Mass production method of pluripotent stem cell stock

Abstract

The purpose of the present invention is to produce a large amount of a high-quality pluripotent stem cell stock.　A mass production method of a pluripotent stem cell stock that comprises thawing starting cells followed by adhesion culture to thereby stabilize the cell conditions, and then growing the cells to a cell count that enables suspension culture. Subsequently, the cells are suspension cultured while precisely controlling the culture environment to thereby grow a large number of high-quality cells. Then, a stock is prepared at a low temperature from the cells having been grown by the suspension culture.

Description

Methods for mass production of pluripotent stem cell stocks

[0001] The present invention relates to a method for large-scale production of high-quality pluripotent stem cell stocks by suspension culture, and a method for improving the quality of pluripotent stem cells.

[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 therapies involving the transplantation of somatic cells induced to differentiate from pluripotent stem cells has the potential to fundamentally revolutionize treatments for intractable diseases and lifestyle-related diseases. For example, technologies have already been developed to induce differentiation from pluripotent stem cells in vitro into a wide variety of somatic cells, including neurons, cardiomyocytes, blood cells, and retinal cells. Furthermore, efforts are underway to mass-cultivate iPS cells that are HLA-homozygous, which avoid immune rejection in many people when transplanted from differentiated somatic cells, and universal iPS cells that have been gene-edited to avoid immune rejection in all people, and to create a common source of iPS cells for clinical use.

[0003] However, there are still several challenges to overcome in creating such a clinical iPS cell stock, and one of the biggest challenges is the efficient mass culture of iPS cells. For example, for the treatment of heart disease, approximately 1 × 10 iPS cells per patient are required. ８ ~1 x 10 ９ It is said that 1000 cells are needed, but with current general technology, there is a limit to the number of cells that can be handled at one time due to handling difficulties, so only enough cells for about 5-10 patients can be produced from one vial of iPS cell stock in one production run. Based on this premise, to produce therapeutic cells for 10,000 patients, 1,000-2,000 vials of iPS cell stock would be required (i.e., 1,000-2,000 production runs would be required). Meanwhile, pluripotent stem cell culture methods are broadly divided into adhesion culture, in which cells are cultured by adhering them to a flat substrate, and suspension culture, in which cells are cultured suspended in a liquid medium. General adhesion culture can produce, for example, 1 x 10 cells. ９ To culture 10 cells, the total adhesion area is 10 ４ cm ２The above substrates are required, which is equivalent to approximately 100-200 standard 10 cm dishes. Handling such a large number of substrates in a clinical cell manufacturing environment is unrealistic due to the significant manual labor required. Thus, it is extremely difficult to use adherent culture on a substrate surface, where the number of cells obtained depends on the culture area, as a method for mass-cultivating pluripotent stem cells such as iPS cells as a common source of cells used in regenerative medicine.

[0004] On the other hand, in suspension culture, cells are cultured while suspended in a liquid medium, so the number of cells obtained depends on the volume of the medium. Therefore, compared to adherent culture, scaling up suspension culture does not require a significantly larger culture area, and culture can be performed in a small area (cell preparation environment). This makes suspension culture practical and suitable for mass production of cells. For example, Non-Patent Document 1 discloses a method of using a spinner flask as a cell culture vessel for suspension culture to culture pluripotent stem cells in suspension while stirring the liquid medium. Non-Patent Document 2 discloses a method for improving cell proliferation using a suspension culture method with a medium perfusion method. However, all of these methods assume that high-quality cells are used as the starting material for culture.

[0005] Furthermore, since repeated passages in the culture of pluripotent stem cells can lead to a decline in quality, it is said that it is preferable to culture and expand the cells with as few passages as possible (Non-Patent Document 3).

[0006] In preparing a cell stock of iPS cells for clinical use, it is important to produce a large stock, but it is also extremely important that the stock is composed of iPS cells of high quality and consistent quality. Patent Document 1 discloses a method for preparing a high-quality pluripotent stem cell stock after confirming that the cells are in a suitable state by monitoring the morphology of cell colonies. However, as mentioned above, this method utilizes an adherent culture method, which has limitations in terms of scaling up the cell stock. Furthermore, the monitoring method described cannot be applied to suspension culture methods, making it virtually impossible to achieve large-scale culture.

[0007] Although various iPS cell lines exist, including research lines, clinical lines in particular are often not of sufficient quality, such as survival rate or adhesion rate, due to factors such as the fact that they are established in restrictive manufacturing environments and conditions in order to meet clinical standards, making it difficult to produce a sufficient amount of iPS cell stock suitable for clinical use. Therefore, it is even more difficult to mass-produce iPS cell stocks for use in generating therapeutic somatic cells using these iPS cell lines as raw materials.

[0008] Olmer R. et al. , Tissue Engineering: Part C, Volume 18(10):772-784 (2012) Kropp C. et al. , Stem Cells Translational Medicine, 5:1289-1301 (2016) Bai Q. et al. , Stem Cells And Development, 24(5):653-662 (2015)

[0009] Patent Publication No. 2020-120613

[0010] As described above, methods for mass-culturing pluripotent stem cells using suspension culture have been reported, but the inventors' investigations have revealed that the methods disclosed above are not suitable for efficiently culturing and amplifying pluripotent stem cells (raw material cells) for clinical use that are of poor quality in terms of survival rate, adhesion rate, etc. In other words, a technology that can produce high-quality pluripotent stem cell stocks from poor-quality pluripotent stem cells (raw material cells) for clinical use using a suspension culture method that is suitable for scale-up has not yet been established, and there is a need for the early development of such a technology and manufacturing process.

[0011] As a result of intensive research to solve the above-mentioned problems, the present inventors have surprisingly found that high-quality cell stocks can be produced extremely efficiently by thawing cryopreserved starting cells and then first carrying out an adhesion culture step for at least one passage, preferably at least two passages, followed by a suspension culture step. Furthermore, they have found that by carrying out each of the above adhesion culture and suspension culture steps under appropriate conditions, and by preparing the cell stock after production but before freezing under specific conditions, it is possible to scale up and mass-produce the cell stock while maintaining high quality, and have thus completed the present invention.

[0012] The present invention encompasses the following: (1) A method for producing a pluripotent stem cell stock, comprising: (a) a step of thawing frozen cells that will serve as the source material for the cell stock to be produced; (b) a step of performing adhesion culture on the thawed source cells; (c) a step of performing suspension culture on the adherent cultured cells; (d) a step of dispensing the suspension cultured cells into a container for stock storage; and (e) a step of freezing the cells dispensed into the container. (2) A method for producing a pluripotent stem cell stock, comprising: (a) a step of thawing frozen cells that will serve as the source material for the cell stock to be produced; (b) a step of performing adhesion culture on the thawed source cells; (c) a step of performing suspension culture on the adherent cultured cells; (d) a step of dispensing the suspension cultured cells into a container for stock storage; and (e) a step of freezing the cells dispensed into the container. ６ cells or less, and the number of cells at the end of the culture in the step (c) is 1 x 10 ８ (3) The method of (1) or (2), wherein the adhesion rate of the starting cells is 70% or less. More preferably, the method of (1) or (2), wherein the adhesion rate of the starting cells when seeded in step (b) is 70% or less. (4) In the adherent culture of step (b), the adhesion rate of the starting cells is 3 x 10 ３ cells / cm ２ (5) The method according to any one of (1) to (3), wherein the step (b) comprises subculturing. (6) The method according to (4), wherein the subculturing is subculturing into a vessel having a larger surface area. More preferably, the adherent culture in the step (b) comprises subculturing the thawed cells at a density of 3 x 10 or more. ３ cells / cm ２(7) The method for producing the cells according to any one of (1) to (3), wherein the cells are seeded and cultured at a density equal to or higher than 1000 kJ / cm, and then subcultured in a vessel with a larger surface area, followed by further adhesion culture. (7) The method for producing the cells according to any one of (1) to (6), wherein the suspension culture in step (c) is performed by perfusion of the medium. (8) The method for producing the cells according to (7), wherein the method for perfusion of the medium comprises increasing the perfusion rate of the medium from any time point in accordance with cell growth. (9) The method for producing the cells according to (7) or (8), wherein the method for perfusion of the medium comprises controlling the perfusion rate of the medium so as to maintain the pH of the culture solution between 6.5 and 9.0. More preferably, the method for producing the cells according to (7) or (8), wherein the method for perfusion of the medium maintains the lactic acid concentration in the culture solution at 12 mM or less. (10) The method for producing the cells according to any one of (1) to (9), wherein the suspension culture in step (c) comprises varying the concentration of carbon dioxide gas supplied within a range of 10 to 0% as the culture progresses. (11) The production method according to any one of (1) to (10), wherein the suspension culture in the step (c) is suspension agitation culture. (12) The production method according to (11), wherein the suspension agitation culture includes decreasing the agitation speed during the culture period. More preferably, the production method according to (11), wherein the agitation speed of the suspension agitation culture is decreased as the size of the cell aggregates increases. (13) The production method according to any one of (1) to (12), wherein the volume of the culture medium in the suspension culture in the step (c) is 100 mL or more. (14) The production method according to any one of (1) to (12), wherein the specific growth rate of the cells at the end of the step (c) is 0.70 day －１ More preferably, the specific growth rate of the cells in the 24 hours immediately before the transition from step (c) to step (d) is 0.7 day or more. －１(15) The method for producing pluripotent stem cells according to any one of (1) to (14), wherein the step (c) comprises disaggregating cell aggregates into single cells. (16) The method for producing pluripotent stem cells according to any one of (1) to (15), wherein the disaggregation comprises enzymatic treatment in the presence of a ROCK inhibitor. (17) The method for producing pluripotent stem cells according to any one of (1) to (16), wherein in the step (d), at least one of the container and the cell suspension is maintained at 10°C or below. More preferably, the step (d) is carried out in a state where the container or the cell suspension is maintained on a low-temperature substrate at 10°C or below, or in a low-temperature environment at 10°C or below. (18) The method for producing pluripotent stem cells according to any one of (1) to (17), wherein a stock of 100 or more pluripotent stem cells is produced. More preferably, the number of stock storage containers into which the cells are dispensed in the step (d) is 100 or above. (19) The manufacturing method according to any one of (1) to (18), wherein the dispensing in step (d) is performed using a multiple-barrel pipette. More preferably, the dispensing of cells in step (d) is performed simultaneously using a multiple-barrel pipette. (20) The manufacturing method according to any one of (1) to (19), wherein the adherent cells are subjected to enzymatic treatment in the presence of a ROCK inhibitor at the transition from step (b) to step (c). (21) The manufacturing method according to any one of (1) to (20), wherein the medium used for culturing in step (c) contains a ROCK inhibitor. More preferably, the liquid medium in step (c) contains a ROCK inhibitor. (22) The manufacturing method according to (21), wherein the ROCK inhibitor is Y-27632. (23) The method according to any one of (1) to (22), wherein the culture medium used for the culture in the steps (b) and (c) contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate. More preferably, the method according to any one of (1) to (22), wherein the liquid culture medium in the steps (b) and (c) contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.(24) The production method according to any one of (1) to (23), wherein the culture medium used in the culture in steps (b) and (c) contains FGF2 and / or TGF-β1. More preferably, the liquid culture medium in steps (b) and (c) contains FGF2 and / or TGF-β1. (25) The production method according to any one of (1) to (24), wherein, among the pluripotent stem cells constituting the stock, the proportion of cells positive for OCT4 is 90% or more and the proportion of cells positive for TRA-1-60 is 90% or more. (26) A method for improving the quality of pluripotent stem cells, comprising: (f) a step of thawing cryopreserved pluripotent stem cells and subsequently culturing them in adhesion; and (g) a step of suspension culturing the cells after adhesion culture. (27) The method according to (26), wherein the suspension culture in step (g) is performed by perfusion of the medium, and the amount of perfusion of the medium is increased in accordance with cell growth. More preferably, the method according to (26) is performed in the suspension culture of step (g) by perfusion of the medium, and includes increasing the perfusion rate of the medium from any time point in accordance with cell growth. (28) The production method according to (27), which includes controlling the perfusion rate of the medium so as to maintain the pH of the culture solution between 6.5 and 9.0 by perfusion of the medium. More preferably, the method according to (27), in which the lactic acid concentration in the culture solution is maintained at 12 mM or less by perfusion of the medium. (29) The method according to any of (26) to (28), in which the suspension culture of step (g) includes varying the concentration of carbon dioxide gas supplied within a range of 10 to 0% as the culture progresses. (30) The method according to any of (26) to (29), in which the quality is the viability of the cell population. (31) The method according to any of (26) to (30), in which the quality is the adhesion rate to the culture substrate. (32) The method according to (31), wherein the adhesion rate of the pluripotent stem cells subjected to adhesion culture in step (f) is 70% or less. (33) A pluripotent stem cell stock having a cell survival rate of 90% or more after thawing, and in which, when subjected to adhesion culture after thawing, the number of adherent cells at 24 hours of culture is 0.8 times or more the number of seeded cells.(34) A pluripotent stem cell stock having a cell viability of 90% or more after thawing, and having an aggregate formation rate of 0.8 times or more the number of seeded cells at 24 hours after thawing and subsequent suspension culture. (35) A pluripotent stem cell stock having, with respect to the cell cycle of the contained cells, a proportion of cells in the G2 / M phase that is 1.5 times or more the proportion of cells in the G0 / G1 phase. This specification incorporates the disclosures of Japanese Patent Application Nos. 2021-206065 and 2022-162712, from which the present application claims priority.

[0013] According to the present invention, it is possible to efficiently proliferate poor-quality pluripotent stem cells for clinical use, produce large quantities of high-quality and consistent pluripotent stem cell stocks, and improve the quality of poor-quality pluripotent stem cells for clinical use.

[0014] 1 is a characteristic diagram showing the transition of carbon dioxide concentration when suspension culture was carried out in Production Example 3.

[0034] FIG. 1 is a diagram showing the medium used when differentiating pluripotent stem cells into each of the three germ layers in suspension rotation culture in Evaluation Example 4.

[0035] FIG. 1 is a characteristic diagram showing the three germ layer differentiation potential of the stocks prepared in Examples 1, 2, and 3, measured in Evaluation Example 4. In the diagram, black bars indicate the results for undifferentiated cells before differentiation induction, and "below detection limit" indicates that the expression level of the marker in the undifferentiated cells was below the detection limit.

[0036] Hatched bars indicate the results for cells after differentiation induction.

[0037] FIG. 1 is a characteristic diagram showing the difference in cell viability between the stock prepared in Comparative Example 1 and the stocks prepared in Examples 1, 2, and 3, measured in Evaluation Example 6. In the diagram, error bars indicate standard error, and * indicates a p-value of less than 0.05.

[0038] FIG. 1 is a characteristic diagram showing the difference in adhesion rate between the stock prepared in Comparative Example 1 and the stocks prepared in Examples 1, 2, and 3, measured in Evaluation Example 7, when the cells were thawed and seeded into adherent culture. 1 is a characteristic diagram showing the difference in viability between the stock preparation methods of Examples 4 and 5, measured in Evaluation Example 10. In the figure, the waiting time until freezing is shown in minutes. FIG. 1 is a characteristic diagram showing the difference in aggregate formation rate in suspension culture between the stock prepared in Comparative Example 1 and the stocks prepared in Examples 1, 2, and 3, measured in Evaluation Example 11. FIG. 1 is a characteristic diagram showing the results of cell cycle analysis of the stock prepared in Comparative Example 1 and the stock prepared in Example 1, measured in Evaluation Example 12.

[0015] 1. Method for Producing Pluripotent Stem Cell Stocks 1-1. Overview In a preferred method for producing a pluripotent stem cell stock according to the present invention, raw material cells are thawed and seeded at high density for adhesion culture, followed by efficient mass expansion of the cells in suspension culture under controlled carbon dioxide and lactic acid concentrations, and then the resulting cells are prepared at low temperature to a state and form suitable for storage, thereby producing a high-quality pluripotent stem cell stock. The method for producing a pluripotent stem cell stock according to the present invention enables the mass production of high-quality pluripotent stem cell stocks with high post-thaw survival and adhesion rates.

[0016] 1-2. Definitions of Terms The following terms used in this specification are defined below.

[0017] <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 that 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 cells of all types of 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" refers to pluripotent stem cells prepared from early embryos. "EG cells" refer to 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" refer to pluripotent stem cells prepared from testicular cells (particularly spermatogonial stem cells) (Conrad S., 2008, Nature, 456:344-349). "iPS cells" refer to pluripotent stem cells obtained by reprogramming differentiated somatic cells to an undifferentiated state by introducing genes encoding a small number of reprogramming factors into the differentiated somatic cells.

[0018] The pluripotent stem cells referred to herein may be cells derived from a multicellular organism. They are preferably animal-derived or mammal-derived cells. Examples of mammals 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. For example, human-derived cells can be preferably used.

[0019] As used herein, pluripotent stem cells include naive pluripotent stem cells and primed pluripotent stem cells. Naive pluripotent stem cells are defined as cells in a state close to pluripotency found in the inner cell mass before implantation, while primed pluripotent stem cells are defined as cells in a state close to pluripotency found in the epiblast after implantation. Compared to naive pluripotent stem cells, primed pluripotent stem cells are characterized by a lower contribution to ontogeny, a single transcriptionally active X chromosome, and a high level of transcriptionally repressive histone modifications. Furthermore, the marker for primed pluripotent stem cells is the OTX2 gene, while the marker genes for naive pluripotent stem cells are REX1 and KLF family genes. Furthermore, the colonies formed by primed pluripotent stem cells are flat, while the colonies formed by naive pluripotent stem cells are dome-shaped. Primed pluripotent stem cells are particularly suitable for use as the pluripotent stem cells herein.

[0020] The pluripotent stem cells used herein are preferably cells that can be cryopreserved and can be further propagated after thawing while maintaining pluripotency. The culture conditions used for proliferation after thawing are not particularly limited. Furthermore, as long as there are culture conditions that allow pluripotent stem cells to be propagated while maintaining pluripotency, they can be used in the invention described herein.

[0021] 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.

[0022] When the iPS cells used herein are commercially available or research strains, they include, but are not limited to, the 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, TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, and hiPSC 38-2 strain, MSC-iPSC1 strain, BJ-iPSC1 strain, RPChiPS771-2, WTC-11 strain, 1231A3 strain, 1383D2 strain, 1383D6 strain, 1210B2 strain, 1201C1 strain, 1205B2 strain, etc. can be used.

