Method for producing pluripotent stem cells

Abstract

The death of a portion of the pluripotent stem cells during the suspension culture of pluripotent stem cells is avoided.　During the suspension culture of pluripotent cells, a liquid culture medium that substantially does not contain a PKCβ inhibitor is used for the single cell state, and, after cell masses have formed, a liquid culture medium is used that contains a PKCβ inhibitor and a WNT inhibitor.

Description

Method for producing pluripotent stem cells

[0001] The present invention relates to a method for producing pluripotent stem cells by suspension culture.

[0002] Pluripotent stem cells, such as ES cells and iPS cells, have the ability to proliferate indefinitely and differentiate into various somatic cells. The practical application of a treatment involving transplantation of somatic cells induced to differentiate from pluripotent stem cells has the potential to fundamentally revolutionize the treatment of intractable diseases and lifestyle-related diseases. For example, technology has already been developed to induce differentiation from pluripotent stem cells into a wide variety of somatic cells, including nerve cells, cardiomyocytes, blood cells, and retinal cells, at the test tube level.

[0003] On the other hand, regenerative medicine using pluripotent stem cells has many challenges to overcome before it can be put to practical use. One of these challenges is the productivity of pluripotent stem cells themselves. For example, liver regeneration requires approximately 2 × 10 １１ It is said that 10 cells are needed to culture pluripotent stem cells. The culture methods are roughly divided into adhesion culture, in which cells are cultured by adhering them to a flat culture substrate (plate), and suspension culture, in which cells are cultured by suspending them in a liquid medium. To culture the above number of cells by adhesion culture, 10 ６ cm ２ This requires a culture substrate (plate) of at least 1000 cells, equivalent to approximately 20,000 standard 10 cm dishes. Thus, because the number of cells obtained in adherent culture on the surface of a culture substrate (plate) depends on the culture area, scaling up requires a vast amount of surface area, making it difficult to supply the amount of cells required for regenerative medicine. On the other hand, in suspension culture, cells are cultured in a liquid medium while suspended in the medium, and the number of cells obtained depends on the medium volume. Therefore, suspension culture is relatively feasible to scale up and is considered suitable for mass cell production. For example, Non-Patent Document 1 discloses a method of suspension culture of pluripotent stem cells using a spinner flask as a cell culture vessel for suspension culture while stirring the liquid medium.

[0004] Another challenge facing the practical application of regenerative medicine is the productivity of target somatic cells. Efforts to efficiently produce target somatic cells include culturing high-quality pluripotent stem cells to improve differentiation induction efficiency, and various methods have been reported. For example, Non-Patent Document 2 discloses a method for suppressing spontaneous differentiation of pluripotent stem cells in adherent culture by adding a protein kinase C (PKC) inhibitor to the medium. Non-Patent Document 3 also discloses a method for suppressing spontaneous differentiation of pluripotent stem cells in adherent culture by adding a tankyrase (TNKS) inhibitor to the medium.

[0005] As mentioned above, various methods have been developed for efficiently producing target somatic cells from pluripotent stem cells in adherent culture.

[0006] Olmer R. et al. , Tissue Engineering: Part C, Volume 18(10):772-784 (2012)M. Kinehara et al. , PLoS One. 2013;8(1):e54122T. Sumi et al. , PLoS One. 2013;8(5):e63378

[0007] As mentioned above, suspension culture is suitable for mass production of pluripotent stem cells, but it has been found that suspension culture has the problem of being more susceptible to unintended differentiation than adherent culture. Therefore, the present inventors attempted to develop a technology that can maintain the undifferentiated state of pluripotent stem cells by constantly applying a PKCβ inhibitor and a TNKS inhibitor, a type of WNT signaling inhibitor, to suspension culture of pluripotent stem cells, thereby suppressing spontaneous differentiation toward all three germ layers. However, when a PKCβ inhibitor and a WNT signaling inhibitor were added, a phenomenon was observed in which some pluripotent stem cells died in suspension culture, resulting in another problem of poor cell culture efficiency.

[0008] Therefore, the inventors conducted extensive research to solve the above problems and discovered that when pluripotent stem cells are cultured in suspension in a single cell state, a decrease in cell culture efficiency can be avoided without spontaneous differentiation by using a medium containing a PKCβ inhibitor at the lowest possible concentration (i.e., a medium that is substantially free of a PKCβ inhibitor) at the stage before cell mass formation, thereby completing the present invention.

[0009] The present invention includes the following: (1) A method for producing pluripotent stem cells, comprising the steps of: suspension culturing unicellular pluripotent stem cells in a first liquid medium substantially free of a PKCβ inhibitor; and, after the pluripotent stem cells have formed cell clusters, suspension culturing the pluripotent stem cells in a second liquid medium containing a PKCβ inhibitor and a WNT inhibitor. (2) The method according to (1), wherein the suspension culturing in the second liquid medium is carried out by adding a PKCβ inhibitor to the first liquid medium after the pluripotent stem cells have formed cell clusters in the suspension culturing in the first liquid medium. (3) The method according to (1), wherein, after the pluripotent stem cells have formed cell clusters, the medium is replaced from the first liquid medium to the second liquid medium. (4) The method for producing pluripotent stem cells according to any one of (1) to (3), further comprising a step of culturing pluripotent stem cells in an adherent manner and a step of detaching and dissociating the adherently cultured pluripotent stem cells into single cells, wherein the single-celled pluripotent stem cells are cultured in suspension in a first liquid medium. (5) The method for producing pluripotent stem cells according to any one of (1) to (4), characterized in that after the step of culturing in suspension in a second liquid medium, the recovered cell masses are dissociated into single cells and cultured in suspension in the first liquid medium and again cultured in suspension in the second liquid medium. (6) The method for producing pluripotent stem cells according to any one of (1) to (5), characterized in that the first liquid medium contains a PKCβ inhibitor at a concentration of less than 25 nM. (7) The method for producing pluripotent stem cells according to any one of (1) to (6), characterized in that the second liquid medium contains a PKCβ inhibitor at a concentration of 25 nM or more and 15 μM or less. (8) The method according to any one of (1) to (7), wherein the first liquid medium is substantially free of a WNT inhibitor, or the first liquid medium and / or the second liquid medium contain a WNT inhibitor at a concentration of 90 nM to 40 μM. (9) The method according to any one of (1) to (8), wherein the second liquid medium contains a PKCβ inhibitor and a WNT inhibitor at a molar ratio of 167:1 to 1:1600.(10) The method for producing a cell culture medium according to any one of (1) to (9), wherein the first liquid medium and the second liquid medium contain at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate. (11) The method for producing a cell culture medium according to any one of (1) to (10), wherein the first liquid medium and the second liquid medium contain FGF2 and / or TGF-β1. (12) The method for producing a cell culture medium according to any one of (1) to (11), wherein the first liquid medium and the second liquid medium contain a ROCK inhibitor. (13) The method for producing a cell culture medium according to (12), wherein the ROCK inhibitor is Y-27632. (14) The manufacturing method according to any one of (1) to (13), wherein the suspension culture in the first liquid medium and the suspension culture in the second liquid medium are performed by stirring, and the tip speed of the stirring impeller in the suspension culture in the second liquid medium is equal to or greater than the tip speed of the stirring impeller in the suspension culture in the first liquid medium. (15) The manufacturing method according to any one of (1) to (14), wherein the suspension culture in the second liquid medium is performed for 48 hours or less. (16) The manufacturing method according to any one of (1) to (15), wherein the suspension culture in the first liquid medium is performed for 48 hours or less. (17) The method for producing the pluripotent stem cells according to any one of (1) to (16), wherein the pluripotent stem cells have a ratio of OCT4-positive cells of 90% or more, a ratio of SOX2-positive cells of 90% or more, and a ratio of NANOG-positive cells of 90% or more. (18) The method for producing the pluripotent stem cells according to any one of (1) to (17), wherein the pluripotent stem cells are ES cells and / or induced pluripotent stem cells. (19) Pluripotent stem cells or a pluripotent stem cell population produced by the method for producing the pluripotent stem cells according to any one of (1) to (18).

[0010] In the method for producing pluripotent stem cells according to the present invention, the step of carrying out suspension culture in the second liquid medium is preferably carried out until the lactic acid concentration in the medium reaches 5 to 15 mM.

[0011] Furthermore, in the method for producing pluripotent stem cells according to the present invention, the step of performing suspension culture in the first liquid medium and / or the step of performing suspension culture in the second liquid medium are preferably performed by agitation culture with a blade tip speed of 0.05 m / s or more and 1.37 m / s or less.

[0012] Furthermore, in the method for producing pluripotent stem cells according to the present invention, examples of the PKCβ inhibitor include Go6983, GF109203X, LY-333531, Enzastaurin, Sotrastaurin, Ro-31-8220-mesylate, Ro-32-0432-hydrochloride, Go6976, Rottlerin, Midostaurin, Daphnetin, Dequalinium Chloride, Baicalein, Quercetin, Luteolin, Bisindolylmaleimide II, Calphostin C, Chelerythrine chloride, and L-threo It may be at least one selected from the group consisting of dihydrosphingosine and melittin.

[0013] Furthermore, in the method for producing pluripotent stem cells according to the present invention, the WNT inhibitor can be at least one selected from the group consisting of tankirase inhibitors (TNKS inhibitors), such as IWR-1-endo, XAV939, G007-LK, G244-LM, and WikiI4.

[0014] Furthermore, the method for producing pluripotent stem cells according to the present invention can be applied to primed pluripotent stem cells as pluripotent stem cells.

[0015] Furthermore, in the method for producing pluripotent stem cells according to the present invention, it is preferable that the first liquid medium and / or the second liquid medium does not contain LIF.

[0016] Furthermore, in the method for producing pluripotent stem cells according to the present invention, it is preferable that the first liquid medium and / or the second liquid medium does not contain a GSK3 inhibitor.

[0017] Furthermore, in the method for producing pluripotent stem cells according to the present invention, it is preferable that the first liquid medium and / or the second liquid medium does not contain a GSK3 inhibitor and a MEK / ERK inhibitor.

[0018] Furthermore, in the method for producing pluripotent stem cells according to the present invention, the PKCβ inhibitor can include a compound having the following structural formula [Formula I]:

[0019] A compound represented by the formula (I) or a salt thereof.

[0020] In Formula I, R １ is a hydrogen atom or an alkoxy group having 1 to 3 carbon atoms (preferably a methoxy group), and R ２ is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms (preferably a methyl group), or —N(R Ａ ) ２ an alkyl group having 1 to 3 carbon atoms (preferably -(CH ２ ) ３ -N(CH ３ ) ２ ) and R Ａ are independently an ethyl group or a methyl group (preferably a methyl group), and R ３ teeth, , , or R is a group represented by Ｂ represents a hydrogen atom, -SC(=NH)(-NH ２ an alkyl group having 2 to 4 carbon atoms (preferably -(CH ２ ) ３ -SC(=NH)(-NH ２ )), or or R ２ and R Ｂ and combine to form the following divalent group: where # is R ２ ## indicates a bond to the bonding position of R Ｂ The configuration of the asymmetric carbon atom contained in the divalent group is not particularly limited, but preferably, and R Ｃ is -N(R Ｄ) ２ an alkyl group having 1 to 3 carbon atoms (preferably -(CH ２ )-N(CH ３ ) ２ ) and R Ｄ are independently an ethyl group or a methyl group (preferably a methyl group). Examples of salts of the compound represented by Formula I include hydrochloride and sulfate. This specification incorporates the disclosure of Japanese Patent Application No. 2021-130903, which is the priority basis of the present application.

[0021] According to the method for producing pluripotent stem cells of the present invention, when producing a pluripotent stem cell population by suspension culture, not only can high-quality pluripotent stem cells be obtained by maintaining an undifferentiated state, but also excellent cell culture efficiency can be achieved by preventing cell death.

[0022] 1 is a characteristic diagram showing the expression levels (a) of the OCT4 gene and the expression levels (b) of the NANOG gene measured by quantitative real-time PCR. FIG. 2 is a characteristic diagram showing the expression levels (a) of the SOX2 gene and the expression levels (b) of the TBXT gene measured by quantitative real-time PCR. FIG. 3 is a characteristic diagram showing the expression levels (a) of the SOX17 gene and the expression levels (b) of the PAX6 gene measured by quantitative real-time PCR. FIG. 4 is a characteristic diagram showing the results of measuring the number of cells in the cell cultures of Reference Examples 6 and 7. FIG. 5 is a characteristic diagram showing a scheme for adding a PKCβ inhibitor in suspension culture. FIG. 6 is a characteristic diagram showing the cell density maintenance rate calculated based on the number of cells in the suspension cultures of Examples 1 and 2, and Comparative Examples 1 and 2. FIG. 7 is a characteristic diagram showing the results of calculating the specific growth rate of the cells suspension cultured in Examples 2 and 3, and Comparative Examples 2 and 3. FIG. 8 is a characteristic diagram showing the transition of the cell density of the cells suspension cultured in Examples 2 and 3, and Comparative Examples 2 and 3. 1 is a characteristic diagram showing the expression levels of the OCT4 gene (a) and the NANOG gene (b) measured by quantitative real-time PCR for cells on days 5 and 10 of culture in Examples 2 and 3 and Comparative Example 2. FIG. 2 is a characteristic diagram showing the expression levels of the SOX2 gene (a) and the TBXT gene (b) measured by quantitative real-time PCR for cells on days 5 and 10 of culture in Examples 2 and 3 and Comparative Example 2. FIG. 3 is a characteristic diagram showing the expression levels of the SOX17 gene (a) and the PAX6 gene (b) measured by quantitative real-time PCR for cells on days 5 and 10 of culture in Examples 2 and 3 and Comparative Example 2. FIG. 4 is a characteristic diagram showing the positive rates of OCT4 (a), SOX2 (b), and NANOG (c) measured by dot plot for cells cultured in Examples 2 and 3 and Comparative Example 2.

