the culture factor is a megakaryocyte differentiation-inducing factor. [E3] The serum-free medium according to [E1] or [E2], wherein the target cells are human megakaryocytes and the culture factor is a megakaryocyte differentiation-inducing factor. [E4] At least one culture factor selected from a growth factor and a differentiation-inducing factor, human serum albumin, and and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin. 1. A serum-free medium for use in the production of a product by a cell of interest, comprising: [E5] The serum-free medium according to [E4], wherein the production of the product by the target cells is production of platelets by megakaryocytes, and the culture factor is a megakaryocyte differentiation-inducing factor.

[0094] [E6] The serum-free medium according to [E4] or [E5], wherein the production of the product by the target cells is production of human platelets by human megakaryocytes, and the culture factor is a megakaryocyte differentiation-inducing factor. [E7] The serum-free medium according to any one of [E1] to [E6], wherein the additive is an Src inhibitor, preferably A419259. [E8] The serum-free medium according to any one of [E1] to [E6], wherein the additive is a PKC activator, preferably prostratin. [E9] The serum-free medium according to any one of [E1] to [E6], wherein the additive is methylcellulose. [E10] The serum-free medium according to any one of [E1] to [E6], wherein the additive is Essential 8 medium.

[0095] [E11] The serum-free medium according to any one of [E1] to [E6], wherein the additive is recombinant human serum albumin or non-human animal serum albumin. [E12] The serum-free medium according to any one of [E1] to [E6] and [E11], wherein the additive is recombinant human serum albumin, preferably recombinant human serum albumin expressed in rice or recombinant human serum albumin expressed in yeast. [E13] The serum-free medium according to any one of [E1] to [E6] and [E11], wherein the additive is a non-human animal serum albumin, preferably bovine serum albumin.[E14] The serum-free medium according to any one of [E1] to [E13], wherein the concentration (final concentration) of human serum albumin in the serum-free medium is 0.01 to 2.0% by mass, preferably 0.05 to 1.5% by mass, and more preferably 0.075 to 0.75% by mass. [E15] The serum-free medium according to any one of [E1] to [E7] and [E14], wherein the additive is a Src inhibitor, preferably A419259, and the concentration (final concentration) of the Src inhibitor in the serum-free medium is 0.001 to 10 μM, preferably 0.01 to 1.0 μM, and more preferably 0.03 to 0.2 μM.

[0096] [E16] The serum-free medium according to any one of [E1] to [E6], [E8] and [E14], wherein the additive is a PKC activator, preferably prostratin, and the concentration (final concentration) of the PKC activator in the serum-free medium is 0.001 to 10 μM, preferably 0.01 to 1.0 μM, and more preferably 0.015 to 0.2 μM. [E17] The serum-free medium according to any one of [E1] to [E6], [E9] and [E14], wherein the additive is methylcellulose, and the concentration (final concentration) of methylcellulose in the serum-free medium is 0.001 to 3.0% by mass, preferably 0.01 to 1.0% by mass, and more preferably 0.018 to 0.3% by mass. [E18] The serum-free medium according to any one of [E1] to [E6], [E10] and [E14], wherein the additive is Essential 8 medium, and the concentration (final concentration) of Essential 8 medium in the serum-free medium is 0.5 to 100% by mass, preferably 0.5 to 50% by mass, and more preferably 0.625 to 30% by mass. [E19] The serum-free medium according to any one of [E1] to [E6], [E11], [E12] and [E14], wherein the additive is recombinant human serum albumin, and the concentration (final concentration) of the recombinant human serum albumin in the serum-free medium is 0.001 to 1.0% by mass, preferably 0.01 to 0.5% by mass, and more preferably 0.015 to 0.3% by mass. [E20] The serum-free medium according to any one of [E1] to [E6], [E11], [E13] and [E14], wherein the additive is non-human animal serum albumin, and the concentration (final concentration) of the non-human animal serum albumin in the serum-free medium is 0.01 to 2.0% by mass, preferably 0.05 to 1.5% by mass, and more preferably 0.125 to 0.75% by mass. EXAMPLES

[0097] [Example 1] Differentiation culture of megakaryocyte progenitor cells into megakaryocytes (1) Method In Example 1, differentiation culture was carried out from human iPS cell-derived megakaryocyte progenitor cells to megakaryocytes. The differentiation culture was carried out using the method described in Paper 2 (Ito et al., 2018, Cell 174, 636-648).