[0023] 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.

[0024] Furthermore, the combination of genes for reprogramming factors introduced into cells during the production of iPS cells used herein is not limited. For example, a 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.), or a 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.) can be used. The method for introducing these genes into cells is not particularly limited, and may include, for example, gene introduction using a plasmid such as an episomal vector, introduction as a nucleic acid such as introduction of synthetic RNA, or introduction as a protein. iPS cells generated by methods using Sendai virus vectors, non-translated RNA such as microRNA, low-molecular-weight compounds, etc. may also be used. Furthermore, universal iPS cells in which the HLA gene has been edited or deleted to suppress immune rejection may also be used.

[0025] 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.

[0026] <Pluripotent Stem Cell Population> As used herein, the term "pluripotent stem cell population" refers to a cell population composed of one or more cells, including at least one pluripotent stem cell. A pluripotent stem cell population may consist solely of pluripotent stem cells, or may contain other cells. Its form is not particularly limited, and examples include tissue, tissue fragment, cell pellet, cell aggregate, cell sheet, cell suspension, cell suspension, and frozen forms thereof. The pluripotent stem cell population used herein can contain multiple smaller pluripotent stem cell populations. The small pluripotent stem cell populations contained in a pluripotent stem cell population do not all need to have the same form. Furthermore, the pluripotent stem cell population used herein may contain cells in a single-cell state. Preferably, the pluripotent stem cell population contains cell aggregates.

[0027] <Cell Aggregates> As used herein, a "cell aggregate" refers to a mass-like cell population formed by cell aggregation in suspension culture, and is also called a spheroid. Cell aggregates usually have a roughly spherical shape. The cells that make up the cell aggregate are not particularly limited as long as they include one or more types of pluripotent stem cells. For example, a cell aggregate composed of pluripotent stem cells such as human pluripotent stem cells or human embryonic stem cells contains cells that express a pluripotent stem cell marker and / or are positive for the pluripotent stem cell marker.

[0028] Pluripotent stem cell markers are genes that are specifically or overexpressed in pluripotent stem cells, and examples thereof include alkaline phosphatase, Nanog, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, SSEA-1, and combinations thereof.

[0029] 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 and the various measurement methods described below in connection with three germ layer markers. For example, when flow cytometry is used as the detection method and a fluorescently labeled antibody is used as the detection reagent, cells that exhibit stronger fluorescence compared to a negative control (isotype control) can be considered "positive" for the marker. The proportion of cells that exhibit positive results for a detection reagent (e.g., a fluorescently labeled antibody analyzed by flow cytometry) is sometimes referred to herein as the "positive rate." Furthermore, when a labeled antibody is used as the detection reagent, any antibody known in the art can be used. For example, fluorescently labeled antibodies include, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc.

[0030] The proportion of pluripotent stem cells constituting a cell aggregate can be determined, for example, by the positive rate of a pluripotent stem cell marker. The positive rate of a pluripotent stem cell marker in the cells constituting the cell aggregate is preferably 80% or more, more preferably 90% or more, for example 91% or more, for example 92% or more, for example 93% or more, for example 94% or more, for example 95% or more, for example 96% or more, for example 97% or more, for example 98% or more, for example 99% or more, or for example 100%. A cell aggregate in which the proportion of cells expressing a pluripotent stem cell marker and / or the proportion of cells positive for a pluripotent stem cell marker is within the above-mentioned range is a highly undifferentiated and more homogeneous cell population.

[0031] The proportion of cells expressing pluripotent stem cell markers among the cells constituting the cell aggregate can be preferably 80% or more, more preferably 90% or more, for example 91% or more, for example 92% or more, for example 93% or more, for example 94% or more, for example 95% or more, for example 96% or more, for example 97% or more, for example 98% or more, for example 99% or more, or for example 100%.

[0032] The proportion of pluripotent stem cells can be determined by detecting the expression of one or more, two or more, or three or more pluripotent stem cell markers. In this case, the types of pluripotent stem cell markers within the above-mentioned ranges are not particularly limited. For example, the pluripotent stem cell markers may be one or more, two or more, three or more, or all of the detected pluripotent stem cell markers.

[0033] <<Adhesion Culture>> "Adhesion culture" is a cell culture method in which cells are cultured by adhering them to an external matrix or the like present on the surface of a culture vessel or the like. In typical adhesion culture, cells are grown in a monolayer. The external matrix is ​​not particularly limited, but examples thereof include laminin, vitronectin, gelatin, collagen, E-cadherin chimeric antibody, or combinations thereof. Cells grown in adhesion culture form dense cell colonies as they grow. Note that the aforementioned pluripotent stem cells can usually be cultured not only in adhesion culture but also in suspension culture.

[0034] "Suspension culture" refers to a cell culture method in which cells are cultured in a suspended state in a liquid medium. As used herein, "suspension state" refers to a state in which cells are not fixed by adhesion to an external matrix on the surface of a culture vessel (e.g., the inner surface of the wall, bottom, or underside of the lid, or the surface of a structure within the culture vessel (e.g., agitator blades, etc.)). "Suspension culture" refers to a method of culturing cells in suspension, in which cells exist as aggregated cell masses in the culture medium. Methods for suspending cells include, but are not limited to, stirring, swirling, shaking, etc. Furthermore, for example, a culture method in which cells are attached to microcarriers and cultured in a suspended state in the culture medium is considered suspension culture in this specification because, although the cells adhere to the microcarriers, the entire cell mass including the microcarriers floats without being fixed to the culture vessel. The aforementioned cells can generally be cultured not only in suspension culture but also in adherent culture.

[0035] <Culture Medium and Medium Exchange Method> As used herein, "culture medium" refers to a liquid or solid substance prepared for culturing cells. In principle, it contains the minimum amount of components essential for cell growth and / or maintenance. Unless otherwise specified, the culture medium referred to in this specification refers to a liquid culture medium for animal cells used to culture animal-derived cells. In this specification, liquid culture medium is often simply referred to as "culture medium."

[0036] As used herein, the term "basal medium" refers to a medium that is the basis for various animal cell culture media. Although culture is possible using the basal medium alone, by adding various culture additives, it is also possible to prepare media according to the purpose, for example, media specific to various types of cells. 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)). Examples of suitable DMEM / F12 media include, but are not limited to, DMEM / Nutrient Mixture F-12 (Ham's F12). It is particularly preferred to use a DMEM / F12 medium in which the weight ratio of DMEM medium to Ham's F12 medium is mixed in the range of 60 / 40 to 40 / 60, for example, 58 / 42, 55 / 45, 52 / 48, 50 / 50, 48 / 52, 45 / 55, or 42 / 58. Media used for culturing human iPS cells and human ES cells can also be suitably used. Preferred media that can be used in the present invention include serum-free media, i.e., serum-free media.

[0037] As used herein, the term "culture additive" refers to a substance other than serum and gaseous components 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, or combinations thereof. Insulin, transferrin, and cytokines may be naturally occurring proteins isolated from tissues or serum of animals (e.g., 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, or combinations thereof. Examples of antibiotics that can be used include, but are not limited to, penicillin, streptomycin, amphotericin B, or combinations thereof. Growth factors such as FGF2 and / or TGF-β1 can be suitably used as culture additives for the medium used in the present invention.

[0038] Furthermore, 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 a non-adherent state of pluripotent stem cells to a substrate or other cells and / or under high shear stress can be significantly suppressed. However, in adherent culture, continuous addition of Y-27632 causes cells to become dysmorphic, so it is preferable to use a medium that does not contain Y-27632 after the cells have formed colonies.

[0039] Furthermore, in order to maintain or improve the undifferentiated state of pluripotent stem cells, the medium preferably contains a protein kinase C β (PKC β) inhibitor and / or a WNT inhibitor. PKC β inhibitors include, for example, LY333531, Go6983, and GF109203X. Furthermore, WNT inhibitors include IWR-1-endo, XAV939, WNT-C59, IWP-2, and IWP-3. Addition of these inhibitors can suppress spontaneous differentiation and deterioration of the quality of pluripotent stem cells, thereby stabilizing the cells during culture.

[0040] 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, or both. A medium that does not contain any of LIF, a GSK3 inhibitor, or a MEK / ERK inhibitor allows primed pluripotent stem cells to be cultured without naivetizing them and while maintaining their undifferentiated state.

[0041] 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.

[0042] Culture additives can be added to the medium as they are, or 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 these culture additives, for example, 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), Hybridoma-SFM (Life Technologies Japan), eRDF Dry Powdered Media (Life Technologies Japan), 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.), StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.), mTeSR1 (Veritas Inc.), and TeSR2 (Veritas Inc.).

[0043] As used herein, the term "medium exchange method" refers to a method of supplying cells with medium as a nutrient source for cell survival and proliferation, and a method of removing the medium in which nutrients have been consumed by cells and metabolic products have accumulated. Medium exchange methods are not particularly limited, but include, for example, a batch method and a perfusion method. A batch method refers to replacing a given amount (e.g., all or half) of the medium in the culture system (often referred to herein as "culture solution") with new medium at any given culture time interval. A perfusion method refers to continuous medium exchange by continuously removing and separately supplying the medium in the culture system, and the amount of medium removed and supplied per unit time is referred to as the medium perfusion rate. Medium perfusion may be performed continuously or intermittently in multiple batches. In suspension culture, medium exchange is preferably performed using a perfusion method.

[0044] <Gas Supply> As used herein, "gas supply" refers to supplying oxygen and carbon dioxide necessary for cell survival and / or proliferation to the culture solution by aerating a gas through the culture solution during cell culture. Gas components used for gas supply include oxygen, nitrogen, carbon dioxide, and other gas components present in the atmosphere. Regarding the proportion of each component in the supply gas, the lower limit of the oxygen proportion is preferably 1%, 2%, 3%, 4%, 5%, 10%, or 20%, and the upper limit is preferably 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%. The lower limit of the carbon dioxide proportion is preferably 5%, 4%, 3%, 2%, 1%, or 0%, and the upper limit is preferably 20%, 10%, 9%, 8%, 7%, 6%, or 5%. The proportions of oxygen and carbon dioxide can be independently selected at any ratio. The method for adjusting the proportions of oxygen and carbon dioxide is not particularly limited. For example, the oxygen and carbon dioxide concentrations in the gas can be adjusted by adding nitrogen as a component other than oxygen and carbon dioxide. The method for preparing the feed gas is not particularly limited. For example, purified oxygen, carbon dioxide, and nitrogen may be mixed, or air may be mixed with oxygen, carbon dioxide, and nitrogen. Examples of the ratio of oxygen, carbon dioxide, and nitrogen in the feed gas include, but are not limited to, 20:5:75, 20:4:76, 20:3:77, 20:2:78, 20:1:79, 20:0:80, 5:5:90, 5:0:95, 40:5:55, and 50:0:50. The ratio does not need to be constant during culture and may be changed as needed. Gas supply methods include actively pumping and aerating gas from a tube connected to a bioreactor or the like, or filling an incubator with a gas of desired composition and supplying it to the culture vessel by natural diffusion. Furthermore, the gas supplied to the cell culture medium is preferably sterile, and although not limited to this, it is preferable to supply it to the culture medium through a filter. In this specification, "carbon dioxide" may be referred to as "carbon dioxide gas," and "carbon dioxide concentration in the supplied gas" may be referred to as "carbon dioxide concentration." Furthermore, "carbon dioxide concentration in the liquid medium" may be referred to as "dissolved carbon dioxide concentration."

[0045] <Cell Stock> As used herein, the term "cell stock" refers to a state in which a population of pluripotent stem cells of the same strain and origin has been aliquoted and stored in any desired amount. The containers used for aliquoting and storing the cells are not particularly limited. For example, vials, bags, etc. can be used. In cell stocks, the cells are suspended in a preservation solution. The composition of the preservation solution is not particularly limited; for example, it may be a medium to which DMSO has been added to give a final concentration of 10%, or a commercially available preservation solution or other preservation solutions may be used. Examples of commercially available preservation solutions include STEM-CELLBANKER (registered trademark) GMP grade (Xenogen Pharma), CryoStor (registered trademark) CS10 (Hemakea), CP-5E (Kyokuto Pharmaceutical Industries), Ringer's solution, and lactated Ringer's solution. The form of the preservation solution in which the cells are suspended is not particularly limited. For example, it may be liquid, viscous liquid, or gel. When the cell stock is stored by cryopreservation, it may be frozen into a solid state. The cells may be in the form of single cells, clumps in which multiple cells are adhered to each other, or cell aggregates, but preferably a mixture of single cells and clumps, or a single cell state. The storage state of the cell stock is not particularly specified, but may be refrigerated or frozen. The method of frozen storage is not particularly limited, but may be, for example, stored in a -80°C freezer, in the vapor phase of liquid nitrogen, or in the liquid phase of liquid nitrogen.

[0046] In this specification, "cell stock" may be referred to as "pluripotent stem cell stock," "stock," or "frozen stock," and dividing cells into multiple containers may be referred to as "filling" or "dispensing," and suspending cells in a preservative solution and filling them into containers may be referred to as "preparing a cell stock."

[0047] 1-3. Method for Producing Cell Stock The method of this embodiment essentially includes a step of adherent culture of the starting cells, a subsequent step of suspension culture, and a subsequent step of preparing the cell stock. The method of this embodiment may also include a freezing step. Each step will be explained below.

[0048] 1-3-1. Adhesion culture process The "adhesion culture process" is a process for growing raw material cells (e.g., rare cells) to a number of cells that can be seeded into a suspension culture on a scale sufficient for efficient production of a cell stock, while recovering from damage caused by storage conditions. Adhesion culture can utilize animal cell culture methods known in the art. For example, adhesion culture may be performed in which cells are cultured while adhering to a culture substrate such as a container or carrier.

[0049] (Starting Cells) The cells used as the starting material in this step (starting cells) are cells that can be cultured in an adherent manner and can aggregate in a suspension culture, as described below. As described above in the section "Pluripotent Stem Cells" in "1-2. Definition of Terms," ​​animal cells, for example, human cells, are preferred. Furthermore, pluripotent stem cells such as iPS cells and ES cells can be suitably used as the cell type. Specifically, for example, the QHJI14 strain, which is an iPS cell line with the highest frequency of HLA homozygotes in Japanese people, can be used. The pluripotent stem cells used in this step may be a cell population (pluripotent stem cell population) consisting of multiple cells. When the pluripotent stem cells are a pluripotent stem cell population, the percentage (proportion) of cells in the cell 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%. The cells used in this step may be one type or multiple types. Furthermore, the cells may be one specific type of cell line or a mixture of multiple types of cell lines.

[0050] Furthermore, raw material cells that have been preserved in a frozen state or the like can be used. Furthermore, in the present invention, raw material cells can be used in which the adhesion rate of live cells to the culture substrate after seeding in the adherent culture, which is the first culture step, is inherently low due to the properties of the cell line, or which has decreased due to some factor; the adhesion rate at the start of culture may be, for example, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less. Furthermore, pluripotent stem cells used as raw material are often rare, and the number of cells that can be used as raw material is often small. However, in the present invention, even very small amounts of cells can be used as raw material. There is no particular upper limit on the number of raw material cells, but specifically, the number of raw material cells is 1.5 x 10 ６ Below cells, 1.2×10 ６ Below cells, 1.0×10 ６ cells or less, 0.8 x 10 ６ Below cells, 0.6×10 ６ Cells or less, 0.5 x 10 ６ Below cells, 0.4×10 ６ cells or less, 0.3×10 ６ cells or less, or 0.2 x 10 ６ There is no particular lower limit on the number of raw material cells, and even a very small number of raw material cells can be used as raw material cells in the method of the present invention. For example, 0.01 × 10 ６ cells or more, 0.05 x 10 ６ cells or more, 0.1 x 10 ６ cells or more, 0.125 x 10 ６ cells or more, 0.14 x 10 ６ cells or more, 0.15 x 10 ６ cells or more, 0.16 x 10 ６ cells or more, 0.175 x 10 ６ cells or more, or 0.2 x 10 ６ Furthermore, in the present invention, clinical strains that not only have a small number of cells but also have unstable quality can be used as starting cells.

[0051] The above-mentioned raw material cells are usually commercially available, distributed, or stored in a frozen state. Therefore, in the present invention, when frozen cells are used as raw material cells, the raw material frozen cells must be thawed before being subjected to the adhesion culture process. The thawing conditions in this case are not particularly limited, but it is preferable to thaw the cells by rapid heating. Thawing cells by rapid heating refers to raising the cell temperature above 0°C within a predetermined time. Specifically, for example, the cell temperature is raised above 0°C within 5 minutes, 3 minutes, 2 minutes, 1.5 minutes, or 1 minute. The method of rapid heating is not particularly limited, but examples include a method in which a container filled with frozen cells is placed in a water bath, ethanol bath, or dry bath maintained at approximately 37°C and heated. Alternatively, a cell thawing device such as ThawSTAR (BioLife Solutions) may be used.

[0052] (Culture vessel) The culture vessel used for adhesion culture is not particularly limited, but a vessel that has not been treated to suppress protein adsorption on the inner surface of the vessel is preferred, and one that can be coated with an external matrix is ​​also preferred. For example, a vessel coated with an external matrix, a vessel that has been treated for cell adhesion by a method other than external matrix coating, or a vessel made of a material that allows cells to adhere, such as plastic, may be used. The shape of the culture vessel is not particularly limited, but examples include culture vessels in the shape of a dish, flask, well, or bag. For example, a cell culture flask (TPP) can be used as the culture vessel.

[0053] The capacity of the culture vessel to be used can be selected appropriately 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 (i.e., the bottom area) is 0.32 cm ２ , 0.65 cm ２ , 1.9 cm ２ , 3.0 cm ２ , 3.5 cm ２ , 9.0 cm ２ , or 9.6 cm ２ , or 10.0 cm ２ , 15.0 cm ２ , 20.0 cm２ , 21.0 cm ２ , 22.5 cm ２ , 24.0 cm ２ or 25.0 cm ２ And the upper limit is 1000 cm ２ , 500 cm ２ , 400 cm ２ , 300 cm ２ , 200 cm ２ , 150cm ２ , 100 cm ２ , 75cm ２ , 50 cm ２ , or 25 cm ２ It is preferable that:

[0054] The capacity of the culture vessel to be used can be selected appropriately and is not particularly limited, but the lower limit of the volume that can accommodate the culture medium and be cultured is preferably 0.5 mL, 1 mL, 2 mL, 4 mL, 10 mL, 20 mL, 30 mL, 50 mL, or 100 mL, and the upper limit is preferably 1 L, 500 mL, 200 mL, or 150 mL.