[0023] 1. Method for Producing Pluripotent Stem Cells 1-1. Overview The method for producing pluripotent stem cells according to the present invention is characterized by suppressing differentiation of pluripotent stem cells by culturing the pluripotent stem cells in the presence of a protein kinase C β (PKCβ) inhibitor and a WNT inhibitor, and by suspension culturing the pluripotent stem cells in a single-cell state in a liquid medium (first liquid medium) that is substantially free of a PKCβ inhibitor. In particular, the method for producing pluripotent stem cells according to the present invention comprises a two-step process: culturing the pluripotent stem cells in a liquid medium (first liquid medium) that is substantially free of a PKCβ inhibitor until they form cell clusters, and then, after the pluripotent stem cells have formed cell clusters, culturing the pluripotent stem cells in suspension in a liquid medium (second liquid medium) that contains a PKCβ inhibitor and a WNT inhibitor.

[0024] According to the method for producing pluripotent stem cells of the present invention, cell death of pluripotent stem cells at a stage prior to the formation of cell masses can be prevented, and pluripotent stem cells or pluripotent stem cell populations that maintain an undifferentiated state can be efficiently produced.

[0025] 1-2. Definitions of Terms The following terms used in this specification are defined below.

[0026] <Cells> As used herein, the term "pluripotent stem cells" refers to cells that have the multipotency (pluripotency) to differentiate into all types of cells that constitute a living organism and can continue to proliferate indefinitely while maintaining pluripotency when cultured in vitro under appropriate conditions. More specifically, pluripotency refers to the ability to differentiate into the germ layers that constitute an individual (in vertebrates, the three germ layers are ectoderm, mesoderm, and endoderm). Examples of such cells include embryonic stem cells (ES cells), embryonic germ stem cells (EG cells), germline stem cells (GS cells), and induced pluripotent stem cells (iPS cells). "ES cells" refer to pluripotent stem cells prepared from early embryos. "EG cells" are pluripotent stem cells prepared from fetal primordial germ cells (Shamblott M.J. et al., 1998, Proc. Natl. Acad. Sci. USA., 95:13726-13731). "GS cells" are pluripotent stem cells prepared from testes (Conrad S., 2008, Nature, 456:344-349). "iPS cells" refer to reprogrammed pluripotent stem cells that are produced by introducing genes encoding a small number of reprogramming factors into differentiated somatic cells, thereby reprogramming somatic cells to an undifferentiated state.

[0027] The pluripotent stem cells referred to herein may be cells derived from a multicellular organism. They are preferably animal-derived cells, more preferably mammalian-derived cells. Examples include rodents such as mice, rats, hamsters, and guinea pigs; livestock or pets such as dogs, cats, rabbits, cows, horses, sheep, and goats; and primates such as humans, rhesus monkeys, gorillas, and chimpanzees. Human-derived cells are particularly preferred.

[0028] As used herein, pluripotent stem cells include naive pluripotent stem cells and primed pluripotent stem cells. Naive pluripotent stem cells are defined as a state close to the pluripotency observed in the inner cell mass before implantation, while primed pluripotent stem cells are defined as a state close to the pluripotency observed in the epiblast after implantation. Compared to naive pluripotent stem cells, primed pluripotent stem cells are characterized by a lower frequency of contribution to ontogeny, a single X chromosome with transcriptional activity, and a high level of transcriptionally repressive histone modifications. Furthermore, the marker gene for primed pluripotent stem cells is OTX2, while the marker genes for naive pluripotent stem cells are REX1 and the KLF family. Furthermore, primed pluripotent stem cells form flat colonies, while naive pluripotent stem cells form dome-shaped colonies. It is particularly preferred to use primed pluripotent stem cells as the pluripotent stem cells used herein.

[0029] The pluripotent stem cells used herein may be commercially available cells, cells provided by a donor, or newly prepared cells. Although not limited thereto, when used in each invention of the present specification, the pluripotent stem cells are preferably iPS cells or ES cells.

[0030] When the iPS cells used herein are commercially available, they are not limited to, for example, 253G1 strain, 253G4 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, TkDN4-M strain, TkDA strain, etc. The following strains can be used: TkDA3-3, TkDA3-4, TkDA3-5, TkDA3-9, TkDA3-20, hiPSC38-2, MSC-iPSC1, BJ-iPSC1, RPChiPS771-2, WTC-11, 1231A3, 1383D2, 1383D6, 1210B2, 1201C1, 1205B2, and the like.

[0031] Furthermore, when the iPS cells used herein are clinical strains, examples that can be used include, but are not limited to, the QHJI01s01 strain, the QHJI01s04 strain, the QHJI14s03 strain, the QHJI14s04 strain, the Ff-114s03 strain, the Ff-114s04 strain, and the YZWI strain.

[0032] Furthermore, when the iPS cells used herein are newly produced cells, the combination of genes for the reprogramming factors to be introduced is not limited, and examples include the combination of the OCT3 / 4 gene, the KLF4 gene, the SOX2 gene, and the c-Myc gene (Yu J, et al. 2007, Science, 318:1917-20.), and the combination of the OCT3 / 4 gene, the SOX2 gene, the LIN28 gene, and the NANOG gene (Takahashi K, et al. 2007, Cell, 131:861-72.). The manner in which these genes are introduced into cells is not particularly limited, and may be, for example, gene introduction using a plasmid, introduction of synthetic RNA, or introduction as a protein. iPS cells produced by methods using microRNA, RNA, low-molecular-weight compounds, etc. may also be used. Needless to say, newly produced clinical-grade iPS cells may also be used.

[0033] When the ES cells used herein are commercially available, for example, but not limited to, KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SEES1 strain, SEES2 strain, SEES3 strain, SEES-4 strain, SEES-5 strain, SEES-6 strain, SEES-7 strain, HUES8 strain, CyT49 strain, H1 strain, H9 strain, HS-181 strain, etc. can be used.

[0034] <Cell clusters> As used herein, a "cell cluster" refers to a mass-like aggregate formed by cell aggregation in suspension culture, and is also called a spheroid. Cell clusters usually have a roughly spherical shape. The cells constituting the cell cluster are not particularly limited as long as they are one or more types of the cells. For example, a cell cluster composed of pluripotent stem cells such as human pluripotent stem cells or human embryonic stem cells expresses a pluripotent stem cell marker and / or contains cells that are positive for the pluripotent stem cell marker. Furthermore, the cell cluster may be formed via a microcarrier.

[0035] Pluripotent stem cell markers are gene markers that are specifically or excessively expressed in pluripotent stem cells, and examples thereof include alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1.

[0036] Pluripotent stem cell markers can be detected by any detection method known in the art. Methods for detecting cell markers include, but are not limited to, flow cytometry. When a fluorescently labeled antibody is used as a detection reagent in flow cytometry, cells that emit stronger fluorescence compared to a negative control (isotype control or FMO control) are determined to be "positive" for the marker. The proportion of cells that test positive for a fluorescently labeled antibody analyzed by flow cytometry is sometimes referred to as the "positive rate." Furthermore, any fluorescently labeled antibody known in the art can be used, including, but not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc.

[0037] When the cells constituting the cell cluster are pluripotent stem cells, the positivity rate for the pluripotent stem cell marker is preferably 80% or more, more preferably 90% or more, more preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and more preferably 100%. Cell clusters in which the proportion of cells expressing pluripotent stem cell markers and / or positive for pluripotent stem cell markers is within the above range are highly undifferentiated and are more homogeneous cell aggregates.

[0038] <Culture and Culture Medium> "Suspension culture" is a cell culture method in which cells are grown in a suspended state in a culture medium. As used herein, "suspension state" refers to a non-adherent state in which cells are not fixed to a culture vessel or the like in a culture medium. For example, a culture method in which cells are attached to microcarriers and cultured in a suspended state in a culture medium can be considered suspension culture, since although the cells adhere to the microcarriers, the cell mass containing the microcarriers floats without adhering to the culture vessel. "Suspension culture" is a method of culturing cells in suspension. In this method, cells generally exist primarily in a single-cell state at the start of culture and then exist as aggregated cell masses in the culture medium as the culture progresses. An alternative culture method to suspension culture is adhesion culture. "Adhesion culture" is a method of culturing cells in an adherent state. "Adhesion culture" refers to growing cells by adhering them to an external matrix such as a culture vessel. In this case, the external matrix to which the cells adhere is not suspended in the culture medium. The above-mentioned adhesive cells can generally be cultured not only in an adherent culture but also in a suspension culture.

[0039] In this specification, the terms "first liquid medium" and "second liquid medium" are used, but the first liquid medium and the second liquid medium differ in that the former is a liquid medium that is substantially free of a PKCβ inhibitor, while the latter contains a PKCβ inhibitor. Note that while a WNT inhibitor is always contained in the second liquid medium, it may or may not be contained in the first liquid medium.

[0040] As used herein, the term "culture medium" refers to a liquid or solid substance prepared for culturing cells. In principle, the medium contains at least the minimum amount of components essential for cell growth and / or maintenance. Unless otherwise specified, the medium used herein refers to a liquid culture medium for animal cells used to culture animal-derived cells.

[0041] As used herein, the term "basal medium" refers to a medium that is the basis for various animal cell culture media. It can be used alone for culture, and can also be prepared into a medium specific to various cells depending on the purpose by adding various culture additives. The basal medium used herein includes BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof (e.g., DMEM / F12 medium (Dulbecco's Modified Eagle's Medium)). A medium such as DMEM / Nutrient Mixture F-12 (Ham's) can be used, but is not particularly limited thereto. As the DMEM / F12 medium, a medium in which DMEM medium and Ham's F12 medium are mixed at a weight ratio of preferably 60 / 40 or more and 40 / 60 or less, for example, 58 / 42, 55 / 45, 52 / 48, 50 / 50, 48 / 52, 45 / 55, or 42 / 58, is particularly used. In addition, media used for culturing human iPS cells and human ES cells can also be suitably used. The medium used in the present invention is preferably a medium that does not contain serum, i.e., a serum-free medium.

[0042] As used herein, the term "culture additive" refers to a substance other than serum that is added to a culture medium for the purpose of culture. Specific examples of culture additives include, but are not limited to, L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, growth factors, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, antibiotics, etc. Insulin, transferrin, and cytokines may be naturally derived substances isolated from tissues or serum of animals (preferably humans, mice, rats, cows, horses, goats, etc.), or may be recombinant proteins produced by genetic engineering. Furthermore, examples of growth factors that can be used include, but are not limited to, FGF2 (basic fibroblast growth factor-2), TGF-β1 (transforming growth factor-β1), Activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF, and IGFBP-7. Examples of antibiotics that can be used include, but are not limited to, penicillin, streptomycin, amphotericin B, etc. Particularly preferred growth factors as culture additives for the medium used in the present invention are FGF2 and / or TGF-β1.

[0043] The medium preferably contains a ROCK inhibitor. Examples of ROCK inhibitors include Y-27632. By including a ROCK inhibitor in the medium, cell death in suspension culture of pluripotent stem cells can be significantly suppressed, and the strength of the cell aggregates can be increased, improving their resistance to physical damage. For example, the lower limit of the concentration of the ROCK inhibitor in the medium can be 1 μM, 2 μM, 3 μM, 5 μM, 7 μM, or 10 μM, and the upper limit can be 50 μM, 40 μM, 30 μM, 20 μM, or 10 μM.

[0044] Furthermore, when culturing primed pluripotent stem cells, the medium preferably does not contain LIF. Furthermore, when culturing primed pluripotent stem cells, the medium preferably does not contain either a GSK3 inhibitor or a MEK / ERK inhibitor, preferably both. A medium that does not contain LIF, a GSK3 inhibitor, or a MEK / ERK inhibitor allows primed pluripotent stem cells to be cultured without naivetization and while maintaining an undifferentiated state.

[0045] The medium used in the present invention may contain one or more of the above culture additives. The medium to which the above culture additives are added is generally, but not limited to, the basal medium.

[0046] Culture additives can be added to the medium in the form of a solution, derivative, salt, mixed reagent, or the like. For example, L-ascorbic acid may be added to the medium in the form of a derivative such as magnesium 2-ascorbate phosphate, and selenium may be added to the medium in the form of a selenite (sodium selenite, etc.). Insulin, transferrin, and selenium can also be added to the medium in the form of an ITS reagent (insulin-transferrin-selenium). Commercially available media supplemented with at least one selected from L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate can also be used. Commercially available media supplemented with insulin and transferrin include CHO-S-SFM II (Life Technologies Japan, Inc.), Hybridoma-SFM (Life Technologies Japan, Inc.), eRDF Dry Powdered Media (Life Technologies Japan, Inc.), and UltraCULTURE. ＴＭ (BioWhittaker), UltraDOMA ＴＭ (BioWhittaker), UltraCHO ＴＭ (BioWhittaker), UltraMDCK ＴＭ (BioWhittaker), STEMPRO (registered trademark) hESC SFM (Life Technologies Japan), Essential8 ＴＭ(Life Technologies Japan, Inc.), StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.), mTeSR1 (Veritas, Inc.), TeSR2 (Veritas, Inc.), ReproMed (ReproCELL, Inc.), and StemScale (Thermo Fisher Scientific Inc.).

[0047] The most preferred medium for use in the present invention is a serum-free medium containing L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, and at least one growth factor, and particularly preferred is a serum-free DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, and at least one growth factor (preferably FGF2 and TGF-β1).