[0098] IMDM (Iscove's Modified Dulbecco's Medium) (Sigma-Aldrich, #I3390) was supplemented with 10U heparin sodium (Yoshindo Inc., #(01)14987476163428), 50ng / ml SCF (Stem Cell Factor) (Wako, #193-15513), 200ng / ml TA-316 (Nissan Chemical), 15μM KP-457 (Kaken Pharmaceutical), 0.5μM GNF-351 (Calbiochem), and 0.5μM Y-39983 (MedChemExpress) (concentrations are final concentrations). Human plasma (HP) (Cosmo Bio) was added to the resulting medium to a concentration of 5% by mass to prepare a positive control medium, and the medium to which human plasma was not added was used as a negative control (NC) medium.

[0099] In addition, various serum substitutes were added to the medium instead of human plasma: human serum albumin (HSA) (Sigma-Aldrich), rice-derived human recombinant albumin (R) (Sigma-Aldrich), yeast-derived human recombinant albumin (Y) (Wako), bovine serum albumin (BSA) (Sigma-Aldrich), Essential 8 medium (E) (Invitrogen), A419259 compound (A) (Wako), prostratin (P) (Wako), methylcellulose (M) (R&D Systems), or combinations of these.

[0100] Using these media, megakaryocyte differentiation culture was performed. Specifically, human iPS cell-derived megakaryocyte progenitor cells were cultured for 7 days in 2 wells of a 96-well plate (Corning) in a medium volume of 100 μl per well.

[0101] After differentiation culture, the cells were analyzed using a confocal quantitative image cytometer CQ1 (Yokogawa Electric Corporation). Nuclear staining reagent DRAQ5 TM The cell nuclei were stained with (Cosmo Bio), and the number of cells with a nucleus size of 20 μm or more (i.e., megakaryocytes) was counted in each well.

[0102] (2) Results The results are shown in Figures 1, 2, 3, and 4A to 4E. Figures 1 to 3 are graphs showing the number of megakaryocytes differentiated in each medium. Figures 4A to 4E are photographs showing megakaryocytes stained with a nuclear staining reagent (DRAQ5).

[0103] (Figure 1) FIG. 1 shows the results when the following media were used, from left to right: Negative control: NC medium Positive control: medium containing 5% by mass of human plasma (HP) Example 1A: Culture medium containing 0.25% by weight human serum albumin (HSA) Example 1B: Culture medium containing 0.75% by weight human serum albumin (HSA) Example 1C: Medium containing 0.25% by weight bovine serum albumin (BSA) Example 1D: Medium containing 0.75% by weight bovine serum albumin (BSA) Example 1E: Medium containing 0.03% by weight of rice-derived human recombinant albumin (R) Example 1F: Medium containing 0.1% by weight of rice-derived human recombinant albumin (R) Example 1G: Medium containing 0.25 μM of A419259 compound (A) Example 1H: Medium containing 0.75 μM of A419259 compound (A) Example 1I: Medium containing 0.03 μM prostratin (P) Example 1J: Medium containing 0.1 μM prostratin (P) Example 1K: Medium containing 3% by mass of 3% methylcellulose solution (M) (i.e., medium containing 0.09% by mass of methylcellulose) Example 1L: Medium containing 10% by mass of 3% methylcellulose solution (M) (i.e., medium containing 0.3% by mass methylcellulose) Example 1M: Medium containing 10% by mass of Essential 8 medium (E) Example 1N: Medium containing 30% by weight of Essential 8 medium (E)

[0104] In Examples 1A to 1N, HSA, BSA, R, A, P, M, and E were added alone as serum substitutes. In all of Examples 1A to 1N, the number of megakaryocytes increased compared to the negative control (NC), but tended to be lower than the positive control (HP5%).