[0055] (External Matrix) The external matrix used for adhesion culture may be any matrix to which iPS cells can adhere, such as laminin or vitronectin. Commercially available products include iMatrix-511 (Matrixsome) and Vitronectin-N (Thermo Fisher Scientific). The external matrix used when seeding the starting cells is preferably laminin, such as iMatrix-511 (Matrixsome). Laminin has strong adhesive properties to iPS cells, and when seeding a small number of unstable cells, it allows a larger number of cells to adhere and survive.

[0056] On the other hand, when adherent culture is performed for two or more passages (one or more passages), it is preferable to use vitronectin as the external matrix in the adherent culture immediately before transition to suspension culture. Vitronectin has weaker adhesive strength to iPS cells than laminin, allowing iPS cells to be detached from the culture substrate with less stimulation and transition to suspension culture with less damage. Furthermore, when vitronectin is used, weak adhesion to the culture substrate results in stronger adhesion between cells, resulting in a state relatively similar to cell aggregation in suspension culture, which is thought to enable a smooth transition to suspension culture.

[0057] (Culture Medium) The medium used for adherent culture may be any of the media described above in "1-2. Definition of Terms," ​​and is not limited as long as it is capable of growing and / or maintaining pluripotent stem cells. It is particularly preferable to use a medium that does not contain leukemia inhibitory factor. Furthermore, the medium used in the present invention is preferably a liquid medium containing L-ascorbic acid, insulin, transferrin, selenium, and / or sodium bicarbonate. Furthermore, a liquid medium containing at least one growth factor is preferred, and a liquid medium containing FGF2 and / or TGF-β1 as growth factors is more preferred. For example, a serum-free DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate, as well as FGF2 and TGF-β1, can be suitably used. Furthermore, it is preferable to use a medium containing a ROCK inhibitor from the time of cell seeding until the seeded cells adhere to the container or until the seeded cells form colonies. The concentration of the ROCK inhibitor is not particularly limited. The upper limit of the concentration can be, for example, 40 μM, 30 μM, or 20 μM, and the lower limit can be, for example, 2 μM, 2.5 μM, 3 μM, 5 μM, 7 μM, 8 μM, 9 μM, or 10 μM. The concentration of the ROCK inhibitor may be constant or varied during culture. The period during which the medium containing the ROCK inhibitor is used is not particularly limited. For example, the lower limit of the period during which the medium containing the ROCK inhibitor is used can be 12 hours, 16 hours, 20 hours, or 24 hours after cell seeding, and the upper limit can be 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, or 56 hours. Adherent culture in the medium containing the ROCK inhibitor can suppress cell death during the subsequent transition to suspension culture. Furthermore, the ROCK inhibitor may be added only during a portion of the adherent culture period. For example, a medium containing the ROCK inhibitor may be used for only one, two, or three or more days immediately before transferring to suspension culture.

[0058] When a PKCβ inhibitor is used, the lower limit of the concentration of the PKCβ inhibitor is not particularly limited, and can be selected within a range that can suppress deviation from the undifferentiated state.

[0059] For example, the final concentration of the PKCβ inhibitor in the liquid 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. It can also be, for example, 900 nM or more, 1 μM or more, or 1.1 μM or more. The upper limit of the concentration of the PKCβ inhibitor is not particularly limited and can be determined depending on conditions such as a range that does not cause cell death, a range that does not exhibit toxicity to pluripotent stem cells, and the solubility of the PKCβ inhibitor.

[0060] For example, the final concentration of the PKCβ inhibitor in the liquid medium can be 15 μM or less, 10 μM or less, 5 μM or less, 3 μM or less, or 1 μM or less.

[0061] On the other hand, the WNT inhibitor may be one type or a combination of two or more different types. The lower limit of the concentration of the TNKS inhibitor is not particularly limited and can be determined depending on the range that does not cause cell death.

[0062] For example, the final concentration of the WNT inhibitor in the liquid 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, 900 nM or more, or, for example, 10 μM or more, 15 μM or more, 18 μM or more, 20 μM or more, or 25 μM or more.

[0063] The upper limit of the concentration of the WNT inhibitor is not particularly limited, and can be determined depending on the range that does not cause cell death, the range that does not exhibit toxicity to pluripotent stem cells, the solubility of the TNKS inhibitor, etc.

[0064] For example, the final concentration of the WNT inhibitor in the liquid medium can be 40 μM or less, 35 μM or less, 30 μM or less, 25 μM or less, 20 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.

[0065] The method for adding the PKCβ inhibitor and the TNKS inhibitor is not particularly limited as long as the concentrations of the PKCβ inhibitor and the TNKS inhibitor in the medium at the start of this step are within the above-mentioned ranges. For example, the medium may be prepared by directly adding one or more PKCβ inhibitors and one or more TNKS inhibitors to the medium so that the total amount of the PKCβ inhibitor and the TNKS inhibitor falls within the above-mentioned concentration ranges.

[0066] In adherent culture, the amount of medium and culture solution may be adjusted appropriately depending on the culture vessel used. Preferably, the height of the liquid surface from the bottom of the vessel is 2 mm. For example, 300 cm ２ When using a culture flask, the volume of the medium or culture solution can be, for example, 60 mL. The volume of the medium or culture solution can be, for example, 1 mL or more, 2 mL or more, 3 mL or more, 4 mL or more, 5 mL or more, 10 mL or more, 20 mL or more, 30 mL or more, 40 mL or more, 60 mL or more, 80 mL or more, or 90 mL or more. Furthermore, when cells grow and colonies grow, the volume of the culture solution may be increased to increase the supply of nutrients and reduce the concentration of accumulated waste products. For example, the height of the liquid level from the bottom of the container may be 2.5 mm, 3.0 mm, 3.5 mm, or 4.0 mm. The volume of the medium or culture solution may be constant or variable during the culture.

[0067] (Seeding Density) In adhesive culture, the density of cells to be seeded onto a substrate such as a new culture vessel (seeding density) can be appropriately adjusted taking into consideration the state of the cells used for seeding, the culture time in this step, 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, 0.5 × 10 ３ cells / cm ２ , or 1 × 10 ３ cells / cm ２, and the upper limit is, for example, 5×10 ４ cells / cm ２ , or 10 x 10 ４ cells / cm ２ In particular, the seeding density when seeding the raw material cells and starting the culture is preferably as high as possible to increase the stability of the cells, and is preferably in the range of 2 × 10 ３ cells / cm ２ That's it, 3 x 10 ３ cells / cm ２ That's it, 4 x 10 ３ cells / cm ２ That's it, 5 x 10 ３ cells / cm ２ or more, or 6 x 10 ３ cells / cm ２ Furthermore, the seeding density when seeding the raw material cells and starting the culture is preferably 3 × 10 or more, because the raw material cells are rare and scarce, and if the density is too high, the culture area available for seeding becomes small, and extra passages are required to grow the cells. ４ cells / cm ２ Below, 2 x 10 ４ cells / cm ２ Below, 1 x 10 ４ cells / cm ２ or less, or 0.8 x 10 ４ cells / cm ２ The following is preferred:

[0068] (Culture Period) The culture period for adherent culture can be adjusted appropriately taking into account the number of starting cells, the quality and characteristics of the starting cells, such as their adhesion rate and proliferation rate, the seeding density, and the number of cells required to start suspension culture. Similarly, the number of passages for adherent culture can be adjusted appropriately. Here, "passage" refers to detaching and recovering cells from adherent culture and seeding them into a new adherent culture or suspension culture. Furthermore, "one passage period" refers to the time from seeding the cells to culturing and recovery. The lower limit of one passage period is not particularly limited, as long as it is long enough for the seeded cells to form colonies and grow, and may be 2, 2.5, or 3 days. The upper limit of one passage period may be any period during which cell colonies become dense and / or spread over a wide area of ​​the culture vessel and no deterioration in quality such as proliferation, viability, or undifferentiated state occurs, and may be, for example, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days.

[0069] The number of passages in this step is not particularly limited. For example, passages can be performed 0 times, 1 or more times, 2 or more times, or 3 or more times. The upper limit is not particularly limited, but is, for example, 5 or less times, 4 or less times, or less. The culture conditions (e.g., container, medium composition, medium amount, etc.) can be changed before and after passaging. For example, the cells may be passaged into a larger container than before passaging, the medium amount may be increased, or the seeding density may be lower than before passaging. In terms of increasing productivity, it is preferable to perform one or more passages in adherent culture and, at the time of passaging, to passage the cells into a larger container than before passaging or to divide the cells into multiple containers and passage them.

[0070] (Culture conditions) Culture temperature, time, CO ２ The culture conditions such as the concentration are not particularly limited. The culture may be carried out within the range of a common method in the art. For example, the culture temperature may be 20°C or 35°C at the lower limit and 45°C or 40°C at the upper limit, preferably 37°C. The CO in the gas phase during culture may be ２ The concentration can be, for example, a lower limit of 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, or 4.5% or more, and an upper limit of 10% or less, or 5.5% or less, more preferably 5%. ２The concentration does not need to be constant and may be changed and / or varied during the culture. ２ The concentration can be, for example, a lower limit of 3% or more, or 5% or more, and an upper limit of 21% or less, or 20% or less, with 21% being more preferred.

[0071] Furthermore, in adherent culture, medium changes can be performed at an appropriate frequency. The frequency of medium changes varies depending on the cell line and cell density being cultured, but can be, for example, at least once every four days, at least once every three days, at least once every two days, at least once a day, or at least twice a day. The frequency of medium changes does not need to be constant. For example, the frequency of medium changes can be reduced during the low cell density period in the first half of the first passage period, and increased during the high cell density period in the second half of the first passage period to maintain cell viability and quality. Furthermore, medium may be continuously supplied and discharged using a perfusion method rather than a batch method. The method of medium change is not particularly limited; for example, all or part of the medium can be replaced. Specifically, for example, since cells adhere to a substrate in adherent culture, the culture supernatant (culture solution) can be directly removed from the culture vessel without any special cell separation procedures, and fresh medium can be added and spread over the entire culture surface, followed by re-culture. The method of medium change, etc., is not limited to the above-mentioned frequency and method, and the optimal method can be adopted as appropriate. The number of times the medium is exchanged is not particularly limited, and can be, for example, 0 times, 1 or more times, 2 or more times, or 3 or more times, or can be, for example, 5 or less times, 4 or less times, or 3 or less times.

[0072] (Culture Method) In adhesion culture, the state of flow of the medium during culture is not important. That is, adhesion culture may be static culture or fluid culture.

[0073] "Static culture" refers to culturing in a culture vessel with the medium remaining stationary. This type of culture is usually used for adherent culture. "Flow culture" refers to culturing in a flowing medium.

[0074] In this adhesion culture step, the thawed raw material cells are cultured at a high density, for example, 3×10 ３ cells / cm ２After seeding at a density above 1000 kJ / s and culturing for one passage, the cells are preferably detached and recovered, then passaged in a container with a larger surface area than that used for the adherent culture, and then further cultured in adherent culture (adherent culture for the second passage). This process may also be repeated for a third or subsequent passage. While it is generally believed that the fewer the number of passages, the higher the quality of the cells. Surprisingly, the present invention has demonstrated that by culturing for multiple passages in this manner, it is possible to grow higher quality cells without excessive stress on the cells, up to a cell number sufficient for the subsequent suspension culture step.

[0075] In this adhesion culture step, the number of cells obtained by proliferation can be set as desired. The desired number of cells and cell state can be appropriately determined depending on the cell line to be cultured, the seeding density of the suspension culture, the scale of the suspension culture, the type of medium, and the culture conditions. For example, the degree of cell proliferation and cell state are not particularly limited in terms of the occupancy rate relative to the culture area of ​​the culture vessel, but the lower limit may be 10%, 20%, 30%, 40%, or 50%. Meanwhile, the upper limit may be 100%, 90%, 80%, 70%, or 60%. In particular, it is preferable to proliferate cells so that the occupancy rate relative to the culture area of ​​the culture vessel is a state in which the lower limit is 50% and the upper limit is 80%. Furthermore, the number of cells at the end of the adhesion culture step is not particularly limited, but is preferably 1.5 x 10 ６ cells or more, 3.0 x 10 ６ cells or more, 6.0 x 10 ６ More than 10 x 10 cells ６ cells or more, 16 x 10 ６ cells or more, 20 x 10 ６ cells or more, 21 x 10 ６ cells or more, 22 x 10 ６ cells or more, 25 x 10 ６ cells or more, 30 x 10 ６ cells or more, 32 x 10 ６ cells or more, 35 x 10 ６ More than cell, 36×10 ６ cells or more, 38 x 10 ６ cells or more, 40 x 10 ６cells or more, 41 x 10 ６ cells or more, 45 x 10 ６ cells or more, 48 x 10 ６ cells or more, or 64 x 10 ６ Cells or more are preferred.

[0076] It is preferable to increase the starting cells to a certain multiple or more by this adherent culture. Specifically, it is preferable that, for example, 142-fold, 143-fold, 145-fold, 150-fold, 160-fold, 170-fold, 175-fold, 180-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, or 274-fold or more cells can be recovered at the end of the adherent culture compared to the seeded starting cells.

[0077] In this adherent culture process, a portion of the pluripotent stem cells can be removed during culture to confirm the cell number and whether the cells maintain an undifferentiated state. For example, by measuring the expression level of pluripotent stem cell markers expressed in pluripotent stem cells removed during culture, such as during passaging, it can be confirmed whether the cells maintain an undifferentiated state. 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.

[0078] 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 pluripotent stem cells maintain an undifferentiated state.

[0079] Furthermore, in this step, the maintenance of an undifferentiated state 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 culture. That is, if 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 an undifferentiated state has been maintained. Alternatively, it can be determined that an undifferentiated state has been maintained if the expression levels of these markers are below a certain level compared to the expression levels of each marker in the cell population after differentiation induction. Specifically, for example, if the expression level in the cell population after differentiation induction is less than 1 / 10, 1 / 50, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, or 1 / 600, it can be determined that the cell population maintains an undifferentiated state.

[0080] Endodermal cell markers are genes specific to endodermal cells, and examples thereof include SOX17, FOXA2, CXCR4, AFP, GATA4, EOMES, etc. Endodermal cells form tissues of organs such as the digestive tract, lung, thyroid gland, pancreas, and liver, cells of secretory glands that open into the digestive tract, peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, urinary tract (bladder, most part of the urethra, and part of the ureter), etc.

[0081] 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 form 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, gonads (testes, uterus, and gonadal epithelium), and the like.

[0082] 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), lenses, the peripheral nervous system, etc. Furthermore, a portion of the ectoderm invaginates into a groove during development to form a neural tube, which also serves as the source of neurons and melanocytes of the central nervous system, such as the brain and spinal cord.

[0083] 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 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 or bAct) gene.

[0084] (Cell recovery from adhesion culture) After the adhesion culture step, cells are recovered for subsequent suspension culture. This recovery procedure is the same as the procedure for subculture from adhesion culture to adhesion culture in the adhesion culture step. In the step of recovering pluripotent stem cells from adhesion culture, the culture medium and pluripotent stem cells are separated by conventional methods, and the separated pluripotent stem cells are recovered. At this time, it is preferable to recover the pluripotent stem cells as single cells by detachment or dispersion treatment from the external matrix or adjacent pluripotent stem cells. Note that a single cell is sufficient as long as it is a single cell (single cell) that has been released from an adherent cell colony; all cells do not need to be in a single, released state; multiple cells may be present in an adherent state.

[0085] For the dissociation into single cells, an enzyme detachment agent and / or a chelating agent can be used. The enzyme detachment agent is not particularly limited, and any enzyme that can detach cells adhered to a culture vessel from the culture vessel and dissociate them into single cells can be used, even if it is not a commercially available detachment agent. For example, trypsin, collagenase, pronase, hyaluronidase, elastase, as well as commercially available Accutase (registered trademark), Accumax (registered trademark), TrypLE, etc. ＴＭ Express Enzyme (Life Technologies Japan), TrypLE ＴＭSelect Enzyme (Life Technologies Japan, Inc.), Dispase (registered trademark), etc. can be used. The chelating agent is not particularly limited, but examples include EDTA and EGTA. For example, when trypsin is used for single cell dissociation, the lower limit of the concentration in the solution is not particularly limited as long as it is a concentration that can disperse the pluripotent stem cell population, but it can be, for example, 0.15 vol%, 0.18 vol%, 0.20 vol%, or 0.24 vol%. 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 can be 0.30 vol%, 0.28 vol%, or 0.25 vol%. Furthermore, although the treatment time depends on the concentration of trypsin, the lower limit is not particularly limited as long as the pluripotent stem cell population is sufficiently dispersed by the action of trypsin, and it can be, for example, 2 minutes, 3 minutes, 5 minutes, 8 minutes, 10 minutes, 12 minutes, or 15 minutes. On the other hand, the upper limit of the treatment time is not particularly limited as long as it is a time that does not cause the cells themselves to be lysed by the action of trypsin, and may be, for example, 30 minutes, 28 minutes, 25 minutes, 22 minutes, 20 minutes, 18 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 8 minutes, 7 minutes, 6 minutes, or 5 minutes. When using a commercially available enzyme release agent, it should be used at a concentration that can disperse cells into a single-cell state, as described in the attached protocol. For example, when using EDTA for single-cell dissociation, the lower limit of the concentration in the solution is not particularly limited as long as it is a concentration that can disperse the pluripotent stem cell population, but for example, 0.01 mM, 0.1 mM, or 0.5 mM is preferred. 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 cause the cells themselves to be lysed, but 100 mM, 50 mM, 10 mM, or 5 mM is preferred. It is preferable to use at least one type of enzyme release agent and one type of chelating agent for single-cell dissociation. Furthermore, the enzyme detachment agent and chelating agent used to treat the cells during the cell dissociation preferably contain a ROCK inhibitor. Pluripotent stem cells in the single-cell state are known to be unstable and prone to cell death, but cell death can be suppressed by applying a ROCK inhibitor simultaneously with the cell dissociation.The upper limit of the ROCK inhibitor concentration can be, for example, 40 μM, 30 μM, or 20 μM, and the lower limit can be, for example, 2 μM, 2.5 μM, 3 μM, 5 μM, 8 μM, 9 μM, or 10 μM. After treatment with the enzyme detachment agent and / or chelating agent, applying mild stress to the adherent cell colonies or adherent cell colonies detached from the substrate can promote dissociation into single cells. The stress application method is not particularly limited, but possible examples include physical stimulation such as pipetting the cells together with the solution multiple times, spraying a solution such as a buffer solution onto the adherent cells, using a cell scraper, or tapping the adherent culture vessel. Furthermore, if necessary, the cells may be passed through a strainer or mesh.