[0048] <PKCβ Inhibitor> As used herein, the term "protein kinase Cβ (PKCβ) inhibitor" refers to a substance that inhibits or suppresses the activity of PKCβ. Protein kinases have a catalytic domain on the C-terminal side and a regulatory domain on the N-terminal side. The catalytic domain contains a sequence that recognizes phosphorylated residues on a substrate protein and a regulatory domain that recognizes ATP / Mg ２＋ The regulatory region is composed of the C1 domain and the C2 domain.

[0049] PKC includes conventional isozymes such as PKCα, PKCβI, PKCβII, and PKCγ, novel isozymes such as PKCδ, PKCε, PKCθ, and PKCη, and atypical isozymes such as PKCζ, PKCλ, and PKCμ.

[0050] As used herein, the term "PKCβ" refers to both PKCβI and PKCβII, or to either PKCβI or PKCβII. Furthermore, as used herein, the term "PKCβ inhibitor" refers to a substance that inhibits at least PKCβI and / or PKCβII among the conventional, novel, and atypical isozymes. In other words, the term "PKCβ inhibitor" includes substances that inhibit or suppress only the activity of PKCβI, substances that inhibit or suppress only the activity of PKCβII, and substances that inhibit or suppress the activities of PKCβI and PKCβII.

[0051] The PKCβ inhibitor may be a substance that specifically inhibits or suppresses only the activity of PKCβ, or may be a substance that inhibits or suppresses the activity of other isozymes in addition to PKCβI or PKCβII. For example, the PKCβ inhibitor may be a substance that inhibits or suppresses the activity of all of the conventional, novel, and atypical isozymes described above, including PKCβI and PKCβII. The PKCβ inhibitor may also be a substance that inhibits or suppresses the activity of the conventional isozymes PKCα and PKCγ in addition to PKCβI and PKCβII. Furthermore, the PKCβ inhibitor may be a substance that inhibits or suppresses the activity of the novel isozymes PKCδ, PKCε, PKCθ, and PKCη in addition to PKCβI and PKCβII.

[0052] The PKCβ inhibitors include compounds that act directly or indirectly on PKCβ, antisense nucleic acids against the gene encoding PKCβ, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof. An example of a PKCβ inhibitor is a compound having the following structural formula [Formula I]:

[0053] A compound represented by the formula (I) or a salt thereof.

[0054] In Formula I, R １ is a hydrogen atom or an alkoxy group having 1 to 3 carbon atoms (preferably a methoxy group), and R ２ is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms (preferably a methyl group), or —N(R Ａ) ２ an alkyl group having 1 to 3 carbon atoms (preferably -(CH ２ ) ３ -N(CH ３ ) ２ ) and R A are independently an ethyl group or a methyl group (preferably a methyl group), and R ３ teeth, , , or R is a group represented by Ｂ represents a hydrogen atom, -SC(=NH)(-NH ２ an alkyl group having 2 to 4 carbon atoms (preferably -(CH ２ ) ３ -SC(=NH)(-NH ２ )), or or R ２ and R Ｂ and combine to form the following divalent group: where # is R ２ ## indicates a bond to the bonding position of R Ｂ The configuration of the asymmetric carbon atom contained in the divalent group is not particularly limited, but preferably, and R Ｃ is -N(R Ｄ ) ２ an alkyl group having 1 to 3 carbon atoms (preferably -(CH ２ )-N(CH ３ ) ２ ) and R Ｄ are independently an ethyl group or a methyl group (preferably a methyl group). Examples of salts of the compound represented by formula I include hydrochlorides and sulfates.

[0055] Specific examples of PKCβ inhibitors having the above structural formula [Formula I] include Go6983, GF109203X, LY-333531, Enzastaurin, Sotrastaurin, Ro-31-8220-mesylate, Ro-32-0432-hydrochloride, Go6976, Rottlerin, Midostaurin, Daphenetin, Dequalinium Chloride, Baicalein, Quercetin, Luteolin, Bisindolylmaleimide II, Calphostin C, Chelerythrine chloride, and L-threo Examples of the PKCβ inhibitor include a compound selected from the group consisting of dihydrosphingosine and melittin. In particular, among the PKCβ inhibitors having the above structural formula, it is preferable to use a compound selected from the group consisting of Go6983, GF109203X, and LY-333531.

[0056] The structural formula of Go6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione) is shown below.

[0057]

[0058] The structural formula of GF109203X (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide) is shown below.

[0059]

[0060] The structural formula of LY-333531 ((9S)-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-dimethenodibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione, monohydroxychloride) is shown below.

[0061]

[0062] The structural formula of Enzastaurin (3-(1-methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione) is shown below.

[0063]

[0064] The structural formula of sotrastaurin (3-(1H-indol-3-yl)-4-(2-(4-methylpiperazin-1-yl)quinazolin-4-yl)-1H-pyrrole-2,5-dione) is shown below.

[0065]

[0066] The structural formula of Ro-31-8220-mesylate (3-[3-[2,5-dihydro-4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-1H-pyrrol-3-yl]-1H-indol-1-yl]propylcarbamimidothioic acid ester mesylate) is shown below.

[0067]

[0068] The structural formula of Ro-32-0432-hydrochloride (3-[(8S)-8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl]-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione hydrochloride) is shown below.

[0069]

[0070] <WNT inhibitor> As used herein, the term "WNT signaling (WNT) inhibitor" includes all substances that inhibit or reduce Wnt signaling. Wnt is a secreted intercellular signaling protein known to be involved in intracellular signaling. Signaling pathways involving Wnt control functions such as cell proliferation and differentiation, movement, body axis formation during early embryonic development, and organogenesis. Signaling pathways involving Wnt include several different intracellular signaling mechanisms that are activated by the action of Wnt on cells. Signaling pathways involving Wnt include the β-catenin pathway, which controls gene expression via β-catenin, the PCP (planar cell polarity) pathway, which controls the planar polarity of cells, and the Ca ２＋ Ca that promotes intracellular mobilization of ２＋ As used herein, the WNT inhibitor may inhibit any pathway included in the signal transduction pathway involving Wnt.

[0071] Examples of WNT inhibitors include proteins such as secreted Frizzled-related proteins, Dickkopf (DKK) proteins, sclerostin, Cerberus, and Kremen. Specific examples of WNT inhibitors include, but are not limited to, IWR-1-endo, IWP-2, IWP-3, WNT-C59, XAV-939, CCT251545, KY1220, iCRT14, iCRT3, LF3, PNU-74654, KYA1797K, Capmatinib (INCB28060), KY02111, RCM-1, Resibufogenin, MSAB, PRI-724, Tegatrabetan (BC-2059), JW55, Adavivint (SM04690), Tryptonide, Isocercitrin, IQ-1, Salinomycin, and FH535. Furthermore, examples of WNT inhibitors include Wnt receptor inhibitors, soluble Wnt receptors, Wnt antibodies, casein kinase inhibitors, and dominant-negative Wnt proteins.

[0072] These WNT inhibitors include so-called tankirase (TNKS) inhibitors, and in the present invention, it is particularly preferable to use a TNKS inhibitor as the WNT inhibitor.

[0073] <TNKS Inhibitor> As used herein, the term "tankyrase (TNKS) inhibitor" refers to a substance that inhibits or suppresses the activity of tankyrase. Tankyrase (sometimes referred to as tankyrase) belongs to the poly(ADP-ribosyl)transferase (PARP) family, which poly(ADP-ribosyl)ates target proteins, and tankyrase-1 (tankyrase-1 / PARP-5a) and tankyrase-2 (tankyrase-2 / PARP-5b) are known. Tankyrase is known to poly(ADP-ribosyl)ate the telomeric protein TRF1 and release it from telomeres, thereby promoting telomere elongation by telomerase.

[0074] As used herein, the term "TNKS" refers to both tankyrase 1 and tankyrase 2, or to either tankyrase 1 or tankyrase 2. Furthermore, as used herein, the term "TNKS inhibitor" refers to a substance that inhibits tankyrase 1 and / or tankyrase 2. That is, the term "TNKS inhibitor" includes substances that inhibit or suppress only the activity of tankyrase 1, substances that inhibit or suppress only the activity of tankyrase 2, and substances that inhibit or suppress the activity of tankyrase 1 and tankyrase 2.

[0075] TNKS inhibitors include compounds that act directly or indirectly on TNKS, antisense nucleic acids against the gene encoding TNKS, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof.

[0076] As an example, the TNKS inhibitor may include a compound selected from the group consisting of IWR-1-endo, XAV939, G007-LK, G244-LM, MSC2504877, and WikiI4. Among the TNKS inhibitors, it is particularly preferable to use IWR-1-endo and / or XAV939.

[0077] The structural formula of IWR-1-endo (4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide) is shown below.

[0078]

[0079] The structural formula of XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one) is shown below.

[0080]

[0081] The structural formula of G007-LK (4-[5-[(1E)-2-[4-(2-chlorophenyl)-5-[5-(methylsulfonyl)-2-pyridinyl]-4H-1,2,4-thiazol-3-yl]ethenyl]-1,3,4-oxadiazol-2-yl]-benzonitrile) is shown below.

[0082]

[0083] The structural formula of G244-LM (3,5,7,8-tetrahydro-2-[4-[2-(methylsulfonyl)phenyl]-1-piperazinyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one) is shown below.

[0084]

[0085] The structural formula of WIKI4 (2-[3-[[4-(4-methoxyphenyl)-5-(4-pyridinyl)-4H-1,2,4-thiazol-3-yl]thio]propyl]-1H-benzo[de]isoquinoline-1,3(2H)-dione) is shown below.

[0086]

[0087] 1-3. Configuration The second liquid medium used in the method for producing pluripotent stem cells according to the present invention contains the PKCβ inhibitor and WNT inhibitor defined in 1-2 above. Note that the second liquid medium can also be prepared using a pluripotent stem cell differentiation inhibitor containing a PKCβ inhibitor and a WNT inhibitor as active ingredients.

[0088] Here, the PKCβ inhibitor contained in the second liquid medium may be one type or a combination of two or more different types.

[0089] The lower limit of the concentration of the PKCβ inhibitor is not particularly limited, and can be determined depending on the range in which it has an inhibitory effect on differentiation of pluripotent stem cells.

[0090] For example, the final concentration of the PKCβ inhibitor in the second liquid culture medium can be 25 nM or more, 30 nM or more, 50 nM or more, 80 nM or more, 100 nM or more, 150 nM or more, 200 nM or more, 500 nM or more, or 700 nM or more.

[0091] The upper limit of the concentration of the PKCβ inhibitor is not particularly limited, and can be determined depending on the range in which it has a differentiation-inhibiting effect on pluripotent stem cells, the range in which it is not toxic to pluripotent stem cells, the solubility of the PKCβ inhibitor, etc.

[0092] For example, the final concentration of the PKCβ inhibitor in the second liquid culture medium can be 15 μM or less, 10 μM or less, 5 μM or less, 3 μM or less, or 1 μM or less.

[0093] On the other hand, the WNT inhibitor contained in the second liquid medium may be one type or a combination of two or more different types.

[0094] The lower limit of the concentration of the WNT inhibitor is not particularly limited, and can be determined depending on the range in which it has an inhibitory effect on differentiation of pluripotent stem cells.

[0095] For example, the final concentration of the WNT inhibitor in the second liquid culture medium can be 90 nM or more, 100 nM or more, 150 nM or more, 200 nM or more, 300 nM or more, 400 nM or more, 500 nM or more, 600 nM or more, 700 nM or more, 800 nM or more, or 900 nM or more.

[0096] The upper limit of the concentration of the WNT inhibitor is not particularly limited, and can be determined depending on the range in which it has a differentiation-suppressing effect on pluripotent stem cells, the range in which it is not toxic to pluripotent stem cells, the solubility of the WNT inhibitor, etc.

[0097] For example, the final concentration of the WNT inhibitor in the second liquid medium can be 40 μM or less, 35 μM or less, 30 μM or less, 15 μM or less, 10 μM or less, 5 μM or less, 3 μM or less, 1.5 μM or less, or 1 μM or less.

[0098] Furthermore, the lower limit of the ratio of the concentration (molar concentration) of the PKCβ inhibitor to the WNT inhibitor in the second liquid culture medium is not particularly limited, but can be, for example, 167:1 or more, 111:1 or more, 56:1 or more, 33:1 or more, 11:1 or more, 7.8:1 or more, 5.6:1 or more, 2.2:1 or more, 1.7:1 or more, or 1.1:1 or more. The upper limit of the ratio of the concentrations (molar concentrations) of the PKCβ inhibitor to the WNT inhibitor in the second liquid medium is not particularly limited, but can be, for example, 1:1600 or less, 1:1400 or less, 1:1200 or less, 1:600 ​​or less, 1:400 or less, 1:200 or less, 1:120 or less, 1:60 or less, 1:40 or less, 1:36 or less, 1:32 or less, 1:28 or less, 1:24 or less, 1:20 or less, 1:16 or less, 1:12 or less, 1:8 or less, 1:6 or less, or 1:4 or less. The ratio of the concentrations (molar concentrations) of the PKCβ inhibitor to the WNT inhibitor in the second liquid medium is not particularly limited, but can be, for example, in the range of 167:1 or more and 1:1600 or less. Furthermore, the ratio of the concentration (molar concentration) of the PKCβ inhibitor to the WNT inhibitor in the second liquid culture medium can preferably be in the range of 111:1 or more and 1:1600 or less, 56:1 or more and 1:1600 or less, 33:1 or more and 1:1600 or less, 11:1 or more and 1:1600 or less, 7.8:1 or more and 1:1600 or less, 5.6:1 or more and 1:1600 or less, 2.2:1 or more and 1:1600 or less, 1.7:1 or more and 1:1600 or less, or 1.1:1 or more and 1:1600 or less.Furthermore, the ratio of the concentration (molar concentration) of the PKCβ inhibitor to the WNT inhibitor in the second liquid medium can be in the range of 167:1 or more and 1:1400 or less, 167:1 or more and 1:1200 or less, 167:1 or more and 1:600 ​​or less, 167:1 or more and 1:400 or less, 167:1 or more and 1:200 or less, 167:1 or more and 1:120 or less, 167:1 or more and 1:60 or less, or 167:1 or more and 1:40 or less. , can be in the range of 167:1 or more and 1:36 or less, can be in the range of 167:1 or more and 1:32 or less, can be in the range of 167:1 or more and 1:28 or less, can be in the range of 167:1 or more and 1:24 or less, can be in the range of 167:1 or more and 1:20 or less, can be in the range of 167:1 or more and 1:16 or less, can be in the range of 167:1 or more and 1:12 or less, can be in the range of 167:1 or more and 1:8 or less, can be in the range of 167:1 or more and 1:6 or less, can be in the range of 167:1 or more and 1:4 or less.