[0105] (Figure 2) FIG. 2 shows the results when the following media were used, from left to right: Negative control: NC medium Positive control 1: Medium containing 0.5% human plasma (HP) by mass Positive control 2: medium containing 5% by mass human plasma (HP) Example 2A: Culture medium containing 0.125% by weight human serum albumin (HSA) Example 2B: Medium containing 0.125% by weight human serum albumin (HSA) and 0.03% by weight human recombinant albumin (R) derived from rice Example 2C: Medium containing 0.125% by weight human serum albumin (HSA) and 0.125% by weight yeast-derived human recombinant albumin (Y) Example 2D: Medium containing 0.125% by weight human serum albumin (HSA) and 0.125% by weight bovine serum albumin (BSA) Example 2E: Culture medium containing 0.125% by weight of human serum albumin (HSA) and 0.06 μM of A419259 compound (A) Example 2F: Medium containing 0.125% by weight human serum albumin (HSA) and 0.03 μM prostratin (P) Example 2G: Culture medium containing 0.125% by weight human serum albumin (HSA) and 1.25% by weight of a 3% methylcellulose solution (M) (i.e., 0.0375% by weight methylcellulose). Example 2H: Medium containing 0.125% by weight human serum albumin (HSA) and 1.25% by weight Essential 8 medium (E)

[0106] In Example 2A, 0.125% by mass of HSA was added alone as a serum substitute, and in Examples 2B to 2H, 0.125% by mass of HSA was added in combination with each additive (R, Y, BSA, A, P, M, or E) as a serum substitute. In Examples 2B to 2H, the number of megakaryocytes tended to increase compared to the case of HSA alone (Example 2A), and the number of megakaryocytes obtained was equivalent to that of positive control 2 (HP5%).

[0107] (Figure 3) FIG. 3 shows the results when the following media were used, from left to right: Negative control: NC medium Positive control 1: Medium containing 0.5% human plasma (HP) by mass Positive control 2: medium containing 5% by mass human plasma (HP) Example 3A: Culture medium containing 0.25% by weight human serum albumin (HSA) Example 3B: Medium containing 0.25% by weight human serum albumin (HSA) and 0.03% by weight human recombinant albumin (R) derived from rice Example 3C: Medium containing 0.25% by weight human serum albumin (HSA) and 0.125% by weight yeast-derived human recombinant albumin (Y) Example 3D: Medium containing 0.25% by weight human serum albumin (HSA) and 0.125% by weight bovine serum albumin (BSA) Example 3E: Culture medium containing 0.25% by weight of human serum albumin (HSA) and 0.06 μM of A419259 compound (A) Example 3F: Medium containing 0.25% by weight human serum albumin (HSA) and 0.03 μM prostratin (P) Example 3G: Culture medium containing 0.25% by weight human serum albumin (HSA) and 1.25% by weight of a 3% methylcellulose solution (M) (i.e., 0.0375% by weight methylcellulose). Example 3H: Medium containing 0.25% by weight human serum albumin (HSA) and 1.25% by weight Essential 8 medium (E)

[0108] In Example 3A, 0.25% by mass of HSA was added alone as a serum substitute, and in Examples 3B to 3H, 0.25% by mass of HSA was added in combination with each additive (R, Y, BSA, A, P, M, or E) as a serum substitute. In Examples 3B to 3H, the number of megakaryocytes tended to increase compared to the case of HSA alone (Example 3A), and the number of megakaryocytes obtained was equivalent to that of positive control 2 (HP5%).

[0109] (Figures 4A to 4E) 4A-4E show DRAQ5 staining images of the negative control, positive control 2, Example 2A, Example 2B, and Example 2E, respectively.