[0086] The single-celled cells can be recovered by removing the supernatant containing the detachment agent by standing or centrifugation, etc. The recovered cells can be used as is, or, if necessary, suspended in a buffer (including PBS buffer), physiological saline, or medium (preferably the medium or basal medium used in the next step) and then subjected to the next step. The time required for the recovered cells to be subjected to the next step (passage or suspension culture step) is not particularly limited. However, from the viewpoint of maintaining cell quality, it is preferable to proceed to the next step promptly. The waiting time (the time from completion of recovery to the start of seeding in passaging or the start of seeding in suspension culture) is, for example, 24 hours or less, 18 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. If the waiting time is long due to the process schedule, it is recommended to store the recovered cells at a low temperature (e.g., 10°C or less, 5°C or less). It is preferable that the cells are not frozen during the period from the adherent culture step to the suspension culture step.

[0087] In the present invention, by carrying out the adhesion culture step under the above-mentioned preferred conditions, even a small amount of pluripotent stem cells can be grown to a number of cells suitable for suspension culture, and for example, in the subsequent suspension culture step, it is possible to control the culture environment using various sensors, etc. Furthermore, even when cells of unstable quality are used as the starting material, by undergoing the adhesion culture step of the present invention, cell aggregates can be efficiently formed during suspension culture and cell death can be suppressed.

[0088] 1-3-2. Suspension culture step The "suspension culture step" is a step of culturing a pluripotent stem cell population in order to proliferate the population while maintaining the undifferentiated state. Suspension culture can be performed using animal cell culture methods known in the art. For example, the method may be a suspension culture method in which cells are stirred in a liquid medium in a non-cell-adhesive container.

[0089] (Cells) The cells used in this step are the cells cultured and recovered in "1-3-1. Adherent culture step," and are pluripotent stem cells capable of cell aggregation in suspension culture. The pluripotent stem cells used in this step are typically a cell population consisting of multiple cells (pluripotent stem cell population), and the percentage (proportion) of cells in the cell population that express pluripotent stem cell markers (e.g., OCT4, SOX2, Nanog) and / or are positive for the pluripotent stem cell markers 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%.

[0090] (Culture vessel) The culture vessel used for suspension culture is not particularly limited, but is preferably a culture vessel whose inner surface has been treated to suppress protein adsorption. Also preferred are vessels with ports that can be fitted with sensors such as a pH sensor, DO sensor, and temperature sensor. Also preferred are vessels with ports that can supply gas and ports that can supply and aspirate culture medium. The shape and type of the culture vessel are not particularly limited, but examples include dish-shaped, flask-shaped, cylindrical, well-shaped, bag-shaped, and spinner flask-shaped vessels, as well as bioreactors equipped with stirring blades. For example, the BioBLU 1c Single-Use Vessel (Eppendorf) can be used as a culture vessel for a bioreactor.

[0091] The capacity of the culture vessel used can be selected appropriately and is not particularly limited, but the lower limit of the volume capable of accommodating and culturing the medium is preferably 1 mL, 2 mL, 4 mL, 10 mL, 20 mL, 30 mL, 50 mL, 100 mL, or 200 mL, and the upper limit is preferably 1000 L, 100 L, 50 L, 20 L, 10 L, 5 L, 3 L, 1 L, or 500 mL. However, a large culture scale is preferred for culturing a large amount of cells to prepare a cell stock, and it is particularly preferred that the volume of culture medium per passage period for recovering cells used to prepare the cell stock be 100 mL or more. Furthermore, when using an agitator-type reactor of any capacity, the working volume can be within the range specified by the manufacturer of the reactor.

[0092] In this specification, the volume of the medium actually contained in the culture vessel and used for cell culture is referred to as the culture volume or the amount of culture solution.

[0093] (Culture Medium) The medium used for suspension culture is a medium that preferably contains a ROCK inhibitor in addition to the basal medium described above in "1-2. Definition of Terms." The inclusion of a ROCK inhibitor increases the strength of cell aggregates against shear stress, enabling more stable suspension culture. Furthermore, the medium used for suspension culture preferably contains a PKCβ inhibitor and / or a WNT inhibitor. The inclusion of a PKCβ inhibitor and / or a WNT inhibitor further suppresses spontaneous differentiation and deterioration in quality of pluripotent stem cells, and in some cases, makes it possible to improve quality. Furthermore, the medium used in the present invention is preferably a liquid medium containing at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate. Furthermore, the liquid medium preferably contains at least one growth factor, and more preferably a liquid medium containing FGF2 and / or TGF-β1 as growth factors. Particularly preferred is serum-free DMEM / F12 medium containing L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, FGF2, and TGF-β1.

[0094] Furthermore, in the present invention, the medium exchange in this step is preferably performed by a perfusion method, in which the medium is perfused. By exchanging the medium by a perfusion method, the culture environment can be continuously controlled. When a perfusion method is used as the medium exchange method in this step, the culture additive composition of the medium used in this step does not need to be constant. Specifically, the culture additive composition of the medium at the start of the culture in this step may be different from the culture additive composition of the medium used for the medium exchange by the perfusion method during the culture in this step. Furthermore, multiple types of medium may be used for the medium exchange by the perfusion method during the culture in this step, or the medium used for the medium exchange by the perfusion method may be switched to one with a different culture additive composition at any point during the culture. Alternatively, the culture additive composition of the liquid medium used for perfusion can be changed during the culture. By changing the culture additive composition of the medium in this way, it is possible to continuously control the concentration of any culture additive or medium component in the culture system in accordance with various medium perfusion schemes (e.g., medium perfusion rate per unit time) and achieve an appropriate concentration transition. In the perfusion method, it is preferable to increase the perfusion rate of the medium at any time point in accordance with the growth of the cells. More preferable perfusion rates of the medium are described below.

[0095] The concentration of the ROCK inhibitor in the medium in this step can have a lower limit of, for example, 0 μM, 1 μM, 2 μM, 2.5 μM, 3 μM, 5 μM, 7 μM, 8 μM, 9 μM, or 10 μM as the final concentration in the liquid medium at the start of the culture in this step.

[0096] The upper limit of the concentration of the ROCK inhibitor in the liquid medium at the start of culture in this step is not particularly limited, and can be determined depending on conditions such as a range that does not cause cell death, a range that does not cause deviation from an undifferentiated state, and the solubility of the ROCK inhibitor.

[0097] For example, the upper limit of the concentration of the ROCK inhibitor in the liquid medium in the perfusion method of this step can be 50 μM, 40 μM, 30 μM, or 20 μM as the final concentration in the liquid medium at the start of culture.

[0098] Furthermore, although not particularly limited, when a ROCK inhibitor is used, the concentration of the ROCK inhibitor in the liquid medium used for medium exchange by the perfusion method in this step is preferably lower than the concentration of the ROCK inhibitor in the liquid medium at the start of culture in this step.

[0099] The upper limit of the concentration of the ROCK inhibitor as the final concentration in the liquid medium used for medium exchange by the perfusion method in this step is not particularly limited, and can be determined depending on conditions such as a range that does not cause cell death, a range that does not cause deviation from an undifferentiated state, and the solubility of the ROCK inhibitor.

[0100] For example, the upper limit of the final concentration of the ROCK inhibitor in the liquid medium used for medium exchange by perfusion in this step can be 50 μM, 40 μM, 30 μM, or 20 μM.

[0101] Furthermore, the lower limit of the final concentration of the ROCK inhibitor in the liquid medium used for medium exchange by the perfusion method in this step can be 0 μM, 1 μM, 2 μM, 2.5 μM, 3 μM, 5 μM, 7 μM, 8 μM, 9 μM, or 10 μM.

[0102] The method for adding the ROCK inhibitor is not particularly limited as long as the concentration of the ROCK inhibitor in the medium is within the above-mentioned range. For example, the ROCK inhibitor may be directly added to the medium so as to achieve a total concentration within the above-mentioned range, or a solution of the ROCK inhibitor diluted with another solvent may be added by mixing it with the medium.

[0103] Furthermore, in this step, the lower limit of the final concentration of the PKCβ inhibitor in the liquid medium at the start of the culture in this step can be 0 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 0.9 μM, 1 μM, or 1.1 μM.

[0104] The upper limit of the concentration of the PKCβ inhibitor in the liquid medium at the start of the culture in this step is not particularly limited, and can be determined depending on conditions such as the range that does not cause cell death, the range that does not cause deviation from the undifferentiated state, and the solubility of the PKCβ inhibitor.

[0105] For example, the upper limit of the final concentration in the liquid medium at the start of the culture in this step can be 10 μM, 5 μM, 2 μM, 1.5 μM, or 1 μM.

[0106] When cells recovered from adherent culture are seeded into suspension culture to initiate culture, it is preferable not to add a PKCβ inhibitor until the seeded cells form cell aggregates, as there is a risk that a PKCβ inhibitor may slightly induce cell death in pluripotent stem cells in a single-cell state immediately after adherent culture.

[0107] Furthermore, although not particularly limited, when a PKCβ inhibitor is used, it is preferable that the concentration of the PKCβ inhibitor in the liquid medium used for medium exchange by perfusion in this step be equal to or higher than the concentration of the PKCβ inhibitor in the liquid medium at the start of culture in this step.

[0108] The upper limit of the concentration of the PKCβ inhibitor as the final concentration in the liquid medium used for medium exchange by the perfusion method in this process is not particularly limited, and can be determined depending on conditions such as the range that does not cause cell death, the range that does not cause deviation from the undifferentiated state, and the solubility of the PKCβ inhibitor.

[0109] For example, the upper limit of the final concentration of the PKCβ inhibitor in the liquid medium used for medium exchange by perfusion in this step can be 10 μM, 5 μM, 2 μM, 1.5 μM, or 1 μM.

[0110] Furthermore, the lower limit of the final concentration of the PKCβ inhibitor in the liquid medium used for medium exchange by perfusion in this step can be 0 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 0.9 μM, 1 μM, or 1.1 μM.

[0111] The method for adding the PKCβ inhibitor is not particularly limited as long as the concentration of the PKCβ inhibitor in the medium is within the above-mentioned range. For example, the PKCβ inhibitor may be directly added to the medium so as to achieve a total concentration within the above-mentioned range, or a solution of the PKCβ inhibitor diluted with another solvent may be added by mixing it with the medium.

[0112] Furthermore, in this step, the lower limit of the final concentration of the WNT inhibitor in the liquid medium at the start of the culture in this step can be 0 μM, 1 μM, 2 μM, 3 μM, 5 μM, 7 μM, 10 μM, 15 μM, 18 μM, or 20 μM.

[0113] The upper limit of the concentration of the WNT inhibitor in the liquid medium at the start of culture in this step is not particularly limited, and can be determined depending on conditions such as the range that does not cause cell death or the range that does not cause deviation from the undifferentiated state, the solubility of the WNT inhibitor, etc.

[0114] For example, the upper limit of the final concentration in the liquid medium at the start of the culture in this step can be set to 50 μM, 40 μM, 30 μM, 25 μM, or 20 μM.

[0115] Furthermore, although not particularly limited, when a WNT inhibitor is used, the concentration of the WNT inhibitor in the liquid medium used for medium exchange by the perfusion method in this step is preferably equal to or higher than the concentration of the WNT inhibitor in the liquid medium at the start of culture in this step.

[0116] The upper limit of the final concentration of the WNT inhibitor in the liquid medium used for medium exchange by the perfusion method in this step is not particularly limited, and can be determined depending on conditions such as the range that does not cause cell death or the range that does not cause deviation from the undifferentiated state, the solubility of the WNT inhibitor, etc.

[0117] For example, the upper limit of the final concentration of the WNT inhibitor in the liquid medium used for medium exchange by perfusion in this step can be 50 μM, 40 μM, 30 μM, 25 μM, or 20 μM.

[0118] Furthermore, the lower limit of the final concentration of the WNT inhibitor in the liquid medium used for medium exchange by perfusion in this step can be 0 μM, 1 μM, 2 μM, 3 μM, 5 μM, 7 μM, 10 μM, 15 μM, 18 μM, or 20 μM.

[0119] The method for adding the WNT inhibitor is not particularly limited as long as the concentration of the WNT inhibitor in the medium is within the above-mentioned range. For example, the WNT inhibitor may be directly added to the medium so that the total amount of the WNT inhibitor falls within the above-mentioned concentration range, or a solution of the WNT inhibitor diluted with another solvent may be added by mixing it with the medium.

[0120] Furthermore, the medium used for perfusion in the present invention is preferably kept at a refrigerated temperature. For example, it is preferably kept refrigerated until immediately before being used for culture by perfusion. Refrigeration can suppress the decomposition and deterioration of protein components such as growth factors in the medium. The lower limit of the refrigeration temperature may be any temperature at which the medium does not freeze, and is preferably 0°C, 1°C, 2°C, 3°C, or 4°C, and the upper limit is preferably 12°C, 10°C, 8°C, 7°C, 6°C, 5°C, or 4°C, for example.

[0121] In the present invention, the amount of carbon dioxide gas supplied can be reduced as the culture progresses. In other words, the concentration of dissolved carbon dioxide gas in the culture solution can be reduced. On the other hand, when the culture medium is replaced by perfusion, if the concentration of dissolved carbon dioxide gas in the culture medium used for perfusion is higher than the concentration of dissolved carbon dioxide gas in the culture solution, the concentration of dissolved carbon dioxide gas in the culture solution will increase. Therefore, it is preferable that the concentration of dissolved carbon dioxide gas in the culture medium used for perfusion is lower than the concentration of dissolved carbon dioxide gas in the culture solution.

[0122] (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 state of the cells used for seeding, the cell yield in the preceding adhesion culture step, the culture time in this step, and the number of cells required after culture. Although not limited thereto, the lower limit of the seeding density is usually sufficient as long as the cells can form cell aggregates and the state of the cells does not become unstable, for example, 0.01 × 10 ５ cells / mL, 0.1×10 ５ cells / mL, 0.5×10 ５ cells / mL, 1×10 ５ cells / mL, 1.25×10 ５ cells / mL, 1.5×10 ５ cells / mL, or 2 x 10 ５ The upper limit of the cell density may be any cell density that does not cause excessive cell aggregation, injury, or rapid consumption of medium components, for example, 100 × 10 ５ cells / mL, 50×10 ５ cells / mL, 10×10 ５cells / mL, 8×10 ５ cells / mL, 6×10 ５ cells / mL, 4×10 ５ cells / mL, 2×10 ５ cells / mL, 1.5×10 ５ cells / mL or 1.4 x 10 ５ Since the growth efficiency at the initial stage of culture is affected by the seeding density, the lower limit of the seeding density is preferably 1 × 10 ５ cells / mL, upper limit 2 x 10 ５ Furthermore, when subculture is performed in the suspension culture step, the seeding density may be changed during each subculture period, for example, the seeding density may be gradually increased for each subculture.

[0123] (Culture Conditions) Culture conditions such as culture temperature, time, and oxygen concentration are not particularly limited. Culture may be performed within the range of conventional methods in the field. For example, the culture temperature may be 20°C or 35°C as the lower limit and 45°C or 40°C as the upper limit, preferably 37°C. The culture time can be adjusted appropriately depending on the desired number of cells to be obtained as a cell stock, the proliferation of the cell line, the state of the cells recovered by adherent culture, etc. For example, a lower limit of 24, 48, 60, 72, or 75 hours per passage period allows sufficient cell proliferation, and an upper limit of 168, 144, 120, 96, 84, or 78 hours allows culture without a decrease in quality, such as viability or undifferentiated state, due to excessive growth of cell aggregates. The oxygen concentration during culture can be, for example, 3% or 5% as the lower limit and 21% or 20% as the upper limit, with 21% being more preferred.

[0124] (Culture Method) In the suspension culture of this step, any method can be used as a gas supply method, and a standard method used in general culture methods can be used. For example, but not limited to, the supply gas can be supplied by passing it over the liquid surface of the culture solution, by bubbling it in the culture solution using a sparger, or by filling the surroundings of the culture solution with the desired gas and supplying it by natural diffusion. In this step, a method of passing it over the liquid surface of the culture solution can be preferably used.

[0125] When culturing cells in a culture device such as an incubator, the amount of gas supplied should be sufficient to fill the interior of the device. When culturing cells using a vessel such as a bioreactor, aeration is performed through a gas supply port attached to the vessel, and the amount of gas supplied should be appropriately determined taking into consideration the culture volume, the surface area of ​​the culture solution, the gas requirements of the cultured cells, the gas transfer rate in the culture solution, and the like. As an example, when culturing cells in a 320 mL culture solution volume using a BioBLU 1c Single-Use Vessel (Eppendorf), the appropriate gas supply rate is, for example, 0.1 L / min, 0.2 L / min, or 0.3 L / min. The gas supply rate may be increased when the volume of culture solution is increased from the aforementioned volume, or decreased when the volume of culture solution is decreased from the aforementioned volume.

[0126] In this suspension culture process, the concentration of carbon dioxide gas supplied to the liquid medium is variable. In typical cell culture methods, the concentrations of each gas component, including carbon dioxide gas concentration, remain constant throughout the culture. However, these concentrations must be appropriately adjusted to accommodate the changing cell conditions and medium environment during the culture. In the present invention, varying the carbon dioxide gas concentration supplied in this process within a range of 10% to 0% as the culture progresses is preferable because it maintains an appropriate culture environment and enables the production of a cell stock with high quality, such as high viability. In this process, the lower limit of the carbon dioxide gas concentration in the feed gas is preferably 0%, 0.5%, or 1%, and the upper limit is preferably 10%, 9%, 8%, 7%, 6%, or 5%. The amount of carbon dioxide gas supplied to the liquid medium is calculated by multiplying the carbon dioxide gas concentration in the feed gas by the amount of the feed gas. In other words, methods for changing the amount of carbon dioxide gas supplied to the culture solution include changing the carbon dioxide gas concentration in the feed gas, changing the amount of carbon dioxide-containing feed gas supplied, or a combination of these methods.