[0099] The active ingredients, PKCβ inhibitor and WNT inhibitor, can also be added to the medium in combination with a carrier, which includes a solvent and / or excipient.

[0100] Examples of solvents include water, buffers (including PBS), physiological saline, organic solvents (DMSO, DMF, xylene, lower alcohols), and the like.

[0101] Examples of excipients include antibiotics, buffers, thickeners, colorants, stabilizers, surfactants, emulsifiers, antiseptics, preservatives, antioxidants, etc. Antibiotics are not particularly limited, and examples thereof include penicillin, streptomycin, and amphotericin B. Examples of buffers include phosphate buffer, Tris-HCl buffer, and glycine buffer. Examples of thickeners include gelatin and polysaccharides. Examples of colorants include phenol red. Examples of stabilizers include albumin, dextran, methylcellulose, and gelatin. Examples of surfactants include cholesterol, alkyl glycosides, alkyl polyglucosides, alkyl monoglyceryl ethers, glucosides, maltosides, neopentyl glycols, polyoxyethylene glycols, thioglucosides, thiomaltosides, peptides, saponins, phospholipids, fatty acid sorbitan esters, and fatty acid diethanolamides. Examples of emulsifiers include glycerin fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, sucrose fatty acid esters, etc. Examples of preservatives include aminoethylsulfonic acid, benzoic acid, sodium benzoate, ethanol, sodium edetate, agar, dl-camphor, citric acid, sodium citrate, salicylic acid, sodium salicylate, phenyl salicylate, dibutylhydroxytoluene, sorbic acid, potassium sorbate, nitrogen, dehydroacetic acid, sodium dehydroacetate, 2-naphthol, sucrose, honey, isobutyl parahydroxybenzoate, isopropyl parahydroxybenzoate, ethyl parahydroxybenzoate, butyl parahydroxybenzoate, propyl parahydroxybenzoate, methyl parahydroxybenzoate, 1-menthol, eucalyptus oil, etc.Examples of preservatives include benzoic acid, sodium benzoate, ethanol, sodium edetate, dried sodium sulfite, citric acid, glycerin, salicylic acid, sodium salicylate, dibutylhydroxytoluene, D-sorbitol, sorbic acid, potassium sorbate, sodium dehydroacetate, isobutyl parahydroxybenzoate, isopropyl parahydroxybenzoate, ethyl parahydroxybenzoate, butyl parahydroxybenzoate, propyl parahydroxybenzoate, methyl parahydroxybenzoate, propylene glycol, and phosphoric acid. Examples of antioxidants include citric acid, citric acid derivatives, vitamin C and its derivatives, lycopene, vitamin A, carotenoids, vitamin B and its derivatives, flavonoids, polyphenols, glutathione, selenium, sodium thiosulfate, vitamin E and its derivatives, α-lipoic acid and its derivatives, pycnogenol, flavangenol, superoxide dismutase (SOD), glutathione peroxidase, glutathione-S-transferase, glutathione reductase, catalase, ascorbate peroxidase, and mixtures thereof.

[0102] The first and second liquid media may also contain one or more growth factors, such as FGF2 and TGF-β1.

[0103] In the method for producing pluripotent stem cells according to the present invention, it is important to culture unicellular pluripotent stem cells in a liquid medium (first liquid medium) that is substantially free of a PKCβ inhibitor before culturing them in the second liquid medium. The first liquid medium used in the method for producing pluripotent stem cells according to the present invention may or may not contain a WNT inhibitor, but preferably does not contain a WNT inhibitor.

[0104] When the first liquid medium contains a WNT inhibitor, the concentration range of the WNT inhibitor is not necessarily limited, but may be the same as that of the second liquid medium described above.

[0105] 1-4. Effects According to the method for producing pluripotent stem cells of the present invention, unicellular pluripotent stem cells are cultured in a first liquid medium substantially free of a PKCβ inhibitor, and after the pluripotent stem cells form cell clusters, the pluripotent stem cells are suspension-cultured in a second liquid medium containing a PKCβ inhibitor and a WNT inhibitor, thereby forming cell clusters that maintain the undifferentiated state of the pluripotent stem cells while preventing cell death. Therefore, the method for producing pluripotent stem cells of the present invention makes it possible to efficiently produce large quantities of high-quality pluripotent stem cells.

[0106] 2. Further detailed explanation of the culturing process 2-1. Overview Below, the process of performing suspension culture using a first liquid medium, the subsequent process of performing suspension culture using a second liquid medium, and any other optional processes included in the method for producing pluripotent stem cells according to the present invention will be described.

[0107] 2-2. Method The method of this embodiment includes a suspension culture step using a first liquid medium and a suspension culture step using a second liquid medium as essential steps, and a maintenance culture step and a recovery step as optional steps. In the following description, the terms "suspension culture" and "suspension culture step" simply refer to the culture from the start of the suspension culture step using the first liquid medium to the end of the suspension culture step using the second liquid medium.

[0108] 2-2-1. Maintenance Culture Step The "maintenance culture step" is a step of culturing a cell population before the suspension culture step, or a cell mass obtained after the suspension culture step or the subsequent recovery step, in order to proliferate the cells while maintaining their undifferentiated state. Maintenance culture can utilize animal cell culture methods known in the art. For example, the cells may be cultured by adhesion to a culture substrate such as a container or carrier, or by suspension culture.

[0109] Hereinafter, although not limited thereto, examples of animal cell culture methods used in this step and in the suspension culture step described below will be given and explained.

[0110] (Cells) The cells used in this step are cells capable of cell aggregation in suspension culture. As described above in the section "Culture and Medium" in "1-2. Definition of Terms," ​​animal cells are preferred, and human cells are more preferred. The type of cells is pluripotent stem cells, and pluripotent stem cells such as iPS cells and ES cells are particularly preferred.

[0111] (Culture vessel) The culture vessel used for culturing is preferably a vessel with low cell adhesion to the inner surface of the vessel. Examples of vessels with low cell adhesion to the inner surface of the vessel include plates that have been hydrophilically treated with a biocompatible substance. For example, Nunclon ＴＭ Sphera (Thermo Fisher Scientific) can be used as a culture vessel.

[0112] The shape of the culture vessel is not particularly limited, and examples thereof include culture vessels in the shape of a dish, flask, well, bag, spinner flask, etc.

[0113] The capacity of the culture vessel to be used can be appropriately selected and is not particularly limited, but the lower limit of the area of ​​the bottom surface of the part containing the culture medium when viewed in plan is 0.32 cm ２ More than 0.65 cm ２ More than 1.9 cm ２ Above, 3.0cm ２ Above, 3.5cm ２ Above, 9.0cm ２ or more, or 9.6 cm ２ Above, the upper limit is 1000 cm ２ Below, 500cm ２ Below, 300cm ２ Below, 150cm ２ Below, 75cm ２ Below, 55cm ２ Below, 25cm ２ Below, 21cm ２ Below, 10cm ２ Less than or equal to 3.5 cm ２ It is preferable that:

[0114] The capacity of the culture vessel to be used can be selected appropriately and is not particularly limited, but it is preferable that the lower limit of the volume capable of accommodating the culture medium and culturing is 1 mL, 2 mL, 4 mL, 10 mL, 20 mL, 30 mL, 50 mL, 100 mL, 200 mL, 500 mL, 1 L, 3 L, 5 L, 10 L, or 20 L, and the upper limit is 100 L, 50 L, 20 L, 10 L, 5 L, 3 L, 1 L, 500 mL, 200 mL, 100 mL, 50 mL, or 30 mL.

[0115] (Culture medium) The amount of culture medium and culture solution may be adjusted appropriately depending on the culture vessel used. Although not particularly limited, examples are given below. For example, in a 12-well plate (where the area of ​​the well bottom in a plan view is 3.5 cm per well), ２ When using a 6-well plate (where the area of ​​the well bottom in a plan view is 9.6 cm per well), the amount per well can be 0.5 mL or more and 1.5 mL or less, preferably about 1.3 mL. ２When using a 125 mL Erlenmeyer flask (a 125 mL Erlenmeyer flask), the lower limit of the amount per well can be 1.5 mL or more, 2 mL or more, or 3 mL or more, and the upper limit can be 6 mL or less, 5 mL or less, or 4 mL or less. Furthermore, when using a 125 mL Erlenmeyer flask (a 125 mL Erlenmeyer flask), the lower limit of the amount per container can be 10 mL or more, 15 mL or more, 20 mL or more, 25 mL or more, or 30 mL or more, and the upper limit can be 50 mL or less, 45 mL or less, or 40 mL or less. Furthermore, when using a 500 mL Erlenmeyer flask, the lower limit of the amount per container can be 100 mL or more, 105 mL or more, 110 mL or more, 115 mL or more, or 120 mL or more, and the upper limit can be 150 mL or less, 145 mL or less, 140 mL or less, 135 mL or less, 130 mL or less, or 125 mL or less. Furthermore, when a 1000 mL Erlenmeyer flask is used, the lower limit of the amount per container can be 250 mL or more, 260 mL or more, 270 mL or more, 280 mL or more, or 290 mL or more, and the upper limit can be 350 mL or less, 340 mL or less, 330 mL or less, 320 mL or less, or 310 mL or less. Furthermore, for example, when using a disposable culture bag with a capacity of 2 L, the amount per bag can be 100 mL or more, 200 mL or more, 300 mL or more, 400 mL or more, 500 mL or more, 600 mL or more, 700 mL or more, 800 mL or more, 900 mL or more, or 1000 mL or more, and the amount per bag can be 2000 mL or less, 1900 mL or less, 1800 mL or less, 1700 mL or less, 1600 mL or less, 1500 mL or less, 1400 mL or less, 1300 mL or less, 1200 mL or less, or 1100 mL or less. Furthermore, when using a disposable culture bag with a capacity of 10 L, the amount per bag can be 500 mL or more, 1 L or more, 2 L or more, 3 L or more, 4 L or more, or 5 L or more, and the amount per bag can be 10 L or less, 9 L or less, 8 L or less, 7 L or less, or 6 L or less.When a single-use reactor with a capacity of 1 L is used, the lower limit of the amount per reactor can be 300 mL or more, 350 mL or more, 400 mL or more, 450 mL or more, or 500 mL or more, and the upper limit can be 1 L or less, 900 mL or less, 800 mL or less, 700 mL or less, or 600 mL or less. When a stirring impeller-type reactor of any capacity is used, the working volume can be within the range specified by each manufacturer.

[0116] (Seeding density) In suspension culture, the density of cells to be seeded in a new medium (seeding density) can be appropriately adjusted taking into consideration the culture time, the state of the cells after culture, and the number of cells required after culture. Although not limited thereto, the lower limit is usually, for example, 0.01 × 10 ５ cells / mL or more, 0.1×10 ５ cells / mL or more, 1×10 ５ cells / mL or more, or 2 x 10 ５ cells / mL or more, and the upper limit is, for example, 20 x 10 ５ cells / mL or less, or 10 x 10 ５ The range is less than 1000 cells / mL.

[0117] (Culture conditions) Culture temperature, time, CO ２ The culture conditions such as concentration are not particularly limited. They may be within the range of conventional methods in the field. For example, the culture temperature may be 20°C or higher or 35°C or higher at the lower limit and 45°C or lower or 40°C or lower at the upper limit, preferably 37°C. The culture time may be 0.5 hours or higher or 6 hours or higher at the lower limit and 7 days or shorter, 120 hours or shorter, 96 hours or shorter, 72 hours or shorter, or 48 hours or shorter at the upper limit. CO during culture ２ The CO concentration may be 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, or 4.5% or more at the lower limit, or 10% or less, or 5.5% or less at the upper limit, preferably 5%. ２The concentration does not need to be constant and may be changed or varied during the culture. Furthermore, medium exchange can be performed at an appropriate frequency. The frequency of medium exchange varies depending on the cell type being cultured, but may be, for example, at least once every 5 days, at least once every 4 days, at least once every 3 days, at least once every 2 days, at least once a day, or at least twice a day, but is not limited to these. Furthermore, medium exchange can be performed continuously using a perfusion method. After collecting the cells using a method similar to the collection step, fresh medium can be added, the cell masses can be gently dispersed, and the cells can be cultured again. When medium exchange is performed using a perfusion method, the cells and medium can be separated using a filter to retain the cells in the culture system, and the medium can then be exchanged. The frequency and method of medium exchange are not limited to those described above, and the optimal method can be adopted as appropriate.

[0118] The timing of culture termination and medium replacement can also be determined based on, for example, the lactic acid concentration in the medium. Lactic acid is produced by cells during culture and accumulates in the medium. Lactic acid produced by cells or lactic acid originally contained in the medium is known to damage cells, thereby inhibiting the maintenance of undifferentiated state of pluripotent stem cells and adversely affecting proliferation, particularly cell proliferation ability after subculture. Therefore, by determining the timing of culture termination and / or the timing and amount of medium replacement based on the lactic acid concentration in the medium, it is possible to avoid the inhibitory effect of lactic acid on the maintenance of undifferentiated state and the decrease in cell proliferation ability caused by lactic acid.