[0110] Megakaryocytes with large nuclei stained with DRAQ5 were increased in Example 2B (HSA 0.125% + R 0.03%) and Example 2E (HSA 0.125% + A 0.06 μM) compared to the negative control and Example 2A (HSA 0.125%), which is a cell number close to that of the positive control 2 (HP 5%).

[0111] (3) Discussion The results of Figures 1, 2, 3, and 4A-4E show that when megakaryocyte progenitor cells are differentiated and cultured using a combination of human serum albumin and specific additives as a serum substitute, megakaryocytes can be produced with the same differentiation efficiency as when human plasma is used. Since serum substitutes do not require blood components, they can reduce the production cost of megakaryocytes and improve the lot stability of megakaryocytes.

[0112] In addition, the present inventors have demonstrated that megakaryocytes can be produced using the medium described below with the same differentiation efficiency as when human plasma is used. A medium containing 0.5% by mass of human serum albumin (HSA) and 0.015% by mass of rice-derived human recombinant albumin (R). A medium containing 0.5% by mass of human serum albumin (HSA) and 0.03 μM of A419259 compound (A) A medium containing 0.5% by mass of human serum albumin (HSA) and 0.015 μM of prostratin (P) A medium containing 0.5% by weight of human serum albumin (HSA) and 0.625% by weight of a 3% methylcellulose solution (M) (i.e., 0.01875% by weight of methylcellulose). A medium containing 0.5% by mass of human serum albumin (HSA) and 0.625% by mass of Essential 8 medium (E). A medium containing 0.125% by mass of human serum albumin (HSA) and 0.015% by mass of rice-derived human recombinant albumin (R). A medium containing 0.125% by mass of human serum albumin (HSA) and 0.03 μM of A419259 compound (A). A medium containing 0.125% by mass of human serum albumin (HSA) and 0.015 μM of prostratin (P). A medium containing 0.125% by weight of human serum albumin (HSA) and 0.625% by weight of a 3% methylcellulose solution (M) (i.e., 0.01875% by weight of methylcellulose). A medium containing 0.125% by mass of human serum albumin (HSA) and 0.625% by mass of Essential 8 medium (E).

[0113] [Example 2] Platelet production by megakaryocytes (1) Method Using the combination of human serum albumin (HSA) and rice-derived human recombinant albumin (R) and the combination of HSA and A419259 compound (A), which were effective in megakaryocyte differentiation in Example 1, flask shaking culture was performed on a 25 ml scale in the same manner as in Example 1, thereby producing megakaryocyte-derived platelets.

[0114] After the culture, the expression of platelet markers was analyzed by FACS (fluorescence-activated cell sorting) using a flow cytometer (Sony SA3800).

[0115] Specifically, platelets were stained with CD41 and CD42b antibodies to analyze the CD41-positive and CD42b-positive cell rates in platelets. To investigate platelet aggregation function, the increase in the PAC1-positive cell rate by adding 0.2 μM PMA (phorbol 12-myristate 13-acetate) and the increase in the PAC1-positive cell rate by adding 100 μM ADP (adenosine diphosphate) were analyzed.

[0116] (2) Results The results are shown in Figures 5 and 6.

[0117] (Figure 5) The CD41- and CD42b-positive cell rates are shown in Figure 5. Figure 5 shows the results when the following media were used, from the left: Example 5A: Culture medium containing 5% by weight of human plasma (HP) Example 5B: Culture medium containing 0.25% by weight human serum albumin (HSA) Example 5C: Medium containing 0.25% by weight human serum albumin (HSA) and 0.05% by weight human recombinant albumin (R) derived from rice Example 5D: Culture medium containing 0.25% by weight human serum albumin (HSA) and 0.05 μM of A419259 compound (A)

[0118] Compared with Example 5B (HSA 0.25%), the CD41-positive cell rate and CD42b-positive cell rate were increased in Example 5C (HSA 0.25% + R 0.05%) and Example 5D (HSA 0.25% + A 0.05 μM), and the CD41-positive cell rate and CD42b-positive cell rate were equivalent to those of the positive control, Example 5A (HP 5%).