[0127] As the culture progresses, the cells grow, which is thought to increase the total amount of oxygen consumed by the cells themselves and the carbon dioxide emitted. For this reason, the culture environment can be controlled more consistently by changing the amount of carbon dioxide supplied from the outside. In addition, the impact on the culture environment of metabolites other than carbon dioxide emitted by the cells as the culture progresses can also be controlled by reducing the amount of carbon dioxide supplied. That is, in this process, the amount of carbon dioxide supplied can be reduced as the culture progresses. For example, when the amount of feed gas supplied is constant, it is preferable to reduce the carbon dioxide concentration as the culture progresses. However, the reduction does not have to be monotonous; a method of gradually reducing the carbon dioxide concentration by adjusting the balance by raising and lowering the carbon dioxide concentration is also acceptable.

[0128] The carbon dioxide concentration can also be reduced in stages. For example, the carbon dioxide concentration can be reduced to a first range within a first period from the start of the reduction, and then reduced to a second range within a second period. Specifically, for example, the carbon dioxide concentration can be reduced to a range of 0% to 2.5% within 1.5 days from the start of the reduction, and then reduced to a range of 0% to 1% within 2 days from the start of the reduction.

[0129] Furthermore, the amount of carbon dioxide gas supplied to the liquid medium can be changed based on one or more indicators. Examples of indicators for reducing the carbon dioxide gas concentration as the culture progresses include pH, cell density, lactate concentration, and the rate at which the cells produce lactate. This indicator can be selected independently of the culture variable used to control the medium perfusion rate, or in relation to that culture variable. The amount of carbon dioxide gas supplied can be reduced in proportion to or inversely proportional to one or more of these indicators. Therefore, the mathematical formulas described below for the culture variables can also be used for these indicators.

[0130] In this case, the sign of the correction coefficient M is usually reversed from that used for the culture variables. For example, when cell density, cell density growth rate, cell number, or size or volume of cell aggregates is used as an index, it is generally preferable to reduce the carbon dioxide concentration as these variables increase, so a negative value is used for M. On the other hand, when pH is used as an index instead of cell density, it is generally preferable to reduce the carbon dioxide concentration as the pH decreases, so a positive value is used for M.

[0131] For example, the pH of the culture solution can be used as one of the indicators. In this case, the carbon dioxide supply amount can be changed (particularly reduced) by changing the carbon dioxide concentration in the supply gas so as to suppress a decrease in pH. Specifically, for example, the carbon dioxide concentration in the supply gas can be set to be proportional to the pH value.

[0132] The timing for starting to decrease the carbon dioxide concentration is arbitrary. Furthermore, unlike the timing for starting perfusion of the culture medium described below, the timing for starting to decrease the carbon dioxide concentration may be before the cells form cell aggregates, or the carbon dioxide concentration may be started to decrease from the start of culture. The timing for starting to decrease the carbon dioxide concentration may be, for example, when the pH of the culture medium falls below an arbitrary reference pH, and the reference pH may be, for example, 7.25, 7.24, 7.23, 7.22, 7.21, 7.20, 7.19, 7.18, 7.17, 7.16, 7.15, 7.14, 7.13, 7.12, 7.11, 7.10, 7.09, 7.08, 7.07, 7.06, 7.05, 7.04, 7.03, 7.02, 7.01, 7.00, 6.99, 6.98, 6.97, 6.96, or 6.95. As described below, when culturing is initiated in a single-cell state, it is not desirable to perfuse the liquid medium before cell aggregates are formed. Therefore, by adjusting the carbon dioxide concentration, it is possible to maintain or improve the proliferation ability (e.g., specific growth rate), viability, and undifferentiated state of the cells even during periods when the culture environment cannot be controlled by perfusion.

[0133] In the suspension culture of this step, the medium is in a flowing state during culture. "Flowing culture" refers to culturing under conditions in which the medium is flowing. In the case of flowing culture, a method in which the medium is flowed is preferred so as to promote aggregation of cells seeded in a single-cell state and to suppress excessive aggregation of the cells. Examples of such culture methods include rotation culture, shaking culture, agitation culture, or a combination thereof. In the present invention, it is preferred that the suspension culture of this step is carried out by an agitation culture method, i.e., this step is suspension agitation culture. Furthermore, in terms of scale-up and process simplicity, it is preferred not to use microcarriers or the like.

[0134] "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, modified circle, or modified ellipse.

[0135] The rotation speed is not particularly limited, but the lower limit can be 1 rpm, 10 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 83 rpm, 85 rpm, or 90 rpm. Meanwhile, the upper limit can be 200 rpm, 150 rpm, 120 rpm, 115 rpm, 110 rpm, 105 rpm, 100 rpm, 95 rpm, or 90 rpm. The amplitude of the shaker used for rotational culture is not particularly limited, but the lower limit can be, for example, 1 mm, 10 mm, 20 mm, or 25 mm. Meanwhile, the upper limit can be, for example, 200 mm, 100 mm, 50 mm, 30 mm, or 25 mm. The rotation radius during rotational culture is also not particularly limited, but the amplitude is preferably set to be within the above range. The lower limit of the rotation radius can be, for example, 5 mm or 10 mm, and the upper limit can be, for example, 100 mm or 50 mm. In particular, when this method is used as a method for producing cell aggregates, which will be described later, it is preferable to set the rotation conditions within the above range, because this makes it easier to produce uniform cell aggregates of an appropriate size.

[0136] "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, 4, 6, 8, or 10 times per minute, while the upper limit is 15, 20, 25, or 50 times per minute. During rocking, it is preferable to rock the culture vessel at a slight angle relative to the vertical plane, i.e., at a rocking angle. The rocking angle is not particularly limited, but for example, the lower limit can be 0.1°, 2°, 4°, 6°, or 8°, while the upper limit can be 20°, 18°, 15°, 12°, or 10°. When this method is used as a method for producing cell aggregates, which will be described later, it is preferable to set the rocking conditions within the above ranges, since this makes it easier to produce cell aggregates of an appropriate size.

[0137] Furthermore, the culture can be performed while stirring by a combination of the above-mentioned rotation and rocking movements.

[0138] "Agitation culture" refers to a culture method in which the culture medium is agitated with an agitator or stirrer, and the cells and / or cell aggregates are dispersed in the culture medium. When the medium is fluidized by agitation with an agitator, the agitation speed is not particularly limited, but the lower limit is 1 rpm, 5 rpm, 10 rpm, 20 rpm, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 65 rpm, 66 rpm, 67 rpm, 68 rpm, 70 rpm, 75 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, or 130 rpm. It is preferable that the lower limit is 0 rpm and the upper limit is 200 rpm, 190 rpm, 180 rpm, 170 rpm, 160 rpm, 150 rpm, 140 rpm, 130 rpm, 120 rpm, 110 rpm, 100 rpm, 90 rpm, 80 rpm, 79 rpm, 78 rpm, 77 rpm, 76 rpm, 75 rpm, 70 rpm, 60 rpm, 50 rpm, 40 rpm, or 30 rpm.

[0139] Furthermore, in "stirring culture," a method of suspension culture using a stirring method such as a reactor with a stirring blade, 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 types of cells. Therefore, if the shear stress applied to cells during stirring culture is too high, the cells may suffer physical damage, resulting in a decrease in proliferation ability, the collapse of cell aggregates and cell death, or, in the case of pluripotent stem cells, the inability to maintain undifferentiated state. On the other hand, if the shear stress applied to cells during stirring culture is too low, the cells may become over-aggregated.

[0140] The shear stress imposed on cells in agitation culture depends on, but is not limited to, the impeller tip speed. The impeller tip speed is the peripheral speed of the impeller tip and can be calculated as impeller diameter [m] x pi x rotation speed [rps] = impeller tip speed [m / s]. Note that when multiple impeller diameters are determined based on the impeller tip shape, the largest distance can be used.

[0141] Furthermore, the tip speed of the blade is not particularly limited, but the lower limit is preferably 0.05 m / s, 0.08 m / s, 0.10 m / s, 0.13 m / s, 0.17 m / s, 0.20 m / s, 0.23 m / s, 0.25 m / s, or 0.30 m / s. By setting the tip speed within this range, it is possible to suppress excessive aggregation of cells while maintaining the undifferentiated state of the pluripotent stem cells.

[0142] Furthermore, the tip speed of the blade is not particularly limited, but the upper limit is preferably 1.37 m / s, 1.00 m / s, 0.84 m / s, 0.50 m / s, 0.42 m / s, 0.34 m / s, or 0.30 m / s. By setting the tip speed within this range, the flow state of the medium in the culture system can be stabilized while maintaining the undifferentiated state of the pluripotent stem cells. Furthermore, when single-cell cells are seeded and cultured with agitation, small-sized cell aggregates, which are preferable in terms of nutrient supply, can be formed.

[0143] Furthermore, the impeller tip speed does not need to be constant during agitation culture and may be changed during culture. For example, since cell aggregates grow larger as cells grow, it is preferable to reduce the agitation speed as the cell aggregate size increases. For example, the impeller tip speed may be changed between the first and second halves of the culture, or may be changed every 24 hours of culture. By changing the impeller tip speed during culture in this way, it may be possible to minimize damage to the cells due to shear stress applied to the cell aggregates.

[0144] Furthermore, in agitation culture, although not particularly limited, when changing the culture scale, the rotation speed of the agitator blades can be determined using the constant Pv formula. Pv is the agitation power required per unit volume, and by making 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.

[0145] It is preferable to start medium exchange using the perfusion method when the cells seeded in the culture solution have adhered to each other and formed cell aggregates. For example, when medium exchange is performed using the perfusion method, it is preferable to start perfusion using the perfusion method after the formation of cell aggregates. This allows the cell aggregates to be retained in the culture solution during medium exchange using a filter that removes cells from the culture solution and only the medium, as described below. Note that not all cells in the culture solution need to form cell aggregates; cells in a single-cell state may also be present. Cells in a single-cell state at the start of perfusion may form cell aggregates under perfusion with the medium. The lower limit of the ratio of the number of cells forming cell aggregates to the number of cells seeded at the start of medium perfusion is not particularly limited, but is preferably 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%, and the upper limit is preferably 300%, 200%, 150%, 140%, 130%, 120%, 110%, 100%, or 90%. Generally, some cells seeded in suspension culture die, temporarily reducing the cell count relative to the seeding amount. However, it is preferable for this rate of decrease to be low, for growth to begin quickly after seeding, and for the growth rate to be high. Furthermore, when medium exchange is performed using the perfusion method, if the ratio of the number of cells forming cell aggregates to the number of seeded cells at the start of perfusion is too high, there is a concern that nutrient depletion will progress before the start of perfusion, adversely affecting the cells. Therefore, it is preferable that this ratio is not too high. For this reason, it is preferable that the lower limit of the range of the ratio of the number of cells forming cell aggregates to the number of seeded cells is 100%.

[0146] Furthermore, when perfusion is used as the medium exchange method in this step, the timing of starting medium exchange by perfusion can be set arbitrarily, as long as the cells in the culture solution are in a state where they have adhered to each other and formed cell aggregates, taking into consideration the number of cells seeded, the efficiency of cell aggregate formation after seeding, the proliferation of the cells, etc. Although not particularly limited, the timing of starting perfusion is preferably, for example, 72 hours or more, 60 hours or more, 48 hours or more, 42 hours or more, 36 hours or more, 30 hours or more, 24 hours or more, 18 hours or more, or 12 hours or more after seeding the cells and starting the culture.

[0147] The medium perfusion rate per unit time at the start of perfusion (often referred to herein as the "reference perfusion rate") can be determined arbitrarily. The reference perfusion rate refers to the medium perfusion rate that replaces 100% of the medium volume in a given time multiplied by a start coefficient based on the culture conditions at the start of culture. The length of the given time is not particularly limited. For example, it can be 1 hour, 3 hours, 5 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 60 hours, or 72 hours. While not particularly limited, for example, if the given time is 24 hours, the reference perfusion rate can be determined based on the value obtained by multiplying the culture volume by the ratio of the length of the unit time to 24 hours. Specifically, for example, if the length of the unit time is 1 hour, the reference perfusion rate when the given time is 24 hours is based on the value obtained by dividing the culture volume by 24.

[0148] The initiation coefficient can be determined as a reference perfusion rate, for example, by multiplying the medium perfusion rate per unit time at the start of culture by an appropriate value depending on the culture conditions, such as the cell seeding density, the ratio of the number of cells forming cell aggregates to the number of cells seeded at the start of perfusion, etc. The lower limit of the initiation coefficient is preferably 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, and the upper limit is preferably 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0.

[0149] The initiation coefficient can be set appropriately depending on the purpose and conditions. For example, the initiation coefficient under specific conditions can be set to 1.0, and a value based on the magnitude of deviation from the specific conditions (e.g., the ratio of the actual cell density to the seeding density of specific cells, or the ratio of the number of cells forming cell aggregates to the number of cells seeded at the start of specific perfusion to the number of cells forming cell aggregates to the number of cells seeded at the start of actual perfusion) can be used as the initiation coefficient. Specific conditions include, for example, standard culture conditions when using the same type of cells, culture conditions recommended by the cell provider, etc. After the start of perfusion, the timing for starting control of the medium perfusion rate per unit time can be set arbitrarily. Control of the medium perfusion rate per unit time can be started simultaneously with the start of perfusion, or can be started 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 66 hours, or 72 hours after the medium has been perfused. It is preferable to start controlling the medium perfusion rate before the culture environment, such as the lactic acid concentration or pH, changes significantly and begins to have adverse effects on the cells.

[0150] The lower limit of the medium perfusion rate per unit time (often referred to herein as "variable perfusion rate") in medium exchange by the perfusion method is preferably 0.1%, 1%, 3%, 5%, 10%, 20%, 30%, 40%, or 50% of the culture volume, and the upper limit is preferably 100%, 90%, 80%, 70%, 60%, or 50%. Note that the medium perfusion rate per unit time here refers to the medium perfusion rate per hour.

[0151] It is preferable to control the variable perfusion rate within the above range as the culture progresses. In other words, in the suspension culture process, it is preferable to control the variable perfusion rate within a range of 1% to 100% of the culture volume using a reference perfusion rate and culture variables based on specific culture conditions. The variable perfusion rate can vary over any period as long as it is controlled by the method of the present invention. For example, perfusion may be constant over a unit time, or the medium perfusion rate may be reduced in the first half of the unit time and increased in the second half. Perfusion may be stopped for only a portion of the unit time, resulting in intermittent perfusion. The variable perfusion rate as the culture progresses is preferably controlled based on one or more culture variables. The culture variables are based on specific culture conditions. Specific examples of the culture variables include cell density, cell number, cell aggregate size or volume, the amount of lactic acid in the culture medium, the pH of the culture medium, and the amount of lactic acid produced by metabolism per cell per unit time. Alternatively, the cell density increase rate, which is the ratio of the cell density to the cell density at the start of control of the medium perfusion rate, can be set as a culture variable, or the cell aggregate volume increase rate, which is the ratio of the volume of the cell aggregate to the volume of the cell aggregate at the start of control of the medium perfusion rate, can be set as a culture variable. For example, if one of the culture variables is the cell density increase rate, the medium perfusion rate can be controlled by increasing the variable perfusion rate based on an increase in the cell density increase rate. Also, for example, if one of the culture variables is the cell aggregate volume increase rate, the medium perfusion rate can be controlled by increasing the variable perfusion rate based on an increase in the cell aggregate volume increase rate.

[0152] The medium perfusion rate per unit time can be changed continuously or intermittently in accordance with changes in one or more of these culture variables. For example, the medium perfusion rate per unit time can be controlled so as to be proportional to one or more of the culture variables. In other words, when based on multiple culture variables, the medium perfusion rate can be controlled so as to be proportional to each culture variable when the other culture variables are constants.

[0153] For example, when cell density is the culture variable, the medium perfusion rate per unit time can be increased as the cell density increases. For example, it can be increased in proportion to the increase in cell density. When pH is the culture variable, the medium perfusion rate per unit time can be controlled to suppress a decrease in pH. Suppressing a decrease in pH means maintaining or slightly increasing the pH value so that it does not decrease, or slowing the rate of decrease in pH. Suppressing a decrease in pH can be achieved by increasing the medium perfusion rate and / or reducing the amount of carbon dioxide gas supplied to the medium, as described below. Therefore, for example, a decrease in pH can be suppressed by increasing the medium perfusion rate per unit time based on the decrease in pH. Furthermore, a decrease in pH can be suppressed by adding substances such as caustic soda or baking soda that increase the pH to a level higher than that of the culture solution. However, this can adversely affect cells due to changes in the osmotic pressure of the medium, and it is difficult to reduce the concentration of lactic acid, a waste product that adversely affects cells. Adjusting the medium perfusion rate is preferable because it can simultaneously suppress a decrease in pH and a decrease in lactic acid concentration.

[0154] Below, we will explain the control of the medium perfusion rate using mathematical formulas, taking the case where cell density, etc. is used as a culture variable as an example. However, this is merely an example, and the medium perfusion rate can be controlled in a similar manner even when other information is used as a culture variable.

[0155] For example, when one of the culture variables is the cell density increase rate, the medium perfusion rate at which the medium perfusion rate per unit time starts to be controlled (i.e., the reference perfusion rate) is set to F ０ The cell density at this time is C ０ If the cell density at any time during each subsequent culture period is denoted by C, the medium perfusion rate per unit time at that time (i.e., variable perfusion rate) F can be expressed as the following equation 1, which is proportional to the rate of increase in cell density:

[0156]

[0157] The C value may be a value that is estimated in advance based on the characteristics of the cells or preliminary studies, or may reflect a value actually measured during culture. For example, the estimated C value may be used in the first half of culture, and the C value actually measured during culture may be applied in the second half of culture, and so on. Although not particularly limited, when culture is performed by maintaining an optimal culture medium environment using the method described in the present invention, the specific proliferation rate of pluripotent stem cells is 0.6 days. －１ That's it, 0.7 days －１ That's it, 0.8 days －１ or more, or 0.9 days －１ Since the above can be assumed, the value of C can be estimated in advance based on this.

[0158] The cell density can also be replaced with the number of cells, the size or volume of the cell aggregates. For example, when the cell density is the volume of the cell aggregates (C is the volume of the cell aggregates at any time during culture, C ... ０ is the cell aggregate volume at the start of control), when one of the culture variables is the cell aggregate volume increase rate, F (variable perfusion rate) can be set to the number 1, which is proportional to the cell aggregate volume increase rate.