[0119] (Culture Method) The state of flow of the medium during culture is not important, and either static culture or flow culture may be used, with flow culture being preferred.

[0120] "Static culture" refers to culturing in a culture vessel in a stationary state. This static culture is usually used in adherent culture.

[0121] "Fluid culture" refers to culturing under conditions in which the medium is flowing. In the case of fluid culture, a method in which the medium is flowed so as to promote cell aggregation is preferred. Examples of such culture methods include rotation culture, rocking culture, agitation culture, and combinations thereof.

[0122] "Rotation culture" (including shaking culture) refers to a culture method in which the medium flows so that cells gather at one point due to stress (centrifugal force, centripetal force) caused by a swirling flow. Specifically, this is done by rotating a culture vessel containing a medium containing cells along a generally horizontal plane, tracing a closed orbit such as a circle, ellipse, flattened circle, or flattened ellipse.

[0123] The rotation speed is not particularly limited, but the lower limit can be, for example, 1 rpm or more, 10 rpm or more, 50 rpm or more, 60 rpm or more, 70 rpm or more, 80 rpm or more, 83 rpm or more, 85 rpm or more, or 90 rpm or more. On the other hand, the upper limit can be, for example, 200 rpm or less, 150 rpm or less, 120 rpm or less, 115 rpm or less, 110 rpm or less, 105 rpm or less, 100 rpm or less, 95 rpm or less, or 90 rpm or less. The amplitude of the shaker used for rotational culture is not particularly limited, but the lower limit can be, for example, 1 mm or more, 10 mm or more, or 20 mm or more. On the other hand, the upper limit can be, for example, 200 mm or less, 100 mm or less, 50 mm or less, or 30 mm or less. The radius of rotation during rotational culture is not particularly limited, but is preferably set so that the amplitude is within the above-mentioned range. The lower limit of the radius of rotation can be, for example, 5 mm or more or 10 mm or more, and the upper limit can be, for example, 100 mm or less or 50 mm or less. In particular, in the cell aggregate production method described below, setting the rotation conditions within the above-mentioned range is preferred because it makes it easier to produce cell aggregates of an appropriate size.

[0124] "Rock culture" refers to a culture method in which a rocking flow is imparted to the culture medium by linear reciprocating motion, such as rocking agitation. Specifically, rocking is performed by rocking a culture vessel containing a cell-containing culture medium in a plane perpendicular to a generally horizontal plane. The rocking speed is not particularly limited, but for example, if one reciprocating motion is considered one cycle, the lower limit is 2 or more, 4 or more, 6 or more, 8 or more, or 10 or more times per minute, while the upper limit is 50 or less, 25 or less, 20 or less, or 15 or less times per minute. During rocking, it is preferable to provide the culture vessel with a slight angle, i.e., an induction angle, relative to the vertical plane. The rocking angle is not particularly limited, but for example, the lower limit can be 0.1° or more, 2° or more, 4° or more, 6° or more, or 8° or more, while the upper limit can be 20° or less, 18° or less, 15° or less, 12° or less, or 10° or less. In the cell aggregate manufacturing method described below, it is preferable to set the rocking conditions within the above range, since this makes it easier to manufacture cell aggregates of an appropriate size.

[0125] Furthermore, the culture can be performed while stirring by a combination of the above-mentioned rotation and rocking movements.

[0126] "Agitation culture" refers to a culture method in which the culture vessel is left stationary while the medium in the vessel is agitated using an agitation means such as a stirrer bar or agitation blade. For example, agitation culture can be achieved by using a spinner flask-type culture vessel equipped with an agitation blade. Such culture vessels are commercially available and can also be used. For commercially available spinner flask-type culture vessels, the amount of cell culture composition recommended by the manufacturer can be suitably used. The agitation speed of the agitation means is not particularly limited, but the lower limit can be 1 rpm or more, 10 rpm or more, 30 rpm or more, 50 rpm or more, 70 rpm or more, 90 rpm or more, 110 rpm or more, or 130 rpm or more. On the other hand, the upper limit can be 200 rpm or less, or 150 rpm or less.

[0127] Furthermore, in the "agitated culture method," it is preferable to control the shear stress applied to the cells during culture. Animal cells, including pluripotent stem cells, are generally more susceptible to physical stress than other cells. Therefore, if the shear stress applied to the cells during agitated culture is too great, the cells may suffer physical damage, resulting in a decrease in their proliferation ability or, in the case of pluripotent stem cells, in the inability to maintain their undifferentiated state.

[0128] Physical damage to cells in agitated culture can be affected by local shear stress around the agitator blade. The local shear stress around the agitator blade is related to, but not limited to, the blade tip speed (the peripheral speed at the largest blade diameter). The blade tip speed can be calculated as blade diameter [m] × pi × rotation speed [rps] = blade tip speed [m / s].

[0129] Specifically, in suspension culture using the second liquid medium, agitation culture can be performed while maintaining the undifferentiated state of pluripotent stem cells, even at very high impeller tip speeds of 0.23 m / s or more.

[0130] Furthermore, the blade tip speed is not necessarily limited, but is preferably 0.05 m / s or more, preferably 0.08 m / s or more, preferably 0.10 m / s or more, preferably 0.13 m / s or more, preferably 0.17 m / s or more, preferably 0.20 m / s or more, preferably 0.23 m / s or more, preferably 0.25 m / s or more, and preferably 0.30 m / s or more. By keeping the blade tip speed within this range, it is possible to suppress excessive aggregation of cells while maintaining the undifferentiated state of the pluripotent stem cells.

[0131] Furthermore, the tip speed of the impeller is not necessarily limited, but is preferably 1.37 m / s or less, preferably 1.00 m / s or less, preferably 0.84 m / s or less, preferably 0.50 m / s or less, preferably 0.42 m / s or less, preferably 0.34 m / s or less, and preferably 0.30 m / s or less. By keeping the tip speed within this range, the medium flow state in the culture system can be stabilized while maintaining the undifferentiated state of the pluripotent stem cells.

[0132] The above-mentioned preferred range of impeller tip speed can also be applied to ABLE's single-use reactors provided for suspension culture of pluripotent stem cells. Furthermore, in agitation culture, although not particularly limited, when changing the culture scale, the rotation speed of the agitator impeller can be determined using the constant Pv formula. Pv refers to the agitation power required per unit volume, and by keeping Pv the same, agitation culture can be performed in the same way between different scales. The constant Pv formula is the number of rotations per unit time [rpm or rps] x (impeller diameter [m]) ２／３ = constant.

[0133] The extent to which the number of cells should be increased in this step and the state of the cells should be adjusted may be determined appropriately depending on the type of cells to be cultured, the purpose of cell aggregation, the type of medium, and the culture conditions.

[0134] Hereinafter, although not limited to, an example of a culture method in which this step is carried out by an adherent culture method will be described. Here, explanations common to the above-mentioned "2-2-1. Maintenance culture step" will be omitted, and only the features characteristic of the adherent culture method will be described in detail.

[0135] In adherent culture, cells are cultured by adhering them to a substrate such as a culture vessel or microcarrier, or an external matrix. The external matrix is ​​not particularly limited, but examples of materials that can be used include laminin, vitronectin, gelatin, collagen, and E-cadherin chimeric antibodies. For example, a peel-off cell culture plate (Sumitomo Bakelite Co., Ltd.) can be used as the culture vessel. The extent to which the number of cells is increased and the state of the cells to be cultured in this process can be determined appropriately depending on the type of cells to be cultured, the type of medium, and the culture conditions. For example, the end point of the culture can be when the coverage of the culture substrate with adherent cells reaches 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0136] (Post-process treatment) After this process, the cells are separated from the culture medium by a conventional method and recovered. At this time, the cells are preferably recovered as single cells by detachment or dispersion treatment. Specific methods will be described in detail in the section on single cell formation in the suspension culture process and in the recovery process below. The recovered cells may be used as is, or, if necessary, washed with a buffer (including PBS buffer), physiological saline, or medium (preferably the medium or basal medium used in the next process) before being subjected to the next process.

[0137] 2-2-2. Suspension culture step As described above, the "suspension culture step" includes a suspension culture step using a first liquid medium and a suspension culture step using a second liquid medium. In this section, the suspension culture step using the first liquid medium and the suspension culture step using the second liquid medium will be described, but they are basically similar to the culture method described in "2-2-1. Maintenance culture step" above. Therefore, explanations common to the method already described for the maintenance culture step will be omitted here, and only the characteristics of these steps will be described in detail.

[0138] (Cells) The cells used in this step are not limited, but are preferably cells prepared after the maintenance culture step. The type of cells is also pluripotent stem cells, as described in the maintenance culture step, and pluripotent stem cells such as iPS cells and ES cells are particularly preferred. Furthermore, the cells are preferably in a single-cell state when seeded in the first liquid medium. Note that a single-cell state does not necessarily mean that the entire cell population is in a single-cell state; the cell population may contain, in addition to single-cell cells, cells in a state where several cells are adhered together.

[0139] Furthermore, the pluripotent stem cells used in this step may be a single cell or a cell population consisting of multiple cells (pluripotent stem cell population). When the pluripotent stem cells are a pluripotent stem cell population, the percentage (proportion) of cells in the population that express a pluripotent stem cell marker (e.g., OCT4, SOX2, NANOG) and / or are positive for the pluripotent stem cell marker is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0140] (First liquid medium) The first liquid medium is a liquid medium that differs from the second liquid medium described below in that it does not substantially contain a PKCβ inhibitor. As the first liquid medium, any liquid medium that can be used for suspension culture of pluripotent stem cells, among the media described in the section on the maintenance culture step above, can be used without limitation.

[0141] Here, in the present specification, the condition that the first liquid medium is substantially free of a PKCβ inhibitor is not limited to cases where the medium does not contain any PKCβ inhibitor or where the PKCβ inhibitor in the medium is below the detection limit, but is also interpreted as including cases where the PKCβ inhibitor is, for example, at a final concentration of less than 25 nM. In other words, the first liquid medium can contain the PKCβ inhibitor at a final concentration of less than 25 nM, less than 23 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, or less than 3 nM. Most preferably, the first liquid medium contains the PKCβ inhibitor at a concentration below the detection limit. If the PKCβ inhibitor in the first liquid medium is below the detection limit or within the above concentration range, the PKCβ inhibitor does not act on cells and can exhibit the effect of preventing cell death.

[0142] For example, if pluripotent stem cells are cultured in a medium containing a PKCβ inhibitor (e.g., a second liquid medium) prior to suspension culture of single-celled pluripotent stem cells in a first liquid medium, and then, if necessary, the single-celled pluripotent stem cells are subjected to the production method of the present invention, because the pluripotent stem cells are cultured in a medium containing a PKCβ inhibitor (e.g., a second liquid medium) in the previous step, there is a possibility that the PKCβ inhibitor will be carried over when the pluripotent stem cells are seeded in the first liquid medium. However, even if a PKCβ inhibitor is carried over when the pluripotent stem cells are seeded, the pluripotent stem cells will be suspension cultured in a first liquid medium that is substantially free of the PKCβ inhibitor, so long as the concentration is within the above-mentioned range.

[0143] Furthermore, depending on the type of pluripotent stem cells cultured in the first liquid medium, the culture conditions, etc., substances that have an inhibitory effect on PKCβ, not limited to the above-mentioned PKCβ inhibitors, may be produced in the medium. Even in this case, the embodiment includes suspension culture of pluripotent stem cells in a first liquid medium that is substantially free of a PKCβ inhibitor, as long as the concentration is within the above-mentioned range.

[0144] The first liquid medium may contain a WNT inhibitor, but preferably does not contain a WNT inhibitor. In this case, "not containing a WNT inhibitor" means "substantially not containing a WNT inhibitor." As with the PKCβ inhibitor, this is not limited to cases where the WNT inhibitor is below the detection limit, but is interpreted to include cases where the WNT inhibitor is, for example, less than 90 nM as a final concentration. In other words, the first liquid medium can contain the WNT inhibitor at a final concentration of less than 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, or 3 nM. Most preferably, the first liquid medium contains a WNT inhibitor at a detection limit or below. If the concentration of the WNT inhibitor in the first liquid medium is below the detection limit or within the above-mentioned concentration range, the WNT inhibitor does not act on the cells, and the effect of further preventing cell death can be achieved.

[0145] Regarding the WNT inhibitor, even if it is introduced into the medium when the pluripotent stem cells are seeded, or even if a substance that inhibits WNT signaling is produced in the medium depending on the type of pluripotent stem cells or the culture conditions, as long as the concentration is within the above-mentioned range, the pluripotent stem cells will be cultured in suspension in the first liquid medium that does not contain the WNT inhibitor.

[0146] (Second Liquid Medium) The second liquid medium is characterized by containing a PKCβ inhibitor and a WNT inhibitor. The type of medium is not limited as long as it contains a PKCβ inhibitor and a WNT inhibitor and is capable of growing and / or maintaining cells.

[0147] The concentrations of the PKCβ inhibitor and WNT inhibitor contained in the second liquid medium are as described in the above section 1-3. Composition. The method of adding the PKCβ inhibitor and WNT inhibitor is not particularly limited, as long as the concentrations of the PKCβ inhibitor and WNT inhibitor at the start of culture using the second liquid medium are within the above-mentioned ranges. For example, the medium may be prepared by directly adding one or more PKCβ inhibitors and WNT inhibitors to the medium so that the total amount falls within the above-mentioned concentration range.