[0119] (Figure 6) The PAC1 positive cell rate after PMA treatment and the PAC1 positive cell rate after ADP treatment are shown in Figure 6. Figure 6 shows the results when the following media were used, from the left: Example 6A: Culture medium containing 5% by weight of human plasma (HP) Example 6B: Culture medium containing 0.25% by weight human serum albumin (HSA) Example 6C: Medium containing 0.25% by weight human serum albumin (HSA) and 0.05% by weight human recombinant albumin (R) derived from rice Example 6D: Culture medium containing 0.25% by weight of human serum albumin (HSA) and 0.05 μM of A419259 compound (A)

[0120] In FIG. 6, "Pre_PAC1" indicates the PAC1-positive cell rate before treatment with PMA or ADP, "PMA_PAC1" indicates the PAC1-positive cell rate after PMA treatment, and "ADP_PAC1" indicates the PAC1-positive cell rate after ADP treatment.

[0121] Compared to Example 6B (HSA 0.25%), the PAC1-positive cell rate after PMA treatment and the PAC1-positive cell rate after ADP treatment were increased in Example 6C (HSA 0.25% + R 0.05%) and Example 6D (HSA 0.25% + A 0.05 μM), and the PAC1-positive cell rate was equivalent to that of the positive control, Example 6A (HP 5%).

[0122] (3) Discussion The results of Figures 5 and 6 show that, when megakaryocyte progenitor cells are cultured using the serum substitute (a combination of human serum albumin and specific additives) that was effective in megakaryocyte differentiation in Example 1, functional platelets can be produced, as in the case of using human plasma. Since serum substitutes do not require blood components, they can reduce platelet production costs and improve platelet lot stability.

[0123] [Example 3] Expansion culture of iPS cells and mesenchymal stem cells (1) Method The proliferation ability of iPS cells was examined for the combination of human serum albumin (HSA) and recombinant human serum albumin, and the combination of HSA and prostratin, which were effective in megakaryocytic differentiation in Example 1. In addition, the proliferation ability of mesenchymal stem cells was examined for the combination of human serum albumin (HSA) and recombinant human serum albumin, and the combination of HSA and the A419259 compound, which were effective in megakaryocytic differentiation in Example 1.

[0124] (iPS cells) For iPS cells, iPS cells (253G1 line) were seeded at 1.0x10e4 / well in 6 wells for each condition onto a 96-well plate (Corning) coated with iMatrix-511 (0.1 μg / well) (Nippi Corporation), and cultured for 5 days in the medium described below.

[0125] Control: DMEM / F12 medium (Sigma-Aldrich) Example 7A (HSA 0.125): DMEM / F12 medium containing 0.125% by weight of human serum albumin (HSA) Example 7B (HSA 0.1 + R 0.025): DMEM / F12 medium containing 0.1% by weight of human serum albumin (HSA) and 0.025% by weight of rice-derived human recombinant albumin (R) Example 7C (HSA 0.075 + Y 0.05): DMEM / F12 medium containing 0.075% by weight of human serum albumin (HSA) and 0.05% by weight of yeast-derived human recombinant albumin (Y) Example 7D (HSA0.125+P0.05): DMEM / F12 medium containing 0.125% by weight human serum albumin (HSA) and 0.05 μM prostratin (P) Example 7E (R0.025): DMEM / F12 medium containing 0.025% by weight of rice-derived human recombinant albumin (R) Example 7F (Y0.05): DMEM / F12 medium containing 0.05% by mass of yeast-derived human recombinant albumin (Y) Example 7G (P0.05): DMEM / F12 medium containing 0.05 μM prostratin (P)

[0126] After incubation, the cell number was measured by analyzing DAPI nuclear staining fluorescence using a confocal quantitative image cytometer CQ1 (Yokogawa Electric Corporation).

[0127] (Mesenchymal stem cells) As for mesenchymal stem cells, bone marrow-derived mesenchymal stem cells (JCRB1154 strain) were seeded onto a 96-well plate (Corning) at 1.0x10e4 / well, 6 wells per condition, and cultured for 5 days in the medium described below.