[0159] Furthermore, the following equation 2 can be obtained by multiplying the equation 1 by M as a correction coefficient for correcting differences in cell characteristics due to the cell line and the culture history of the cell line.

[0160]

[0161] The difference in cell characteristics may include, but is not limited to, tolerance to lactic acid in the culture medium, and can be set to reflect the upper limit of the lactic acid concentration that does not significantly adversely affect the cells. The value of M can also be set to reflect the lower limit of the carbon dioxide concentration adjustment described above. Typically, the lower the lower limit of the carbon dioxide concentration adjustment, the smaller the value of the correction coefficient M can be. Although not particularly limited, for example, the correction coefficient M can be considered to represent the difference in tolerance of cell lines to harsh culture environments. As for the absolute value of the correction coefficient M, the lower limit is preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, and the upper limit is preferably 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0. The value of M may be positive or negative. For example, when cell density, cell density growth rate, cell number, or cell aggregate size or volume is used as a culture variable, it is generally preferable to increase the medium perfusion rate as these variables increase, so a positive value is used for M. On the other hand, when pH is used instead of cell density as a culture variable, it is generally preferable to increase the medium perfusion rate as the pH decreases, so a negative value is used for M.

[0162] For example, the value of the correction coefficient M can be set to the value of the resistance of the cell line to lactic acid in the culture medium when the resistance of a specific strain of human iPS cells (for example, Ff-I14s04 strain) to lactic acid in the culture medium is set to 1.0. The resistance of the cell line to lactic acid can be calculated, for example, by adding lactic acid and culturing the cell line. ５０ The lactic acid tolerance can be determined based on the value of the lactic acid tolerance, or based on the accumulated lactic acid concentration before and after the cell growth starts to decrease when the cell is cultured experimentally. The information on the lactic acid tolerance can be provided by the provider of the cell line or can be obtained by actual measurement.

[0163] Furthermore, the value of the correction coefficient M can be a value indicating the tolerance to low pH in the culture medium. In this case, for example, the value of M can be set to reflect the optimal pH of the cell line used or the lower limit of the carbon dioxide concentration described below. Typically, the higher the optimal pH and / or the lower limit of the carbon dioxide concentration adjustment, the smaller the value of M can be. For example, the value of the correction coefficient M can be set to the tolerance of the cell line used to pH in the culture medium when the tolerance of a particular strain of human iPS cells to pH in the culture medium is set to 1.0. pH tolerance information can be provided by the provider of the cell line or obtained by actual measurement.

[0164] It is also possible to multiply the above-mentioned formula 2 by a variable K, which varies depending on the amount of lactic acid produced by metabolism per cell per unit time, to obtain the following formula 3:

[0165]

[0166] K is the amount of lactic acid produced by metabolism per cell per unit time at a certain point in time, L ０ If the amount of lactic acid produced by metabolism per cell per unit time during each subsequent culture period is L, then the following equation 4 can be obtained.

[0167]

[0168] Here, the amount of lactate produced per cell by metabolism per unit time at a certain point in time refers to the value obtained by dividing the change in the amount of lactate in the culture solution over the unit time up to that point by the average number of cells within that unit time (the value obtained by dividing the change in the lactate concentration in the culture solution over the unit time up to that point by the average cell density within that unit time). Here, the amount or concentration of lactate in the culture solution can be, for example, a value measured directly in the culture solution, a value measured in a small sample taken from the culture solution, or a value measured in the medium removed from the culture system by perfusion.

[0169] The upper limit of the lactate concentration at the time when the change in the medium perfusion rate is initiated is preferably 10 mM, 9 mM, 8 mM, or 7 mM. When it is desired to control the lactate concentration, if the lactate concentration at the time when the change in the medium perfusion rate is initiated is high, the amount of medium used for perfusion is usually increased.

[0170] The amount of lactic acid produced by metabolism per cell per unit time may vary depending on the cell line, culture conditions, etc., so it is preferable to measure and confirm it in advance according to the cells to be used. －１０ mmol / cell / h, 3.0×10 －１０ mmol / cell / h, 5.0×10 －１０ mmol / cell / h, 7.0×10 －１０ mmol / cell / h, 1.0×10 －９ mmol / cell / h, 1.1×10 －９ mmol / cell / h, 1.2×10 －９ mmol / cell / h, or 1.3 x 10 －９ mmol / cell / h, upper limit 2.5 × 10 －９ mmol / cell / h, 2.0×10 －９ mmol / cell / h, 1.9×10 －９ mmol / cell / h, 1.8×10 －９ mmol / cell / h, 1.7×10 －９ mmol / cell / h, 1.6×10 －９ mmol / cell / h, 1.5×10 －９ mmol / cell / h, 1.4×10 －９ mmol / cell / h, or 1.3 x 10 －９ The lactic acid production rate during the suspension culture step is preferably maintained between the above-mentioned lower and upper limits. The change in the amount of lactic acid produced per cell by metabolism per unit time during culture can also be estimated from the expression level of the HK2 gene and its change.

[0171] During culture, it is preferable to basically control the medium perfusion rate according to the above formula. However, if the lactate concentration or pH value in the culture medium measured by any method deviates from the initial expected range, i.e., exceeds a value that does not adversely affect cells, or if it is within the range that does not adversely affect cells but continues to be outside the initial expected range, requiring an extra amount of perfusion, application of the above formula may be temporarily stopped, and the lactate concentration or pH value in the culture medium may be increased, decreased, or maintained by an arbitrary amount to return the lactate concentration or pH value in the culture medium to the expected range. The expected range may be determined appropriately based on factors such as cost and equipment. Preferably, the expected range is set within a lactate concentration or pH value range that does not adversely affect cells. While not particularly limited because it varies depending on the cell line, examples of upper limits of lactate concentrations that do not adversely affect cells include 20 mM, 18 mM, 16 mM, 14 mM, 13 mM, 12 mM, 11 mM, 10 mM, 9 mM, 8 mM, and 7 mM. Furthermore, examples of the lower limit of pH that does not adversely affect cells include 6.5, 6.6, 6.7, 6.8, 6.9, 6.95, 7.0, 7.05, 7.10, and 7.14. Examples of the upper limit of pH that does not adversely affect cells include 9.0, 8.5, 8.0, 7.6, 7.5, 7.4, 7.3, 7.2, and 7.16. The pH during the suspension culture step or at the start of control is preferably maintained at or above the above-mentioned lower limit. On the other hand, the lactic acid concentration in the culture medium during the suspension culture step or at the start of control is preferably maintained at or below the above-mentioned upper limit.

[0172] The cell density was 8.0 × 10 ５ After the cell density reaches 8.0 × 10 cells / mL, the culture environment is prone to change. ５ The medium perfusion rate can be controlled so that the total amount of medium used for medium exchange per 6 hours of any given culture after the cell density of pluripotent stem cells reaches 8.0 × 10 cells / mL is greater than the total amount of medium used for medium exchange per 6 hours of culture immediately before the given 6 hours of culture. In other words, the medium perfusion rate can be controlled so that the cell density of pluripotent stem cells reaches 8.0 × 10 cells / mL. ５ This may include increasing the medium perfusion rate for any 6 hours of culture after reaching 100 cells / mL, compared to the medium perfusion rate for the previous 6 hours of culture.

[0173] By performing perfusion culture using the method described above, it becomes possible to grow high-quality cells with high efficiency, and to produce a high-quality pluripotent stem cell stock.

[0174] In addition, medium replacement using the perfusion method involves continuously removing the culture medium from the container, separating the cells using a filter, etc., while continuing the culture, and continuously adding new medium. The size of the filter openings used need only be smaller than the cell aggregates. Furthermore, the size may be such that dead cells in the culture medium can pass through. While not particularly limited, the lower limit is preferably 0.1 μm, 1 μm, 5 μm, 10 μm, or 20 μm, and the upper limit is preferably 50 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, or 15 μm.

[0175] In this suspension culture process, the number of cells obtained by proliferation can be set as desired. The desired cell number and cell state can be determined appropriately depending on the type of cells to be cultured, the purpose of cell aggregation, the type of medium and culture conditions, and the desired number of cells required for stock preparation. For example, the degree of cell proliferation during one passage period is not particularly limited relative to the cell seeding amount at the start of culture, but the lower limit may be 2x, 3x, 5x, 6x, 7x, 8x, 8.5x, 8.8x, 8.9x, 9x, 9.1x, 9.15x, 10x, 11x, or 11.1x. On the other hand, the upper limit is not particularly set, but can be, for example, 100x, 50x, 40x, 30x, 20x, or 10x. Proliferation of 10x or more is particularly preferred. Furthermore, by repeating multiple passages and culturing in suspension culture, the cells may be expanded, for example, to 500-fold or more, 1000-fold or more, 1500-fold or more, 2000-fold or more, 2500-fold or more, 15000-fold or more, 150000-fold or more, or 1500000-fold or more compared to the number of starting cells. The degree of cell proliferation can be measured, for example, on the first, second, third, fourth, fifth, or sixth day of culture, or thereafter. Measurements may also be performed multiple times on different days.

[0176] In this suspension culture process, some of the pluripotent stem cells can be removed during culture to confirm the cell number and cell aggregate size. Cell aggregates of pluripotent stem cells removed during culture can be loosened into single cells, for example, by enzyme treatment, and the number of viable cells can be measured using methods such as the trypan blue method. Alternatively, the cell number can be estimated from the number and size of cell aggregates of pluripotent stem cells removed during culture. Furthermore, the size or volume of cell aggregates can be measured by, but is not limited to, laser size measurement, image acquisition, and calculation of size from the image, among other methods. Furthermore, the number of cells in suspension culture can also be calculated from the dissolved oxygen concentration in the culture medium.

[0177] The size of the cell aggregates produced in this suspension culture process is not limited, but when observed under a microscope, the average diameter of the maximum width size of the observed image of the cell aggregates in the same culture system can be a lower limit of 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm, while the upper limit can be 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, or 150 μm. Cell aggregates within this range are preferred as a cell growth environment, as they facilitate the supply of oxygen and nutrients to the cells inside. The size of the cell aggregates is particularly preferably 40 μm in lower limit and 250 μm in upper limit. Furthermore, the size of the cell aggregates formed by seeding cells in suspension culture, for example, the size of the cell aggregates after 24 hours, is preferably small to enable maximum growth with high quality and high efficiency within one subsequent culture passage period, and is particularly preferably 100 μm or less. It is not necessary that the size of all cell aggregates in the culture medium is within the above range, but it is sufficient that, for example, the number-average size is within the above range.

[0178] Of the population of cell aggregates produced in this suspension culture process, it is preferable that the lower limit of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% by weight be cell aggregates within the above size range.

[0179] Furthermore, in this suspension culture process, the concentrations of nutrients and metabolic products in the medium can be measured using the medium removed from the culture system by the perfusion method. For example, but not limited to, it is possible to measure the glucose concentration, lactic acid concentration, etc. in the removed medium using a medium component measuring device that uses an enzymatic electrode reaction. This information can be reflected in the control of the medium perfusion rate.

[0180] The glucose concentration in the medium removed from the culture system by the perfusion method is not particularly limited, but preferably has a lower limit of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM and an upper limit of 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, or 11 mM. For example, the lower limit can be 4 mM and the upper limit can be 16 mM. Furthermore, the lactate concentration in the medium removed from the culture system by the perfusion method preferably has a lower limit of 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM, and an upper limit of 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, 11 mM, 10 mM, 9 mM, 8 mM, 7 mM, or 6 mM. For example, the lower limit can be 0 mM and the upper limit can be 12 mM.

[0181] Furthermore, in this suspension culture process using the perfusion method, a portion of the pluripotent stem cells can be removed during culture to confirm the cell number and whether the cells are maintaining an undifferentiated state. For example, by measuring the expression of pluripotent stem cell markers expressed in pluripotent stem cells removed during culture, it is possible to confirm whether the cells are maintaining an undifferentiated state. 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.

[0182] When the positivity rate of the pluripotent stem cell marker among the pluripotent stem cells extracted during culture is, for example, 80% or more, 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% or less, it can be determined that the undifferentiated state is maintained. The positivity rate and determination of undifferentiated state when multiple pluripotent stem cell markers are used are as described above.

[0183] Furthermore, in this step, the maintenance of an undifferentiated state can be confirmed by measuring the expression of three germ layer markers (endodermal cell markers, mesodermal cell markers, and ectodermal cell markers) in pluripotent stem cells removed during the culture. That is, if the positive rates of these endodermal cell markers, mesodermal cell markers, and ectodermal cell markers are all, for example, 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or below the detection limit, it can be determined that an undifferentiated state has been maintained. Alternatively, if the expression levels of these markers are below a certain level compared to the expression levels of each marker in the cell population after differentiation induction, it can be determined that an undifferentiated state has been maintained. Specifically, for example, if the expression level in the cell population after differentiation induction is less than 1 / 10, 1 / 50, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, or 1 / 600, it can be determined that the cell population maintains an undifferentiated state.

[0184] Endodermal cell markers are genes specific to endodermal cells, and examples thereof include SOX17, FOXA2, CXCR4, AFP, GATA4, EOMES, etc. Endodermal cells form 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, urinary tract (bladder, most part of the urethra, and part of the ureter), etc.

[0185] Mesodermal cell markers are genes specific to mesodermal cells, and examples thereof include T(BRACHYURY), MESP1, MESP2, FOXF1, HAND1, EVX1, IRX3, CDX2, TBX6, MIXL1, ISL1, SNAI2, FOXC1, and PDGFRα. Mesodermal cells form 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, gonads (testes, uterus, and gonadal epithelium), and the like.

[0186] 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), lenses, the peripheral nervous system, etc. Furthermore, a portion of the ectoderm invaginates into a groove during development to form a neural tube, which also serves as the source of neurons and melanocytes of the central nervous system, such as the brain and spinal cord.

[0187] The expression 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, the flow cytometry method described in the section on pluripotent stem cell markers, as well as quantitative real-time PCR analysis, RNA-Seq, Northern hybridization, and hybridization methods using DNA arrays. In quantitative real-time PCR analysis, the expression level of the marker to be measured is converted to a relative expression level with respect to the expression level of an internal control gene, and the expression level of the marker can be evaluated based on this relative expression level. Examples of internal control genes include the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and the β-actin (ACTB or bAct) gene. This detection method can also be used in analyzing the expression of the pluripotent stem cell markers described above.

[0188] The lower limit of the specific growth rate of the cells at the end of this step is 0.2 days. －１ , 0.3day －１ , 0.4day －１ , 0.5day －１ , or 0.6 days －１ The upper limit of the specific growth rate is not particularly limited. For example, ―１ , 1.4 days －１ , or 1.3 days －１ The specific growth rate refers to the cell growth rate per unit time, and in this specification, particularly refers to the cell growth rate per day (24 hours). For cells in culture, the specific growth rate at a certain point in time refers to the cell growth rate over the 24 hours immediately preceding that point in time.

[0189] (Recovery of cells from suspension culture) After suspension culture, cells are recovered for subsequent stock preparation. This recovery procedure is equivalent to the procedure used during subculture from suspension culture to suspension culture in the suspension culture process. In the step of recovering suspension-cultured pluripotent stem cells, the culture medium and pluripotent stem cells are separated by conventional methods, and the separated pluripotent stem cells are recovered. At this time, the pluripotent stem cells are preferably recovered as single cells from adjacent pluripotent stem cells by a dispersing process. That is, this step preferably includes a step of dissociating cell aggregates into single cells. Note that single-state cells are sufficient as long as there are single cells (single cells) dispersed from the cell aggregates; all cells do not necessarily need to be in a single, free state; there may also be multiple cells in an adherent state.

[0190] After the suspension culture process, the cells or pluripotent stem cell population remain suspended in the culture medium. Therefore, their recovery can be achieved by removing the liquid components of the supernatant by standing or centrifugation. Alternatively, they can be recovered using a filtration filter or hollow fiber separation membrane. When removing the liquid components by standing, the container containing the culture medium is left standing for approximately 5 minutes, and the supernatant is removed, leaving behind the pluripotent stem cell population, such as the settled cells and cell aggregates. When removing the liquid components by centrifugation, the centrifugal acceleration and processing time should be set so that the cells are not damaged by centrifugal force. For example, the lower limit of the centrifugal acceleration is not particularly limited as long as the cells can be sedimented, but may be, for example, 50 × g, 100 × g, 200 × g, 300 × g, 800 × g, or 1000 × g. On the other hand, the upper limit should be a speed at which the cells are not or are least likely to be damaged by centrifugal force, such as 1200 × g, 1500 × g, or 2000 × g. The lower limit of the treatment time is not particularly limited as long as it is a time that allows the cells to settle due to the centrifugal acceleration, but may be, for example, 30 seconds, 1 minute, 3 minutes, or 5 minutes. The upper limit may be a time that does not or is unlikely to damage the cells due to the centrifugal acceleration, for example, 20 minutes, 10 minutes, 8 minutes, 6 minutes, or 5 minutes. When removing liquid components by filtration and recovering cell aggregates, 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).

[0191] The collected cells can be washed as needed. The washing method is not limited. A buffer (including PBS buffer), physiological saline, or a medium (preferably a basal medium) may be used as a washing solution.

[0192] For the dissociation into single cells, an enzymatic dissociation agent and / or a chelating agent can be used. The enzymatic dissociation agent is not particularly limited, and any enzyme that can dissociate single cells by weakening the bonds between cells in a cell aggregate can be used, even if it is not a commercially available dissociation agent. For example, trypsin, collagenase, pronase, hyaluronidase, elastase, as well as commercially available Accutase (registered trademark), Accumax (registered trademark), TrypLE, etc. ＴＭ Express Enzyme (Life Technologies Japan), TrypLE ＴＭ Select Enzyme (Life Technologies Japan, Inc.), Dispase (registered trademark), etc. can be used. The chelating agent is not particularly limited, but examples include EDTA and EGTA. For example, when trypsin is used for single cell dissociation, the lower limit of the concentration in the solution is not particularly limited as long as it is a concentration that can disperse the pluripotent stem cell population, but it can be, for example, 0.15 vol%, 0.18 vol%, 0.20 vol%, or 0.24 vol%. 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 can be, for example, 0.30 vol%, 0.28 vol%, or 0.25 vol%. Furthermore, although the treatment time depends on the concentration of trypsin, the lower limit is not particularly limited as long as the pluripotent stem cell population is sufficiently dispersed by the action of trypsin, and can be, for example, 2 minutes, 3 minutes, 5 minutes, 8 minutes, 10 minutes, 12 minutes, or 15 minutes. 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, and may be, for example, 30 minutes, 28 minutes, 25 minutes, 22 minutes, 20 minutes, or 18 minutes. When a commercially available enzymatic detachment agent is used, it should be used at a concentration that can disperse the cells into a single-cell state, as described in the attached protocol.