[0148] (Conversion to single cells) As used herein, "conversion to single cells" refers to dispersing cell aggregates in which multiple cells adhere or aggregate to each other, such as monolayer cell fragments or cell clumps, into single, free-standing cells. In suspension culture of pluripotent stem cells using the first liquid medium, the cell aggregates are dispersed, and single, free-standing pluripotent stem cells are cultured in suspension.

[0149] Dissociation of the cells into single cells is performed using a detachment agent and / or a chelating agent. Examples of the detachment agent include, but are not limited to, trypsin, collagenase, pronase, hyaluronidase, elastase, and commercially available Accutase (registered trademark), Accumax (registered trademark), and TrypLE. ＴＭ Express Enzyme (Life Technologies Japan), TrypLE ＴＭ　Select Enzyme (Life Technologies Japan, Inc.), Dispase (registered trademark), etc. can be used. For example, when trypsin is used to disperse single cells, the lower limit of the concentration in the solution is not particularly limited as long as it is a concentration that can disperse cell aggregates, but it may be, for example, 0.15% by volume or more, 0.18% by volume or more, 0.20% by volume or more, or 0.24% by volume or more. On the other hand, the upper limit of the concentration in the solution is not particularly limited as long as it is a concentration that does not affect the cells themselves, such as by lysis, but it may be 0.30% by volume or less, 0.28% by volume or less, or 0.25% by volume or less. Furthermore, although the treatment time depends on the concentration of trypsin, the lower limit is not particularly limited as long as the cell aggregates are sufficiently dispersed by the action of trypsin, and it may be, for example, 5 minutes or more, 8 minutes or more, 10 minutes or more, 12 minutes or more, or 15 minutes or more. On the other hand, the upper limit of the treatment time is not particularly limited as long as the cells themselves are not affected by the action of trypsin, such as being lysed. For example, it may be 30 minutes or less, 28 minutes or less, 25 minutes or less, 22 minutes or less, 20 minutes or less, or 18 minutes or less. When using a commercially available detachment agent, it should be used at a concentration that can disperse the cells into a single cell state, as described in the attached protocol. The formation of single cells can be promoted by gentle physical treatment after treatment with the detachment agent and / or chelating agent. This physical treatment is not limited, but examples include pipetting the cells together with the solution multiple times. Furthermore, if necessary, the cells may be passed through a strainer or mesh.

[0150] The single-cells can be recovered by leaving the cells to stand or centrifuging them to remove the supernatant containing the release agent. The recovered cells may be washed as needed. The centrifugation conditions and washing method may be the same as those described above.

[0151] (Culture Method) In the method for producing pluripotent stem cells according to the present invention, first, single-cell pluripotent stem cells are cultured in suspension in a first liquid medium, and after the pluripotent stem cells form cell clusters, the pluripotent stem cells are cultured in suspension in a second liquid medium. Here, for both the suspension culture using the first liquid medium and the suspension culture using the second liquid medium, the culture method is preferably flow culture, in which the medium is flowed.

[0152] In the suspension culture step using the first liquid medium, the conditions described in the section on the maintenance culture step, such as the culture vessel, medium volume, culture temperature, seeding density, rotation conditions, and stirring conditions, can be applied. The suspension culture using the first liquid medium is carried out until the cultured pluripotent stem cells form cell clusters.

[0153] In this method, whether pluripotent stem cells have formed cell clusters (whether they are in a single state or have formed cell clusters) can be determined by sampling the medium during culture and observing the cells contained in the culture medium using a microscope or other means. Specifically, cell aggregates can be defined as those in which the lower limit of the maximum width of the aggregate is 35 μm or more, 40 μm or more, 45 μm or more, 50 μm or more, 55 μm or more, or 60 μm or more. If the sampled medium contains cell clusters defined within this range, this can be considered the end of the suspension culture process using the first liquid medium. Alternatively, the end of the suspension culture process using the first liquid medium can be reached when, among the observed cell aggregates, the cell masses defined within the above ranges account for 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100% or more by weight compared to the cell mass at the start of culture.

[0154] In other words, in this method, whether or not pluripotent stem cells have formed cell clusters can be determined based on whether or not cell aggregates meeting a predetermined size are observed, or whether or not cell aggregates meeting a predetermined size are present at a rate exceeding a predetermined level. More specifically, the suspension culture using the first liquid medium can be determined to be complete when cell aggregates with a maximum width of 50 μm or more account for 60% or more of the total cell mass by weight.

[0155] Alternatively, whether or not cell clusters are formed in suspension culture of pluripotent stem cells using the first liquid medium can be determined by the time elapsed since the start of culture. This determination is based on the finding that the proliferation rate of pluripotent stem cells and the rate of cell cluster formation are more or less the same depending on the type of pluripotent stem cells to be cultured, the medium components, the culture conditions, etc. Generally, cell clusters as defined above are formed 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 48 hours after seeding of single-cell cells. Therefore, the suspension culture using the first liquid medium can be considered to have ended 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 48 hours after the initiation of suspension culture of pluripotent stem cells in the first liquid medium, and the end of suspension culture using the first liquid medium can be determined. In many cases, the end of suspension culture using the first liquid medium can be determined 24 hours after the initiation of suspension culture of pluripotent stem cells in the first liquid medium.

[0156] As described above, after cell clusters are formed in the suspension culture step using the first liquid medium, the process proceeds to the suspension culture step using the second liquid medium. In particular, in the method for producing pluripotent stem cells according to the present invention, the undifferentiated state of the pluripotent stem cells can be maintained by suspension culturing the pluripotent stem cells in the second liquid medium containing a PKCβ inhibitor and a WNT inhibitor.

[0157] The suspension culture step using the second liquid medium may be initiated by adding a PKCβ inhibitor and, if necessary, a WNT inhibitor to the medium after completion of the suspension culture step using the first liquid medium, or by seeding the cell masses recovered from the first liquid medium into the second liquid medium. The PKCβ inhibitor and WNT inhibitor may also be added so that the concentrations in the culture medium gradually increase.

[0158] At this time, a portion of the pluripotent stem cells can be removed during the culture to confirm whether they maintain an undifferentiated state. For example, by measuring the expression level of pluripotent stem cell markers expressed in pluripotent stem cells removed during culture, it can be confirmed whether they maintain an undifferentiated state. As described above, examples of pluripotent stem cell markers include alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1. As described above, methods for detecting these pluripotent stem cell markers include, for example, flow cytometry.

[0159] When the positive rate of pluripotent stem cell markers among the pluripotent stem cells extracted during culture is preferably 80% or more, more preferably 90% or more, more preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, or more preferably 100%, it can be determined that the cells remain undifferentiated.

[0160] Furthermore, whether the undifferentiated state is maintained can be confirmed by measuring the expression levels of three germ layer markers (endodermal cell markers, mesodermal cell markers, and ectodermal cell markers) in pluripotent stem cells removed during the suspension culture step using the second liquid medium. That is, when the positive rates of these endodermal cell markers, mesodermal cell markers, and ectodermal cell markers are all preferably 20% or less, more preferably 10% or less, more preferably 9% or less, more preferably 8% or less, more preferably 7% or less, more preferably 6% or less, more preferably 5% or less, more preferably 4% or less, more preferably 3% or less, more preferably 2% or less, more preferably 1% or less, and more preferably below the detection limit, it can be determined that the undifferentiated state is maintained.

[0161] Endodermal cell markers are genes specific to endodermal cells, and examples thereof include SOX17, FOXA2, CXCR4, AFP, GATA4, EOMES, etc. Endodermal cells differentiate into tissues of organs such as the digestive tract, lung, thyroid, pancreas, and liver, cells of secretory glands that open into the digestive tract, peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most part of the urethra, and part of the ureter), etc.

[0162] Mesodermal cell markers are genes specific to mesodermal cells, and examples thereof include TBXT (BRACHYURY), MESP1, MESP2, FOXF1, HAND1, EVX1, IRX3, CDX2, TBX6, MIXL1, ISL1, SNAI2, FOXC1, and PDGFRα. Mesodermal cells differentiate into body cavities and the mesothelium lining them, muscles, skeletons, skin dermis, connective tissue, heart, blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidneys, ureters, and gonads (testes, uterus, and gonadal epithelium).

[0163] Ectodermal cell markers are genes specific to ectodermal cells, and examples thereof include FGF5, NESTIN, SOX1, and PAX6. Ectodermal cells form the epidermis of the skin, the epithelium of the distal part of the male urethra, hair, nails, skin glands (including mammary glands and sweat glands), sensory organs (including the epithelium of the distal parts of the oral cavity, pharynx, nose, and rectum, and salivary glands), the lens, and the peripheral nervous system. Furthermore, a portion of the ectoderm invaginates into a groove during development to form the neural tube, which also serves as the source of neurons and melanocytes of the central nervous system, such as the brain and spinal cord.

[0164] The expression levels of these three germ layer markers (endodermal cell markers, mesodermal cell markers, and ectodermal cell markers) can be measured by any detection method known in the art. Methods for measuring the expression of the three germ layer markers (endodermal cell markers, mesodermal cell markers, and ectodermal cell markers) include, but are not limited to, quantitative real-time PCR analysis, RNA-Seq, Northern hybridization, and hybridization using a DNA array. In quantitative real-time PCR analysis, the expression level of the marker gene to be measured is converted to a relative expression level with respect to the expression level of an internal standard gene, and the expression level of the marker gene can be evaluated based on this relative expression level. Examples of internal standard genes include the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and the β-actin (ACTB) gene.

[0165] Incidentally, when pluripotent stem cells are cultured in suspension in the second liquid medium, the presence of a PKCβ inhibitor and a WNT inhibitor can reduce the adverse effects of lactic acid, as described in the section "2-2-1. Maintenance Culture Step" (Culture Conditions). In other words, even at higher concentrations of lactic acid than those used in conventional cell culture, it is possible to maintain undifferentiated state and cell proliferation ability. Specifically, culture can be performed favorably even when the lactic acid concentration in the culture medium reaches 5 mM or higher. Furthermore, culture can be performed favorably even when the lactic acid concentration in the culture medium reaches 7 mM or higher, 9 mM or higher, 10 mM or higher, 11 mM or higher, or 12 mM or higher.

[0166] On the other hand, it is preferable that the concentration of lactic acid accumulated in the culture medium be 15 mM or less. In particular, it is preferable that the concentration of lactic acid accumulated in the culture medium be 14 mM or less, 13 mM or less, 12 mM or less, or 10 mM or less. If the concentration of lactic acid accumulated in the culture medium is within this range, adverse effects on cells can be avoided.

[0167] As described above, in the step of suspension culturing pluripotent stem cells in the second liquid medium, the timing of culture termination or medium replacement can be set to when the lactate concentration accumulated in the medium reaches, for example, 15 mM, 14 mM or less, 13 mM or less, 12 mM or less, or 10 mM or less. In other embodiments, the timing of culture termination or medium replacement can also be set to when the lactate concentration accumulated in the medium is in a low concentration range. Furthermore, when medium replacement is performed using a perfusion method, the perfusion rate can be adjusted so that the lactate concentration accumulated in the medium is maintained at, for example, 14 mM or less, 13 mM or less, 12 mM or less, 11 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, or 7 mM or less.

[0168] Alternatively, the timing of culture termination or the timing and amount of medium change during the process of suspension culturing pluripotent stem cells in the second liquid medium can be determined based on the size of the cell aggregates. The upper limit of the cell aggregate size can be, for example, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 250 μm, or 200 μm. The lower limit of the cell aggregate size can be, for example, 50 μm, 100 μm, or 150 μm. Cell aggregates within this range (especially smaller cell aggregates) are preferable as a cell growth environment because oxygen and nutrients are easily supplied to the cells inside. During suspension culture using the second liquid medium, the medium can be sampled, and if cell aggregates of this size are contained in the medium, this can be considered the end of the suspension culture process using the second liquid medium. Alternatively, the end of the suspension culture process using the second liquid medium can be reached when the cell masses defined within the above ranges account for 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100% of the observed cell masses by volume.

[0169] Alternatively, the suspension culture of pluripotent stem cells using the second liquid medium can be determined based on the time elapsed since the start of culture, as in the suspension culture using the first liquid medium. This determination is also based on the finding that the proliferation rate and cell aggregate formation rate of pluripotent stem cells are more or less the same depending on the type of pluripotent stem cells to be cultured, the medium components, the culture conditions, etc. The end of the suspension culture using the second liquid medium can be determined, for example, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, or 144 hours after the start of suspension culture of pluripotent stem cells in the second liquid medium, when cell aggregates within the above-mentioned size ranges are considered to have formed.

[0170] In suspension culture using the second liquid medium, adverse effects (such as reduced proliferation ability and difficulty in maintaining undifferentiated state) caused by physical damage to the cell mass of pluripotent stem cells can be reduced. In other words, when pluripotent stem cells are cultured in suspension in the presence of a PKCβ inhibitor and a WNT inhibitor, undifferentiated state or cell proliferation ability can be maintained even under conditions that impart greater physical damage than conventional cell culture. In other words, in suspension culture using the second liquid medium, culture can be performed at impeller tip speeds, stirring speeds, stirring power per unit volume (Pv), and the like that are equivalent to or greater than those in suspension culture using the first liquid medium.

[0171] Specifically, in suspension culture using the second liquid medium, agitation culture can be performed while maintaining the undifferentiated state of pluripotent stem cells, even at very high impeller tip speeds of 0.23 m / s or more.