[0128] Control: DMEM / F12 medium (Sigma-Aldrich) Example 8A (HSA0.125): DMEM / F12 medium containing 0.125% by weight of human serum albumin (HSA) Example 8B (HSA0.1+R0.025): DMEM / F12 medium containing 0.1% by weight of human serum albumin (HSA) and 0.025% by weight of rice-derived human recombinant albumin (R) Example 8C (HSA 0.075 + Y 0.05): DMEM / F12 medium containing 0.075% by weight of human serum albumin (HSA) and 0.05% by weight of yeast-derived human recombinant albumin (Y) Example 8D (HSA0.125+A0.05): DMEM / F12 medium containing 0.125% by weight of human serum albumin (HSA) and 0.05 μM of A419259 compound (A). Example 8E (R0.025): DMEM / F12 medium containing 0.025% by weight of rice-derived human recombinant albumin (R) Example 8F (Y0.05): DMEM / F12 medium containing 0.05% by mass of yeast-derived human recombinant albumin (Y) Example 8G (A0.05): DMEM / F12 medium containing 0.05 μM of A419259 compound (A)

[0129] After incubation, the cell number was measured by analyzing DAPI nuclear staining fluorescence using a confocal quantitative image cytometer CQ1 (Yokogawa Electric Corporation).

[0130] (2) Results Figure 7 shows the number of iPS cells after culture under each condition. Compared to the control and Example 7A (HSA alone), a significant increase in the number of iPS cells was observed in Examples 7B and 7C (combination of HSA and recombinant human serum albumin), and Example 7D (combination of HSA and prostratin). On the other hand, no significant increase in the number of iPS cells was observed in Examples 7E and 7F (recombinant human serum albumin alone) and Example 7G (prostratin alone).

[0131] Figure 8 shows the number of MSC cells after culture under each condition. Compared to the control and Example 8A (HSA alone), a significant increase in the number of MSC cells was observed in Examples 8B and 8C (combination of HSA and recombinant human serum albumin, and Example 8D (combination of HSA and A419259). On the other hand, no significant increase in the number of MSC cells was observed in Examples 8E and 8F (recombinant human serum albumin alone) and Example 8G (A419259 alone).

[0132] (3) Discussion The results of Figures 7 and 8 show that when the serum substitute (a combination of human serum albumin and specific additives) that was effective in megakaryocyte differentiation in Example 1 is used to perform expansion culture of iPS cells and expansion culture of mesenchymal stem cells, a synergistic effect is achieved to obtain high expansion efficiency. Since the serum substitute does not require blood components, it is possible to reduce the production cost of cells by expansion culture and to improve the lot stability of the expanded cells.

Claims

1. Growth factors, Human serum albumin, Additives that are PKC activators The method comprises culturing a source cell in a serum-free medium containing the The source cells and the target cells are iPS cells, or the source cells and the target cells are mesenchymal stem cells. Methods for producing target cells.

2. 2. The method of claim 1, wherein the additive is prostratin.

3. The method according to claim 1 , wherein the source cells and the target cells are iPS cells, and the additive is a PKC activator.

4. The method of claim 3, wherein the PKC activator is prostratin.

5. The method according to claim 1 , wherein the source cells and the target cells are mesenchymal stem cells, and the additive is a PKC activator.

6. The method of claim 5, wherein the PKC activator is prostratin.

7. A method for producing a product by target cells, comprising culturing source cells according to the method of any one of claims 1 to 6 to produce target cells and have the target cells produce the product.

8. Growth factors, Human serum albumin, Additives that are PKC activators Including, The source cells and the target cells are iPS cells, or the source cells and the target cells are mesenchymal stem cells. Serum-free media for use in the production of target cells.

9. Growth factors, Human serum albumin, Additives that are PKC activators Including, The source cells and the target cells are iPS cells, or the source cells and the target cells are mesenchymal stem cells. A serum-free medium for use in the production of a product by a cell of interest.

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