[0193] For example, when using EDTA for single cell dissociation, the lower limit of the solution concentration is not particularly limited as long as it is a concentration that can disperse the pluripotent stem cell population, but is preferably 0.01 mM, 0.1 mM, or 0.5 mM. On the other hand, the upper limit of the solution concentration is not particularly limited as long as it is a concentration that does not affect the cells themselves, such as by lysis, but is preferably 100 mM, 50 mM, 10 mM, or 5 mM. It is preferable to use at least one type of enzyme detachment agent and one type of chelating agent for single cell dissociation. Furthermore, it is preferable that the enzyme detachment agent and chelating agent used to treat the cells for single cell dissociation do not contain a ROCK inhibitor. The presence of a ROCK inhibitor strengthens the bonds between cells in the pluripotent stem cell population, such as cell aggregates, making single cell dissociation difficult. After treatment with the enzyme detachment agent and / or chelating agent, mild stress can be applied to the treated pluripotent stem cell population, such as cell aggregates, to promote single cell dissociation. The stress application process is not particularly limited, but may include, for example, pipetting the cells together with the solution multiple times, applying shear stress by generating a Taylor vortex, or physical stimulation such as stirring with a stirring blade. Furthermore, if necessary, the cells may be passed through a strainer or mesh. The process of dissociating the cells into single cells using an enzyme detachment agent is referred to as "enzyme treatment" in this specification.

[0194] The single-cells can be recovered by removing the supernatant containing the detachment agent by standing or centrifugation, etc. The recovered cells can be used as is, or, if necessary, can be suspended in a buffer (including PBS buffer), physiological saline, a cell preservative solution used in the cell stock preparation step, or a medium (preferably containing a ROCK inhibitor) and then subjected to the next step.

[0195] Furthermore, the cell viability at the end of this step is preferably, for example, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0196] Passaging can be performed in this step. In this case, the number of passages is not particularly limited. For example, passages can be performed 0 times, 1 or more times, 2 or more times, 3 or more times, or 4 or more times. The upper limit is not particularly limited. The passaging method is not particularly limited. For example, passage can be performed by recovering a cell population using the above-mentioned method during medium replacement and then reseeding the cells that have been single-celled using the above-mentioned method. By performing one or more passages in this step, the absolute number of cells finally obtained can be increased to any desired value by subculturing the cells in a larger container than before subculturing, increasing the amount of medium used, or dividing the cells into multiple containers for subculturing. The time until subculturing is not particularly limited, but from the perspective of maintaining cell quality, it is preferable to perform the next passage promptly. The waiting time (the time from completion of collection to the start of seeding for subculturing) is, for example, 24 hours or less, 18 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. If the waiting time is long due to the process schedule, it is advisable to store the single-celled cells suspended in the washing solution at a low temperature (for example, 10°C or less, 5°C or less). It is preferable that the cells are not frozen during this waiting period.

[0197] In the present invention, by performing the adhesion culture step and the suspension culture step under the preferred conditions as described above, the number of raw material cells used for adhesion culture can be reduced to, for example, 1 × 10 ６ Even in the case of cells or less, the number of cells at the end of the suspension culture step is 1 x 10 ８ More than 5 x 10 cells, depending on the conditions ８ More than cells, 1 x 10 ９ cells or even 2 x 10 ９ It can be a cell or larger.

[0198] 1-3-3. Cell Stock Preparation Step The "cell stock preparation step" is a step for preparing a cell stock by suspending the cells cultured and recovered in the suspension culture step in a preservation solution and dispensing the desired number of cells into desired containers. Cell preservation methods known in the art can be used to prepare the cell stock. For example, a method may be used in which the cells are suspended in a cryopreservation solution, dispensed into cryovials, and slowly frozen.

[0199] (Cells) The cells used in this step are the cells cultured and recovered in the above-mentioned "1-3-2. Suspension culture step." The pluripotent stem cells used in this step are a cell population (pluripotent stem cell population) consisting of multiple cells, and the pluripotent stem cell population expresses pluripotent stem cell markers (e.g., OCT4, SOX2, NANOG, SSEA-4, TRA-1-60). It is preferable that the pluripotent stem cells used for stock preparation in this step have a proportion of OCT4-positive cells of 90% or more and a proportion of TRA-1-60-positive cells of 90% or more. More preferably, the ratio of cells positive for OCT4 is 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%, and the ratio of cells positive for TRA-1-60 is 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%. －１ That's it, 0.65 days －１ That's all, 0.70 days －１ That's all, 0.75 days －１ That's all, 0.80 days －１ That's all, 0.85 days －１ That's it, 0.86 days －１ That's it, 0.87 days －１ That's it, 0.88 days －１ That's all, 0.89 days －１ That's all, 0.90 days －１ or more, or 0.91 days －１The above is preferable, and such cells have a high survival rate and can be used to produce a high-quality cell stock that has excellent adhesion rate, aggregate formation ability, and proliferation start-up when used in culture.

[0200] Furthermore, the cell viability before preparation as a stock is preferably, for example, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0201] Furthermore, the adhesion rate when seeded in adherent culture after preparation and storage as a stock is preferably, for example, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100%. The adhesion rate is usually calculated as the ratio of cells that have adhered at a predetermined time point after the start of culture to the number of cells seeded. In this case, the predetermined time point is not particularly limited, but the adhesion rate can be calculated, for example, 24 hours after the start of culture. In this case, the cells may have already proliferated by the time from the start of culture, and the adhesion rate may exceed 100%. The adhesion rate when seeded after storage of the stock of the present invention may be, for example, 100% or more, 105% or more, 106% or more, 107% or more, 110% or more, 115% or more, 116% or more, or 117% or more.

[0202] Furthermore, the aggregate formation rate when prepared as a stock, stored, and then seeded into a suspension culture is preferably, for example, 80% or more, 85% or more, or 90% or more. The aggregate formation rate is usually calculated as the ratio of cells forming cell aggregates at a predetermined time point after the start of culture to the number of seeded cells. In this case, the predetermined time point is not particularly limited, but the aggregate formation rate can be calculated, for example, 24 hours after the start of culture. In this case, the cells may have already proliferated by the time from the start of culture, and the aggregate formation rate may exceed 100%. The aggregate formation rate when seeded after storage of the stock of the present invention may be, for example, 100% or more, 105% or more, 110% or more, 111% or more, 112% or more, 115% or more, 120% or more, 123% or more, 125% or more, or 127% or more.

[0203] Furthermore, the cell viability after preparation and storage as a stock is preferably, for example, 92% or more, 93% or more, 94% or more, 95% or more, or 96% or more.

[0204] After preparation and storage as a stock, the ratio of cells in the G0 / G1 phase of the cell cycle is preferably 26% or less, 25% or less, 20% or less, 18% or less, 16% or less, or 15% or less. Alternatively, the ratio of cells in the G2 / M phase to cells in the G0 / G1 phase is preferably 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 2.1-fold or more, 2.2-fold or more, 2.3-fold or more, 2.4-fold or more, 2.5-fold or more, 2.6-fold or more, or 2.65-fold or more. Alternatively, the ratio of cells in G0 / G1 phase to cells in S phase is preferably 0.65 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.39 or less, 0.38 or less, or 0.37 or less.

[0205] (Container) The container into which the prepared cell stock is filled and stored is not particularly limited, but is preferably a container whose inner surface is treated to suppress protein adsorption, is preferably a sealable container, and is preferably a container that can be stored under liquid nitrogen. The container may be in the form of, for example, a vial, bag, or tube, and commercially available storage containers can be used. Examples of commercially available containers that can be used include Nunc cryotubes (Thermo Fisher Scientific), Nalgene cryovials (Thermo Fisher Scientific), and Bi.File jacketed tubes (F.C.R. & Bio Inc.). The volume of the container is not particularly limited, but it should be large enough to hold a sufficient amount of storage solution containing suspended cells. For example, the lower limit can be 0.1 mL, 0.5 mL, or 1.0 mL, and the upper limit can be 1000 mL, 500 mL, 100 mL, 50 mL, 10 mL, or 5 mL.

[0206] (Preservation Solution) As described in the section "Cell Stock" in "1-2. Definition of Terms" above, any freezing preservation solution, refrigerated preservation solution, buffer solution, etc. may be used as the preservation solution for suspending the cells obtained in the suspension culture step and preparing a cell stock. A freezing preservation solution is particularly preferred. The preservation solution may also contain a ROCK inhibitor. The density of the cells suspended in the preservation solution may not be such that it particularly reduces the quality of the cells, such as their viability, and for example, the lower limit is 0.1 x 10 ６ cells / mL, 0.2×10 ６ cells / mL, 0.3×10 ６ cells / mL, 0.4×10 ６ cells / mL, 0.5×10 ６ cells / mL, 0.6×10 ６ cells / mL, 0.7×10 ６ cells / mL, 0.8×10 ６ cells / mL, 0.9×10 ６ cells / mL, or 1.0 x 10 ６ cells / mL is preferred, with an upper limit of 100 x 10 ６ cells / mL, 50×10 ６ cells / mL, 10×10 ６ cells / mL, 9×10 ６ cells / mL, 8×10 ６ cells / mL, 7×10 ６ cells / mL, 6×10 ６ cells / mL, 5×10 ６ cells / mL, 4×10 ６ cells / mL, 3×10 ６ cells / mL, or 2 x 10 ６ cells / mL is preferred.

[0207] A low temperature is preferred when suspending and filling cells in a preservation solution to prepare a cell stock. Preservation solutions often contain components that are toxic to cells, and single-cell cells are unstable and prone to cell death. Therefore, preparing a cell stock at a low temperature while suppressing toxicity and excessive cell activity can prevent a decrease in quality, such as cell viability. In this case, a low temperature is any temperature at which the cell suspension does not freeze, and its lower limit is, for example, 0°C, 1°C, or 2°C. While the upper limit is not particularly limited, for the reasons described above, 12°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, or 4°C is preferred. In the present invention, such preferred filling conditions can be achieved by carrying out this filling step while the container or cell suspension is maintained on a low-temperature substrate at or below 10°C, or in a low-temperature environment at or below 10°C.

[0208] (Filling) The method for filling a storage container with a storage solution containing suspended cells is not particularly limited, and may be, for example, filling using a micropipette, filling using an autopipettor, filling using a syringe, filling using a multiple micropipette, or filling using an automatic dispensing device. Particularly preferred is a method using a multiple micropipette or automatic dispensing device that can efficiently fill a large number of storage containers, and even more preferred is a method using a multiple micropipette to simultaneously fill multiple storage containers.

[0209] The time required for filling is preferably short so that the cells can be stored quickly without deteriorating in quality. The upper limit is not particularly limited, but is preferably, for example, 150 minutes, 120 minutes, 90 minutes, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, or 5 minutes. Within this required time, filling at a low temperature can minimize deterioration in the quality of the cell stock, similar to the above-mentioned suspension of cells in a preservation solution at a low temperature. In this case, a low temperature is any temperature at which the cell suspension does not freeze, and the lower limit is, for example, 0°C, 1°C, or 2°C. The upper limit is not particularly limited, but for the reasons described above, 12°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, or 4°C is preferred. Such a preferred filling temperature can be achieved by holding the container or cell suspension on a low-temperature substrate at 10°C or below, or by performing the filling in a low-temperature environment at 10°C or below. Therefore, regardless of whether filling is required, from the end of cell collection to storage (or the start of freezing if frozen), the cells can be left in the above-mentioned low temperature environment for the above-mentioned time, for example, 180 minutes, 150 minutes, 120 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less.

[0210] The number of storage containers to be filled, i.e., the number of containers for stock storage into which cells are dispensed, may be set as desired, taking into consideration the number of cells recovered in suspension culture, the volume of the liquid to be filled, the cell density in the storage liquid, etc. However, considering the properties of the cell stock, the lower limit of the number is not particularly limited, but may be, for example, 20 cells, 50 or more, 100 or more, 200 or more, or 300 or more cells. On the other hand, depending on the use of the cell stock, for example, 1 x 10 cells per container may be used. ９ In some cases, one to several cell stocks containing more than 100 cells are prepared.

[0211] (Storage) A cell stock can be produced by filling the prepared cell stock solution (a solution in which cells are suspended in a storage solution) into a storage container and storing it. Storage can be, for example, maintaining the cell stock solution in a frozen state, a refrigerated state, or a gel state. Maintaining the cell stock solution in a frozen state is particularly preferred. To achieve a frozen state, the cell stock solution must be frozen. Freezing methods include slow freezing and rapid cooling, with slow cooling being more preferred. Slow cooling is a method in which the temperature is gradually lowered while freezing. The upper limit of the cooling rate is preferably 3°C / min, 2.5°C / min, or 2°C / min, and the lower limit is preferably 0.5°C / min, 1.0°C / min, 1.5°C / min, or 2°C / min. At the maximum crystal nucleation zone temperature, the cooling rate may be temporarily faster than the above range to prevent melting due to heat generation and subsequent refreezing. Furthermore, once the maximum crystal nucleation zone temperature is exceeded and the cell stock solution is completely frozen, the subsequent cooling rate may be outside the above range. For example, after freezing to −80° C. by slow cooling, the storage container may be immediately transferred to liquid nitrogen for rapid cooling.

[0212] In the method for producing a pluripotent stem cell stock of the present invention, it is preferable to continuously perform a series of steps, including (a) a thawing step, (b) an adherent culture step, (c) a suspension culture step, (d) a dispensing step into a storage container, and (e) a cell freezing step. By continuously performing these steps and quickly transitioning from one step to the next, a higher-quality cell stock can be produced. "Continuously performing" as used herein does not necessarily mean performing all steps in the same facility. However, to ensure rapid transition between steps, it is preferable to perform the steps in the same facility or between adjacent or nearby facilities. The time between each step is not particularly limited, but may be, for example, the waiting time before passage or suspension culture, the time between cell collection and storage, or the time required for filling.

[0213] In the method for producing a pluripotent stem cell stock of the present invention, the production time for the series of steps of (a) a thawing step, (b) an adherent culture step, (c) a suspension culture step, (d) a step of dispensing into storage containers, and (e) a step of freezing the cells is not particularly limited, depending on the culture period in the culture step and whether or not subculture is performed, but is, for example, 5 days or more, 7 days or more, 10 days or more, 12 days or more, and 3 months or less, or 2 months or less.

[0214] 1-4. Effects According to the method for producing a pluripotent stem cell stock of the present invention, the quality of the resulting cell stock can be improved by first performing adhesion culture on raw material cells with unstable quality. In particular, unlike conventional techniques in which a smaller number of passages is preferable, the quality is further stabilized and restored by preferably performing adhesion culture for two passages. Then, by performing suspension culture to grow the cells in large quantities up to the number of cells required for preparing the cell stock, it becomes possible to produce a large amount of cell stock from a small number of raw material cells. As a result, the number of cells at the end of suspension culture can be increased to, for example, 1 x 10 ８ cells or more, 2 x 10 ８ cells or more, 5 x 10 ８ cells or more, 1 x 10 ９ cells or more, depending on the conditions, 5 x 10 ９ cells or more, 1 x 10 １０ It is also possible to stock cells in a container containing 1 x 10 cells or more. ７ It is also possible to produce cell stocks of 20 or more, 30 or more, 50 or more, or 100 or more cells per vessel. ９It is also possible to produce cell stocks of 1 or more, 2 or more, or 10 or more cells. Furthermore, by preferably adjusting the carbon dioxide concentration or perfusing the medium so that the culture medium environment during the suspension culture step is favorable for the cells, high-quality cells can be obtained more efficiently. Furthermore, by preferably performing the preparation at a low temperature during cell stock preparation, deterioration in the quality of the cell stock can be minimized. In other words, the method of the present invention makes it possible to easily and efficiently grow rare raw cells for clinical use in large quantities, which was difficult using methods using only adherent culture or suspension culture, and further makes it possible to produce high-quality cell stocks.

[0215] 2. Method for Enhancing the Quality of Pluripotent Stem Cells The present invention also relates to a method for enhancing the quality of pluripotent stem cells, comprising the steps of thawing cryopreserved pluripotent stem cells and subsequently culturing them in an adherent culture, and then culturing the cells in a suspension culture. The quality of pluripotent stem cells is not particularly limited, but examples include the viability of the cell population and / or the rate of adhesion to the culture substrate. Preferred conditions for the adherent culture step, suspension culture step, and other steps can be those described above in "1. Method for Producing Pluripotent Stem Cell Stock." Furthermore, the adhesion rate of pluripotent stem cells subjected to adherent culture is preferably 70% or less. Furthermore, it is preferable that clinical strains of pluripotent stem cells be used.

[0216] 3. Pluripotent stem cell stock The pluripotent stem cell stock obtained by the preferred method for producing a pluripotent stem cell stock of the present invention has properties not found in conventional stem cell stocks and is extremely superior in quality. In other words, the novel pluripotent stem cell stock produced by the method described in Section "1. Method for producing a pluripotent stem cell stock" is also one aspect of the present invention.

[0217] The preservation solution, cell number contained, preservation form, etc. used in producing the pluripotent stem cell stock of the present invention may be as described in Section "1. Method for producing pluripotent stem cell stock," and it is preferable to adopt those and conditions that are considered to be preferable. The pluripotent stem cell stock of the present invention is of significantly higher quality in terms of survival rate and utilization efficiency than cell stocks produced by commonly known conventional methods such as adherent culture.

[0218] The pluripotent stem cell stock of the present invention preferably has a cell viability upon thawing of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, or 96% or more. Furthermore, when the pluripotent stem cell stock is thawed and then cultured in an adherent manner, the percentage of live cells that adhere 24 hours after seeding (cell adhesion rate) relative to the number of cells seeded is preferably 80% or more, 90% or more, 100% or more, 105% or more, 106% or more, 107% or more, 110% or more, 115% or more, 116% or more, or 117% or more. Cell stocks exhibiting such high cell adhesion rates are expected to shorten the culture period and improve differentiation induction efficiency, and are therefore of very high quality.