[0172] As described above, a population of cell clusters produced through a suspension culture step using a first liquid medium and a suspension culture step using a second liquid medium is characterized by a high proportion of live cells (survival rate) among the cells constituting the population. That is, a population of cell clusters produced through suspension culture using a first liquid medium and suspension culture using a second liquid medium can exhibit a superior survival rate compared to a population of cell clusters produced only by suspension culture using the second liquid medium. For example, the survival rate of a population of cell clusters produced through suspension culture using a first liquid medium and suspension culture using a second liquid medium can be 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0173] After suspension culture using the second liquid medium is completed, the obtained cell clusters can be used as they are depending on the purpose, or the obtained cell clusters can be subjected to the above-mentioned "single cell" treatment to convert them into individual cells, and the obtained single cells or cell clusters can be again applied to the method for producing pluripotent stem cells according to the present invention. In other words, the obtained single pluripotent stem cells can be again subjected to suspension culture in the first liquid medium, and once cell clusters have formed, they can be again subjected to suspension culture using the second liquid medium.

[0174] The "recovery step" is a step of recovering cultured cells or cell aggregates from the culture medium after the maintenance culture step, the suspension culture step using the first liquid medium, or the suspension culture step using the second liquid medium, and is a selection step in the method of the present invention.

[0175] As used herein, the term "recovery (of cells)" refers to obtaining cells by separating the cells from the culture medium. The method for recovering cells may be any conventional method used in cell culture methods in the art, and is not particularly limited.

[0176] After the suspension culture process using the first liquid medium and the suspension culture process using the second liquid medium, the cells remain suspended in the culture medium. Therefore, cell recovery can be achieved by removing the liquid components of the supernatant either in a static state or by centrifugation. Cells can also be recovered using a filtration filter, hollow fiber separation membrane, or the like. When removing the liquid components in a static state, the container containing the culture medium can be left in a static state for about 5 minutes, and the supernatant can be removed, leaving the settled cells and cell clumps. Furthermore, centrifugation can be performed at a centrifugal acceleration and processing time that do not damage the cells due to centrifugal force. For example, the lower limit of the centrifugal acceleration is not particularly limited as long as it can settle the cells, but it can be, for example, 100 × g or more, 300 × g or more, 800 × g or more, or 1000 × g or more. On the other hand, the upper limit can be any speed at which the cells are not or are least likely to be damaged by centrifugal force, such as 1600 × g or less, 1500 × g or less, or 1400 × g or less. The lower limit of the treatment time is not particularly limited as long as it allows the cells to settle due to the centrifugal acceleration, but may be, for example, 30 seconds or more, 1 minute or more, 3 minutes or more, or 5 minutes or more. The upper limit may be a time that does not or is unlikely to damage the cells due to the centrifugal acceleration, for example, 10 minutes or less, 8 minutes or less, 6 minutes or less, or 30 seconds or less. When removing liquid components by filtration, for example, the culture medium may be passed through a nonwoven fabric or mesh filter to remove the filtrate, and the remaining cell aggregates may be recovered. When removing liquid components using a hollow fiber separation membrane, for example, the culture medium and cells may be separated and recovered using an apparatus equipped with a hollow fiber separation membrane, such as a cell concentration and washing system (Kaneka Corporation).

[0177] The collected cells and cell aggregates can be washed as needed. The washing method is not limited. For example, the washing method may be the same as that described in the "Post-process treatment" in the maintenance culture process. A buffer (e.g., PBS buffer), saline, or a medium (basal medium is preferred) may be used as the washing solution.

[0178] The method for producing pluripotent stem cells will be explained in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0179] Reference Example 1: Adherent culture of human iPS cell line 201B7 Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) was added at a concentration of 0.5 μg / cm ２ Human iPS cell line 201B7 (Center for iPS Cell Research and Application, Kyoto University) was seeded on a cell culture dish coated with HCl and incubated at 37°C in 5% CO ２ Adherent culture was performed under a humid atmosphere. StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) was used as the medium, and the medium was changed daily. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 10 μM only when the cells were seeded.

[0180] Reference Example 2: Suspension culture of human iPS cell line 201B7. Human iPS cell line 201B7, which had been cultured in adhesion culture using the same procedure as in Reference Example 1, was treated with Accutase for 5 minutes to detach it from the culture surface and dispersed into single cells by pipetting. The cells were suspended in StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 x 10 per mL using StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM. ５ The cell suspension was prepared to contain 1000 cells. 1 mL of the cell suspension was seeded per well in a 12-well suspension culture plate (Sumitomo Bakelite Co., Ltd.). The plate with the seeded cells was rotated on a rotary shaker at 98 rpm along the horizontal plane, drawing a circle with a diameter of 25 mm, and incubated at 37°C and 5% CO ２ Suspension culture was performed under the following conditions.

[0181] The day the cells were seeded was designated day 0 of culture, and subculture was performed on day 5 of culture, followed by suspension culture until day 10 of culture. Medium changes were performed daily. To change the medium, the entire volume of the medium containing the cell clumps was collected in a centrifuge tube and allowed to stand for approximately 5 minutes to allow the cell clumps to settle. The culture supernatant was then removed, and the cells were gently resuspended in StemFit® AK02N medium and returned to the original well.

[0182] When the medium was changed, Y-27632 was added to the medium to a final concentration of 5 μM on days 1 and 6 of culture, and to a final concentration of 2 μM on days 2 and 7 of culture. On day 5 of culture, the cell clumps and culture supernatant were collected from the wells into centrifuge tubes and left to stand for about 5 minutes to allow the cell clumps to settle, after which the culture supernatant was removed.

[0183] 1 mL of Accutase was added to the cell clumps and treated at 37°C for 10 minutes, after which the cells were dispersed into single cells by pipetting. The cells were suspended in StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 × 10 per mL using StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM. ５ The suspension was prepared so as to contain 100 cells, and the cells were seeded in the same manner as on day 0 of culture, and the suspension culture was continued.

[0184] Reference Example 3 Addition of WNT inhibitor in suspension culture of human iPS cell line 201B7 Suspension culture was carried out in the same manner as in Reference Example 2, except that IWR-1-endo was added as a WNT inhibitor to the seeding medium and medium exchange medium for suspension culture at a final concentration of 20 μM.

[0185] Reference Example 4 Addition of a PKCβ inhibitor to suspension culture of human iPS cell line 201B7 Suspension culture was performed using the same procedure as in Reference Example 2, except that GF109203X was added as a PKCβ inhibitor to the seeding medium and medium exchange medium for suspension culture at a final concentration of 3 μM.

[0186] Reference Example 5 Addition of WNT inhibitor and PKCβ inhibitor in suspension culture of human iPS cell line 201B7 Suspension culture was carried out using the same procedure as in Reference Example 2, except that IWR-1-endo as a WNT inhibitor was added to a final concentration of 20 μM, and GF109203X as a PKCβ inhibitor was added to a final concentration of 3 μM to the seeding medium and medium exchange medium for suspension culture.

[0187] <Evaluation Example 1: Quantitative Real-Time PCR Analysis> The cells of Reference Examples 1, 2, 3, 4, and 5 were cultured for 10 days and then treated with TRIzol ＴＭThe samples were lysed using TRIzol Reagent (Thermo Fisher Scientific). ＴＭ Total RNA was isolated and purified from the solution dissolved in the reagent. The concentration of the purified RNA was measured using BioSpec-nano (Shimadzu Corporation), and 500 ng of the purified RNA was collected. 2 μL of ReverTra Ace® qPCR RT Master mix (Toyobo Co., Ltd.) and RNase-Free dH ２ The solution was adjusted to 10 μL by adding 0, and cDNA synthesis was carried out using a SimpleAmp Thermal Cycler (Thermo Fisher Scientific). The reaction conditions for cDNA synthesis were 15 minutes at 37°C, followed by 5 minutes at 50°C and 5 minutes at 98°C, followed by cooling to 4°C. The synthesized cDNA solution was diluted 100-fold with 10 mM Tris-HCl pH 8.0 (Nacalai Tesque) and added to a 384-well PCR plate (Thermo Fisher Scientific) at 5 μL / well. KOD SYBR® qPCR Mix (Toyobo Co., Ltd.), a 50 μM forward primer, a 50 μM reverse primer, and DEPC-treated water (Nacalai Tesque) were mixed in a ratio of 100:1:1:48, and this mixture was added to the 384-well PCR plate at 15 μL / well. Primers used were GAPDH, OCT4, T, SOX17, and PAX6. The 384-well PCR plate was centrifuged to remove air bubbles from the wells, and quantitative real-time PCR analysis was performed using a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The reaction conditions are shown in Table 1.

[0188]

[0189] The base sequences of the primers used in the quantitative real-time PCR analysis are shown below. ACTB (Forward): 5'-CCTCATGAAGATCCTCACCGA-3' (SEQ ID NO: 1) ACTB (Reverse): 5'-TTGCCAATGGTGATGACCTGG-3' (SEQ ID NO: 2) OCT4 (Forward): 5'-AGTGGGTGGAGGAAGCTGACAAC-3' (SEQ ID NO: 3) OCT4 (Reverse): 5'-TCGTTGTGCATAGTCGCTGCTTGA-3' (SEQ ID NO: 4) SOX2 (Forward): 5'-CACCAATCCCATCCACACTCAC-3' (SEQ ID NO: 5) SOX2 (Reverse): 5'-GCAAAGCTCCTACCGTACCAC-3' (SEQ ID NO: 6) NANOG (Forward): 5'-AGCCTCCAGCAGATGCAAGAACTC-3' (SEQ ID NO: 7) NANOG (Reverse): 5'-TTGCTCCACATTGGAAGGTTCCCA-3' (SEQ ID NO: 8) TBXT (Forward): 5'-TCACAAAGAGATGATGGAGGAAC-3' (SEQ ID NO: 9) TBXT (Reverse): 5'-ACATGCAGGTGAGTTGTCAG-3' (SEQ ID NO: 10) SOX17 (Forward): 5'-ATCTGCACTTCGTGTGCAAG-3' (SEQ ID NO: 11) SOX17 (Reverse): 5'-GAGTCTGAGGATTTCCTTAGCTC-3' (SEQ ID NO: 12) PAX6 (Forward): 5'-AGGAATGGACTTGAAACAAGG-3' (SEQ ID NO: 13) PAX6 (Reverse): 5'-GCAAAGCTTGTTGATCATGG-3' (SEQ ID NO: 14)

[0190] The results of measuring gene expression are shown in Figures 1 to 3. The results of measuring gene expression of OCT4 and NANOG are shown in Figures 1(a) and (b), the results of measuring gene expression of SOX2 and TBXT are shown in Figures 2(a) and (b), and the results of measuring gene expression of SOX17 and PAX6 are shown in Figures 3(a) and (b). In Reference Example 1, in which adhesion culture was performed, the expression levels of TBXT (mesodermal marker gene), SOX17 (endodermal marker gene), and PAX6 (ectodermal marker gene) were low, and the undifferentiated state of the pluripotent stem cells was maintained.

[0191] On the other hand, in Reference Example 2, where suspension culture was performed, the expression levels of TBXT, SOX17, and PAX6 were increased compared to Reference Example 1. This indicates that spontaneous differentiation into mesoderm, endoderm, and ectoderm occurred in suspension culture, and the undifferentiated state of pluripotent stem cells was not maintained. Furthermore, in Reference Example 3, where suspension culture was performed with the addition of a WNT inhibitor, the expression levels of TBXT and SOX17 were suppressed to levels acceptable for pluripotent stem cells, as in Reference Example 1, but the PAX6 expression level was higher than in Reference Example 1, and no improvement was possible from Reference Example 2. Furthermore, in Reference Example 4, where suspension culture was performed with the addition of a PKCβ inhibitor, the expression level of PAX6 was suppressed to the same level as in Reference Example 1, but the expression levels of TBXT and SOX17 were higher than in Reference Example 1, and were as high as in Reference Example 2. In Reference Example 5, where both a WNT inhibitor and a PKCβ inhibitor were added, the expression levels of TBXT, SOX17, and PAX6 were all similar to those in Reference Example 1.

[0192] The above results indicate that WNT inhibitors can effectively suppress spontaneous differentiation into mesoderm and endoderm in suspension culture, but have a weak inhibitory effect on ectoderm differentiation. Furthermore, PKCβ inhibitors can suppress spontaneous differentiation into ectoderm in suspension culture, but promote mesoderm and endodermal differentiation. On the other hand, it was revealed that suspension culture with the addition of both a WNT inhibitor and a PKCβ inhibitor can suppress spontaneous differentiation into all of mesoderm, endoderm, and ectoderm.

[0193] In other words, it was revealed that adding only one of a WNT inhibitor and a PKCβ inhibitor is insufficient to effectively suppress the spontaneous differentiation of pluripotent stem cells into mesoderm, endoderm, and ectoderm and maintain the undifferentiated state of pluripotent stem cells; only by adding both a WNT inhibitor and a PKCβ inhibitor can spontaneous differentiation into all of mesoderm, endoderm, and ectoderm be suppressed and the undifferentiated state of pluripotent stem cells be maintained.

[0194] Reference Example 6 Suspension Culture with the Addition of a WNT Inhibitor and a PKCβ Inhibitor Suspension culture was carried out using the same procedure as in Reference Example 2, except that IWR-1-endo as a WNT inhibitor was added to a final concentration of 20 μM, and LY-333531 as a PKCβ inhibitor was added to a final concentration of 1 μM to the seeding medium and medium exchange medium for suspension culture, and that the 1383D6 cell line was used.

[0195] Reference Example 7: Suspension culture of human iPS cell line 1383D6 Suspension culture was carried out in the same manner as in Reference Example 2, except that the 1383D6 cell line was used.

[0196] <Evaluation Example 2: Confirmation of proliferation> The cells on day 4 of culture in Reference Examples 6 and 7 were detached into single cells in the same manner as described in Reference Example 2, and the number of cells was counted. The proliferation fold relative to the number of cells seeded was then calculated. The results are shown in Figure 4. As shown in Figure 4, when the cells were cultured in suspension with the addition of a WNT inhibitor and a PKCβ inhibitor (Reference Example 6), the proliferation rate was shown to be lower than when the WNT inhibitor and the PKCβ inhibitor were not added (Reference Example 7). It is possible that the WNT inhibitor and the PKCβ inhibitor caused cell death or inhibited proliferation.

[0197] Example 1: Seeding of suspension culture in the absence of a PKCβ inhibitor and a WNT inhibitor Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) was added at a concentration of 0.5 μg / cm ２ Human iPS cell line 1383D6 (Center for iPS Cell Research and Application, Kyoto University) was seeded on a cell culture dish coated with HCl and incubated at 37°C and 5% CO ２Adherent culture was performed under a humid atmosphere. StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) was used as the medium, and the medium was changed daily. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 10 μM only when the cells were seeded.

[0198] The human iPS cell line 1383D6, which had been cultured in an adherent culture for 4 days, was treated with Accutase for 5 minutes to detach it from the culture surface and dispersed into single cells by pipetting. The cells were suspended in StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 x 10 per mL using StemFit® AK02N medium (a seeding medium for suspension culture) containing Y-27632 at a final concentration of 10 μM. ５ The cell suspension was prepared to contain 1000 cells. Then, 4 mL of the cell suspension was seeded per well into a 6-well suspension culture plate (Sumitomo Bakelite Co., Ltd.). The plate with the seeded cells was rotated on a rotary shaker at 90 rpm along the horizontal plane, drawing a circle with a diameter of 25 mm, and incubated at 37°C and 5% CO ２ The suspension culture was carried out under the same conditions as above. On the first day of the culture, it was confirmed that cell aggregates were formed.

[0199] The day the cells were seeded was designated day 0 of culture, and subculture was performed on day 5 of culture, followed by suspension culture until day 10 of culture. Medium changes were performed daily. To change the medium, the entire volume of the medium containing the cell clumps was collected in a centrifuge tube and allowed to stand for approximately 5 minutes to allow the cell clumps to settle. The culture supernatant was then removed, and the cells were gently resuspended in StemFit® AK02N medium and returned to the original well.

[0200] When changing the medium, Y-27632 was added to the medium to a final concentration of 5 μM on days 1 and 6 of culture, and to a final concentration of 2 μM on days 2, 3, 4, 7, 8, and 9 of culture. In addition, IWR-1-endo was added to a final concentration of 20 μM and LY333531 was added to a final concentration of 1 μM to the medium used for medium changes from day 1 of culture onwards. On day 5 of culture, the cell clumps and culture supernatant were collected from the wells into centrifuge tubes and left to stand for about 5 minutes to allow the cell clumps to settle, after which the culture supernatant was removed.

[0201] 1 mL of Accutase was added to the cell clumps and treated at 37°C for 10 minutes, after which the cells were dispersed into single cells by pipetting. The cells were suspended in StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of viable cells. The cell suspension was diluted to 2 × 10 per mL using StemFit® AK02N medium containing Y-27632 at a final concentration of 10 μM. ５ The suspension was prepared so as to contain 100 cells, and the cells were seeded in the same manner as on day 0 of culture, and the suspension culture was continued.

[0202] Example 2: Seeding of suspension culture in the presence of a WNT inhibitor Suspension culture was carried out in the same manner as in Example 1, except that IWR-1-endo was added to the seeding medium for suspension culture at a final concentration of 20 μM.

[0203] Comparative Example 1: Seeding of suspension culture in the presence of a PKC inhibitor Suspension culture was carried out in the same manner as in Example 1, except that LY333531 was added to the seeding medium for suspension culture at a final concentration of 1 μM.

[0204] <Comparative Example 2: Seeding of suspension culture in the presence of a PKCβ inhibitor and a WNT inhibitor> Suspension culture was carried out using the same procedure as in Example 1, except that IWR-1-endo was added to the seeding medium for suspension culture at a final concentration of 20 μM and LY333531 at a final concentration of 1 μM.

[0205] <Example 3: Suspension culture in which a PKCβ inhibitor is added after cell aggregate formation> Suspension culture was carried out using the same procedure as in Example 2, except that the final concentration of LY333531 in the medium used for medium replacement on or after day 1 of culture, when cell aggregates were formed in the suspension culture, was gradually increased as shown in Figure 5.

[0206] Comparative Example 3: Suspension culture without addition of PKC inhibitor Suspension culture was carried out in the same manner as in Example 2, except that LY333531 was not added to the medium used for medium replacement after the first day of culture.

[0207] Evaluation Example 3: Confirmation of cell aggregate formation efficiency at the start of suspension culture Cells cultured in Examples 1 and 2 and Comparative Examples 1 and 2 on day 1 of culture were recovered, and the cell aggregates were dissociated into single cells and counted using the same method as described in Reference Example 2. The results are shown in Figure 6. As shown in Figure 6, it was suggested that the inclusion of a PKCβ inhibitor at the time of seeding in suspension culture reduces the cell viability (increases the number of dead cells) during the cell aggregate formation phase and / or suppresses cell division immediately after seeding. In other words, it was confirmed that, in order to improve productivity, it is preferable that a PKCβ inhibitor is not present at the time of seeding in suspension culture, before cell aggregate formation.

[0208] Evaluation Example 4: Evaluation of proliferation during cell proliferation phase Cells cultured in suspension in Examples 2 and 3 and Comparative Examples 2 and 3 were recovered on days 1, 2, 3, and 4 of culture, respectively, and the cell clusters were dissociated into single cells using the same method as described in Reference Example 2, and the cell count was counted. Based on the counted cell counts, the specific growth rate during the cell proliferation phase was calculated. The change in specific growth rate is shown in Figure 7. The value shown as the specific growth rate on day n of culture indicates the specific growth rate from day n-1 of culture to day n of culture. As shown in Figure 7, no difference in specific growth rate was observed under each culture condition, confirming that the presence of a PKCβ inhibitor after cell cluster formation did not induce cell death or growth inhibition, resulting in a decrease in productivity.

[0209] The change in cell density over time is shown in Figure 8. This suggests that the decrease in cell number during the cell cluster formation phase caused by the addition of a PKCβ inhibitor at the start of suspension culture is directly linked to a decrease in cell yield at the end of culture. In other words, it was inferred that not including a PKCβ inhibitor in the medium from the start of suspension culture of singled cells until cell cluster formation is extremely important in avoiding a decrease in cell yield at the end of culture.

[0210] Evaluation Example 5: Quantitative Real-Time PCR Analysis Quantitative real-time PCR analysis was performed on the cells of Examples 2 and 3 and Comparative Example 2 on days 5 and 10 of culture in the same manner as in Evaluation Example 1. The results are shown in Figures 9 to 11. The results of measuring the gene expression of OCT4 and NANOG are shown in Figures 9(a) and (b), the results of measuring the gene expression of SOX2 and TBXT are shown in Figures 10(a) and (b), and the results of measuring the gene expression of SOX17 and PAX6 are shown in Figures 11(a) and (b). Compared to Comparative Example 2, in which a PKCβ inhibitor was added from the start of suspension culture to suppress differentiation, Examples 2 and 3, in which a PKCβ inhibitor was not added at the start of suspension culture to improve productivity and addition of a PKCβ inhibitor was started after cell clusters were formed, also showed equivalent expression levels of undifferentiated markers OCT4, NANOG, and SOX2, and it was confirmed that expression levels of mesoderm differentiation marker TBXT, endoderm differentiation marker SOX17, and ectoderm differentiation marker PAX6 were equally suppressed to low levels. In other words, it was demonstrated that by not adding a PKCβ inhibitor at the start of culture and adding a PKCβ inhibitor after cell cluster formation, productivity can be improved and pluripotent stem cells can be cultured in suspension without deviation from the undifferentiated state.

[0211] Evaluation Example 6: Flow Cytometry Analysis Cells from Examples 2 and 3 and Comparative Example 2 on days 5 and 10 of culture were analyzed by flow cytometry according to the following procedure. Cell clumps were treated with Accutase and dispersed into single cells by pipetting. These cells were washed with phosphate-buffered saline (PBS). After fixation with 4% paraformaldehyde (PFA) at room temperature for 20 minutes, they were washed three times with PBS and permeabilized overnight with cold methanol at -20°C. After washing three times with PBS, they were blocked with 3% fetal bovine serum (FBS) / PBS at room temperature for 1 hour. The cell sample was then divided into two aliquots and resuspended in 50 μL each. Fluorescently labeled anti-OCT4, anti-SOX2, and anti-NANOG antibodies were added to one aliquot and mixed, while fluorescently labeled isotype control antibodies were added to the other aliquot and mixed. The aliquots were stained at 4°C for 1 hour in the dark. The antibodies used and their amounts are shown in Table 2.

[0212]

[0213] After washing once with 3% FBS (fetal bovine serum) / PBS, the cells were passed through a cell strainer and analyzed using a Guava easyCyte 8HT (Lumix). For samples treated with isotype control antibodies, all regions in the cell population extracted by the FSC / SSC dot plot where the percentage of cells with stronger fluorescence intensity was 1.0% or less were selected. For samples treated with anti-OCT4, anti-SOX2, and anti-NANOG antibodies, the percentage of cells contained within the region in the cell population extracted by the FSC / SSC dot plot was calculated, and this was used as the percentage of cells positive for OCT4, SOX2, and NANOG. The results calculated for OCT4, SOX2, and NANOG are shown in Figures 12(a), (b), and (c), respectively. Compared to Comparative Example 2, in which a PKCβ inhibitor was added to inhibit differentiation from the start of suspension culture, Examples 2 and 3, in which a PKCβ inhibitor was not added at the start of suspension culture but addition of a PKCβ inhibitor was initiated after cell cluster formation, also demonstrated high and comparable positive rates of the undifferentiation markers OCT4, SOX2, and NANOG, at over 90%. That is, even when singled pluripotent stem cells were cultured in suspension in a medium substantially free of a PKCβ inhibitor and then cultured in suspension in a medium containing a PKCβ inhibitor after cell cluster formation, it was found that the undifferentiated state of the pluripotent stem cells could be maintained in the same manner as when the singled pluripotent stem cells were cultured in suspension in a medium containing a PKCβ inhibitor prior to cell cluster formation. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A step of culturing pluripotent stem cells in a single cell state in suspension in a first liquid medium that does not contain a PKCβ inhibitor; After the pluripotent stem cells form cell aggregates, a step of culturing the pluripotent stem cells in suspension in a second liquid medium containing a PKCβ inhibitor and a WNT inhibitor; A method for producing pluripotent stem cells, comprising the above steps.

2. The step of culturing in suspension in the second liquid medium is performed by adding a PKCβ inhibitor to the first liquid medium after the pluripotent stem cells form cell aggregates in the step of culturing in suspension in the first liquid medium. The production method according to claim 1, characterized in that.

3. The production method according to claim 1, characterized in that after the pluripotent stem cells form cell aggregates, the medium is exchanged from the first liquid medium to the second liquid medium.

4. The method further includes a step of culturing pluripotent stem cells in adhesion and a step of detaching and single-cellularizing the pluripotent stem cells cultured in adhesion, and culturing the single-cellularized pluripotent stem cells in suspension in a first liquid medium. The production method according to any one of claims 1 to 3, characterized in that.

5. After the step of culturing in suspension in the second liquid medium, the step of single-cellularizing the recovered cell aggregates and culturing in suspension in the first liquid medium and the step of culturing in suspension in the second liquid medium are performed again. The production method according to any one of claims 1 to 3, characterized in that.

6. The first liquid medium is characterized in that the concentration of the PKCβ inhibitor is less than 25 nM. The production method according to any one of claims 1 to 3.

7. The second liquid medium is characterized in that the concentration of the PKCβ inhibitor is 25 nM or more and 15 μM or less. The production method according to any one of claims 1 to 3.

8. The first liquid medium does not contain a WNT inhibitor, or the first liquid medium and / or the second liquid medium is characterized in that the concentration of the WNT inhibitor is 90 nM or more and 40 μM or less. The production method according to any one of claims 1 to 3.

9. The second liquid medium is characterized in that the ratio of the content concentrations (molar concentrations) of the PKCβ inhibitor and the WNT inhibitor is in the range of 167:1 or more and 1:1600 or less. The production method according to any one of claims 1 to 3.

10. The manufacturing method according to any one of claims 1 to 3, wherein the first liquid medium and the second liquid medium contain at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium hydrogen carbonate.

11. The manufacturing method according to any one of claims 1 to 3, wherein the first liquid medium and the second liquid medium contain FGF2 and / or TGF-β1.

12. The manufacturing method according to any one of claims 1 to 3, wherein the first liquid medium and the second liquid medium contain a ROCK inhibitor.

13. The manufacturing method according to claim 12, wherein the ROCK inhibitor is Y-27632.

14. In the step of suspension culture in the first liquid medium and the step of suspension culture in the second liquid medium, suspension culture is performed by a stirring method, and the tip speed of the stirring blade in the step of suspension culture in the second liquid medium is equal to or higher than the tip speed of the stirring blade in the step of suspension culture in the first liquid medium. The manufacturing method according to any one of claims 1 to 3.

15. The manufacturing method according to any one of claims 1 to 3, further comprising a step of collecting cell aggregates of pluripotent stem cells after the step of suspension culture in the second liquid medium.

16. The manufacturing method according to any one of claims 1 to 3, wherein the step of suspension culture in the first liquid medium is within 48 hours.

17. The manufacturing method according to any one of claims 1 to 3, wherein the pluripotent stem cells have a ratio of cells positive for OCT4 of 90% or more, a ratio of cells positive for SOX2 of 90% or more, and a ratio of cells positive for NANOG of 90% or more.

18. The manufacturing method according to any one of claims 1 to 3, wherein the pluripotent stem cells are ES cells and / or induced pluripotent stem cells.

19. A pluripotent stem cell or a pluripotent stem cell population produced by the manufacturing method according to any one of claims 1 to 3.