[0219] Furthermore, when the stock is thawed and then cultured in suspension, the ratio of live cells that form cell aggregates 24 hours after seeding to the number of cells seeded (aggregate formation rate) is preferably 80% or more, 90% or more, 100% or more, 105% or more, 110% or more, 111% or more, 112% or more, 115% or more, 120% or more, 123% or more, 125% or more, or 127% or more. Cell stocks that exhibit such high aggregate formation rates are expected to shorten the number of culture days and improve differentiation induction efficiency, and can be said to be of very high quality.

[0220] The cell viability after thawing the stock is preferably, for example, 92% or more, 93% or more, 94% or more, 95% or more, or 96% or more.

[0221] Furthermore, an investigation of the cell cycle of the cells contained in the pluripotent stem cell stock of the present invention revealed that the ratio of G2 / M phase cells to G0 / G1 phase cells was much higher than in conventional cell stocks. Because the cell cycle is closely related to cell proliferation, viability, and differentiation induction ability, it is possible that the pluripotent stem cell stock of the cell population of the present invention, possessing the above-mentioned cell cycle characteristics, results in highly efficient, high-quality cells.

[0222] In the pluripotent stem cell stock of the present invention, the proportion of cells in the G0 / G1 phase of the cell cycle is preferably 26% or less, 25% or less, 20% or less, 18% or less, 16% or less, or 15% or less.

[0223] In the pluripotent stem cell stock of the present invention, with regard to the cell cycle, the ratio of cells in G2 / M phase to cells in G0 / G1 phase is preferably 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, or 2.65 times or more.

[0224] In the pluripotent stem cell stock of the present invention, with respect to the cell cycle, the ratio of cells in G0 / G1 phase to cells in S phase is preferably 0.65 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.39 or less, 0.38 or less, or 0.37 or less.

[0225] The method for producing a pluripotent stem cell population according to the present invention will be explained in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0226] Production Examples 1 to 7, Comparative Example 1, Examples 1 to 5, and Evaluation Examples 1 to 10 of the present application correspond to the Production Examples, Comparative Examples, Examples, and Evaluation Examples of Japanese Patent Application No. 2021-206065, from which the present application claims priority. Tables 1 to 11 and Figures 1 to 6 of the present application correspond to Tables 1 to 11 and Figures 1 to 6 of Japanese Patent Application No. 2021-206065, from which the present application claims priority.

[0227] (Production Example 1: Adherent culture of human iPS cell line Ff-I14s04) Frozen human iPS cell line Ff-I14s04 (Institute for iPS Cell Research and Application, Kyoto University) was thawed and then added with iMatrix-511MG (Matrixome, Inc.) at a concentration of 0.5 μg / cm ２ Coated 25cm ２ In a culture flask, 6000 cells / cm ２ and inoculated at 37°C and 5% CO ２ Adherent culture was performed under a humid atmosphere. StemFit (registered trademark) AK03N (Ajinomoto Co.) was used as the medium, and the day the cells were seeded was counted as day 0 of culture, with the entire medium replaced on days 1, 3, and 5 of culture. The medium volume was 5 mL. Only at the time of cell seeding, Y-27632 (Fujifilm Wako Pure Chemical Industries) was added to the medium to a final concentration of 10 μM. On day 6 of culture, TrypLE cells supplemented with 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries) were used for subculture. ＴＭ The cells were treated with Select Enzyme (Life Technologies Japan, Inc.) for 15 minutes, and dispersed into single cells by pipetting while detaching the cells from the culture surface. The cells were suspended and collected in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM.

[0228] (Production Example 2: Adherent culture of human iPS cell line Ff-I14s04) The cells cultured and collected in Production Example 1 were cultured in a medium containing Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) at a concentration of 0.5 μg / cm ２ 300cm coated with ２ In a culture flask, 4000 cells / cm ２ and inoculated at 37°C and 5% CO ２Adherent culture was performed under a 500-μM atmosphere. StemFit® AK03N (Ajinomoto Co.) was used as the medium, and the entire medium was replaced on the second and third days of culture. The medium volume was 60 mL from day 0 to day 3 of culture, and 90 mL from day 3 to day 4 of culture. Y-27632 (Fujifilm Wako Pure Chemical Industries) was added to the medium to a final concentration of 10 μM only at the time of cell seeding, and LY333531 (Cayman) was added to a final concentration of 1 μM and IWR-1-endo was added to a final concentration of 20 μM only on the second and third days of culture. TrypLE containing 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries) was used. ＴＭ The cells were treated with Select Enzyme (Life Technologies Japan, Inc.) for 3 minutes, detached from the culture surface by tapping, and dispersed into single cells by pipetting. The cells were suspended and collected in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM.

[0229] (Production Example 3: Suspension culture of human iPS cell line Ff-I14s04) The cells cultured and recovered in Production Example 2 were seeded into suspension culture. A BioBlu 1c Single-Use Vessel (Eppendorf) was used as the culture vessel, and a Bioflo (Eppendorf) was used as the reactor system for controlling the culture. The pH sensor and medium perfusion pump installed in the Bioflo were calibrated according to the method specified by the manufacturer. The culture volume was 320 mL, and the cell density at the start of culture was 1.25 x 10 ５Cells were seeded at a concentration of 1000 cells / mL and culture was initiated. The culture medium used at the time of seeding was StemFit® AK03N (Ajinomoto Co.) supplemented with Y-27632 at a final concentration of 10 μM and IWR-1-endo at a final concentration of 20 μM. During culture, the culture temperature was maintained at 37°C, the supply gas flow rate was 0.2 L / min, and the culture solution was aerated from above. The carbon dioxide concentration in the supply gas was 5% at the start of culture and then reduced while adjusting it up and down to maintain the pH of the culture solution around 7.15 (suppressing pH decrease) as shown in Figure 1. The supply gas was prepared by mixing a desired amount of carbon dioxide with air. The stirring speed was 75 rpm until 48 hours of culture and 68 rpm thereafter. The start of culture was considered time 0, and medium perfusion began at 24 hours of culture. The perfusion rate per unit time was controlled every hour as shown in Table 1 to control the culture environment. The amount of medium perfusion per unit time at the start of perfusion was calculated by dividing the culture volume (320 mL) by 24 hours, F ０ The next point at which the medium perfusion rate per unit time began to be changed was the 35th hour of culture, and the medium perfusion rate per unit time thereafter was calculated using the formula 3 above, C ０ Seeding density 1.25 x 10 ５ cells / mL, the percentage of cells forming cell aggregates at 24 hours of culture for the assumed seeding amount is 150%, and the assumed specific cell growth rate is 0.90 days. －１ The cell number at 35 hours after culturing was calculated from the calculated cell density. C was also calculated from the predicted cell density transition. The constant M for correcting the influence of cell line, etc. was set to 1. The amount of lactic acid produced per cell by metabolism per unit time, L ０The amount of lactic acid produced per cell by metabolism per unit time at each culture time, L, was set from the value for typical pluripotent stem cells to calculate K, and the medium perfusion rate per unit time was set. The composition of the medium used for perfusion was switched between 24 and 48 hours of culture and after 48 hours of culture, with different concentrations of Y-27632: 5 μM for 24 to 48 hours of culture and 2.5 μM after 48 hours of culture. Both media used StemFit® AK03N (Ajinomoto Co., Inc.) supplemented with IWR-1-endo at a final concentration of 20 μM and LY333531 at a final concentration of 1 μM. To remove the cell aggregates from the culture solution and remove only the medium by suction, the medium was removed through a sintered wire mesh filter with a mesh size of 30 μm.

[0230]

[0231] After 75 hours of culture, the entire suspension culture medium was collected, and the cell aggregates were separated from the medium by centrifugation. ＴＭ The cell aggregates were treated with Select Enzyme (Life Technologies Japan, Inc.) for 5 minutes and then pipetted to break them into single cells, after which the cells were suspended in medium and collected.

[0232] (Production Example 4: Suspension culture of human iPS cell line Ff-I14s04) The cells that were cultured in suspension and collected in Production Example 3 were further cultured in suspension. The seeding density was 1.50 × 10 ５ The cells were cultured in the same manner as in Production Example 3, except that the concentration was adjusted to 100 cells / mL, LY333531 was added to the medium at the time of seeding to a final concentration of 1 μM, and the cells were collected 72 hours after culture.

[0233] (Production Example 5: Suspension culture of human iPS cell line Ff-I14s04) The cells that were cultured in suspension and collected in Production Example 4 were further cultured in suspension. The cells were cultured in the same manner as in Production Example 4.

[0234] (Example 1: Preparation of stock of human iPS cell line Ff-I14s04) The cells collected from suspension culture in Production Example 3 were transferred to a STEM-CELLBANKER (Xenogen Pharma) that had been cooled to about 4°C in advance, at a cell density of 1.0 × 10 ６The cells were suspended in a 330 mL solution to give a concentration of 100 cells / mL. The STEM-CELLBANKER (hereinafter referred to as stock solution) containing the suspended cells was then kept cold on a cooling core (Corning Incorporated), and 1 mL of the stock solution was dispensed into 300 vials of NUNC cryotubes (Thermo Fisher Scientific Inc.), which were also kept cold on the cooling core, using an 8-tube electric pipette. The cells were then frozen at a cooling rate of 1°C / min using a programmable freezer to produce a cell stock.

[0235] Example 2: Preparation of a stock of human iPS cell line Ff-I14s04 A cell stock was prepared in the same manner as in Example 1 using the cells collected from suspension culture in Production Example 4.

[0236] Example 3: Preparation of stock of human iPS cell line Ff-I14s04 A cell stock was prepared in the same manner as in Example 2 using the cells collected from suspension culture in Production Example 5.

[0237] (Evaluation Example 1: Cell counting of suspension-cultured cells) Using an NC-200 (MS Techno Systems), the viable cell count and viability (ratio of viable cell count to total cell count) of the cells recovered in Production Examples 3, 4, and 5 were measured. The results are shown in Table 2.

[0238]

[0239] As shown in Table 2, our unique suspension culture method allows stable and large-scale cell proliferation in suspension culture at each period. ２ Approximately 50 dishes' worth of cells were obtained. This demonstrates that the method of the present invention allows for easy mass cell culture. Furthermore, the suspension culture method of the present invention allows for further extension of the culture time, demonstrating its superior ease of use over adherent culture. Furthermore, cells were able to proliferate efficiently at an extremely fast rate of approximately 10 times the rate of cell proliferation in just three days of culture. This demonstrates that the method of the present invention makes it possible to efficiently and easily produce large cell stocks from very small amounts of starting cells. Furthermore, it is clear that the survival rate is very high, and high-quality cells can be cultured.

[0240] (Evaluation Example 2: Confirmation of the positive rate of undifferentiation markers in suspension-cultured cells) The cells collected in Production Examples 3, 4, and 5 were fixed, permeabilized, and blocked using eBioscience Foxp3 Transcription factor staining buffer set (Thermo Fisher Scientific). Thereafter, the cell samples were divided into 50 μL aliquots and resuspended using the buffer included in the eBioscience Foxp3 Transcription factor staining buffer set (Thermo Fisher Scientific). Fluorescently labeled anti-OCT4, anti-SOX2, and anti-NANOG antibodies were added to one of the samples and mixed, while three samples were mixed with the other three fluorescently labeled antibodies (excluding one of each of the three antibodies) to serve as FMO controls. Staining was performed at 4°C for 1 hour in the dark. The antibodies used and the amounts added are shown in Table 3.

[0241]

[0242] 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 the FMO control sample, all regions in the cell population extracted by the FSC / SSC dot plot where the cell population with stronger fluorescence intensity was 0.5% 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 taken as the percentage of cells positive for OCT4, SOX2, and NANOG. The results are shown in Table 4.

[0243]

[0244] As shown in Table 4, the cells obtained in Production Examples 3, 4, and 5 all had a very high proportion of positive cells for the undifferentiated markers OCT4, SOX2, and NANOG, demonstrating that it is possible to culture a cell population that maintains a high level of undifferentiatedness in suspension culture, where it is generally difficult to maintain undifferentiated state, and that the method of the present invention can produce a high-quality cell stock.

[0245] (Evaluation Example 3: Confirmation of inhibition of differentiation of suspension-cultured cells) The cells collected in Production Examples 3, 4, and 5 were incubated with TRIzol ＴＭ The samples were lysed using TRIzol Reagent (Thermo Fisher Scientific), and purified using the PureLink® RNA Mini Kit (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 ２The 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 and mixed. The primers used were ACTB, OCT4, SOX2, NANOG, and HK2. 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 5.

[0246]

[0247] The base sequences of the primers used in quantitative real-time PCR analysis are shown below. ACTB (Forward): 5'-CCTCATGAAGATCCTCACCGA-3' (SEQ ID NO: 1) ACTB (Reverse): 5'-TTGCCAATGGTGAATGACCTGG-3' (SEQ ID NO: 2) PAX6 (Forward): 5'-AGGAATGGACTTGAAACAAGG-3' (SEQ ID NO: 3) PAX6 (Reverse): 5'-GCAAAGCTTGTTGATCATGG-3' (SEQ ID NO: 4) BRACHYURY (Forward): 5'-TCACAAAGAGATGATGGAGGAAC-3' (SEQ ID NO: 5) BRACHYURY (Reverse): 5'-ACATGCAGGTGAGTTGTCAG-3' (SEQ ID NO: 6) SOX17 (Forward): 5'-ATCTGCACTTCGTGTGCAAG-3' (SEQ ID NO: 7) SOX17 (Reverse): 5'-GAGTCTGAGGATTTCCTTAGCTC-3' (SEQ ID NO: 8)

[0248] The measurement results are shown in Table 6.

[0249]

[0250] As shown in Table 6, no differentiation marker genes were detected in any of the iPS cells cultured in each suspension culture of the present invention (detection limit: 1.0 × 10 －５ (See below) It was shown that it is possible to produce a cell stock that maintains undifferentiated state not only from the viewpoint of undifferentiation markers as shown in Evaluation Example 2, but also from the viewpoint of differentiation markers. In other words, it was confirmed that the undifferentiated state of iPS cells produced by the method of the present invention is of high quality according to stricter standards.

[0251] (Evaluation Example 4: Confirmation of the tri-germ layer differentiation ability of suspension-cultured cells) After thawing the cell stocks prepared in Examples 1, 2, and 3, 2.0 × 10 cells were placed in a 6-well plate. ５ The cells were seeded at 1000 cells / mL in a culture medium volume of 4 mL / well, and then subjected to rotational culture at 90 rpm using a rotary shaker to induce differentiation into each of the three germ layers. The media used were as shown in Figure 2. Cells were collected at the end of the culture shown in Figure 2, and the cell stocks prepared in Examples 1, 2, and 3 were also cultured in TRIzolＴＭ The sample was dissolved using Reagent (Thermo Fisher Scientific), and DNA for quantitative real-time PCR was prepared in the same manner as described in Evaluation Example 3, followed by quantitative real-time PCR analysis.

[0252] 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'-TTGCCAATGGTGAATGACCTGG-3' (SEQ ID NO: 2) PAX6 (Forward): 5'-AGGAATGGACTTGAAACAAGG-3' (SEQ ID NO: 3) PAX6 (Reverse): 5'-GCAAAGCTTGTTGATCATGG-3' (SEQ ID NO: 4) PDGFRα (Forward): 5'-GCTGAGCCTAATCCTCTGCC-3' (SEQ ID NO: 9) PDGFRα (Reverse): 5'-ACTGCTCACTTCCAAGACCG-3' (SEQ ID NO: 10) SOX17 (Forward): 5'-ATCTGCACTTCGTGTGCAAG-3' (SEQ ID NO: 7) SOX17 (Reverse): 5'-GAGTCTGAGGATTTCCTTAGCTC-3' (SEQ ID NO: 8)

[0253] The results of measuring gene expression are shown in Figure 3. As shown in Figure 3, the cell stocks of Examples 1, 2, and 3 produced by the method of the present invention all showed significant expression of the three germ layer markers after differentiation induction, confirming that the iPS cell stocks produced by the method of the present invention retain the ability to differentiate into the three germ layers.

[0254] (Evaluation Example 5: Confirmation of specific growth rate immediately before preparation of suspension culture stock) Cell aggregates from Production Examples 3, 4, and 5 after 48 hours of culture were collected, treated with Accutase (Innovative Cell Technologies, Inc.) for 10 minutes, and unicellularized by pipetting. These cells were suspended in StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM, and the number of viable cells was measured using an NC-200. From the results and the cell numbers at the end of culture in Production Examples 3, 4, and 5 measured in Evaluation Example 1, the specific growth rate of the cells immediately before preparation of each suspension culture stock was calculated. The results are shown in Table 7.

[0255]

[0256] As shown in Table 7, the specific growth rate of the suspension-cultured cells in each example was very fast immediately before the stock was prepared. Normally, whether in adherent or suspension culture, the specific growth rate slows down immediately before the cells are harvested after sufficient proliferation, following the completion of the subculture period. However, the method of the present invention monitors and manages changes in the culture environment, preventing a decline in the specific growth rate until the end of the culture. In other words, the cells can be cultured and stocked while maintaining their optimal condition. This makes it possible to produce high-quality cell stocks that are compatible with mass production, something that was previously difficult to achieve.

[0257] Comparative Example 1: Adherent culture of human iPS cell line Ff-I14s04 and preparation of stock. Frozen human iPS cell line Ff-I14s04 (Center for iPS Cell Research and Application, Kyoto University) was thawed and then added with iMatrix-511MG (Matrixome, Inc.) at a concentration of 0.5 μg / cm ２ Coated 25cm ２ In a culture flask, 6000 cells / cm ２ and inoculated at 37°C and 5% CO ２ Adherent culture was performed under a 500-mL atmosphere. StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) was used as the medium. The day the cells were seeded was counted as day 0 of culture, and the entire medium was replaced on days 1, 3, and 5 of culture. The medium volume was 5 mL. Only at the time of cell seeding, Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 10 μM. On day 6 of culture, TrypLE was added for subculture.ＴＭ The cells were treated with Select Enzyme (Life Technologies Japan, Inc.) for 5 minutes, detached from the culture surface with a cell scraper, and dispersed into single cells. The cells were suspended and collected in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM. Thereafter, the cells were placed in a STEM-CELLBANKER (Xenogen Pharma Co., Ltd.) at a cell density of 1.0 × 10 ６ The cells were suspended in STEM-CELLBANKER (hereinafter referred to as stock solution) and the stock solution was dispensed in 1 mL aliquots into NUNC cryotubes (Thermo Fisher Scientific) and frozen at a cooling rate of 1°C / min using a programmed freezer to prepare cell stocks using the conventional adherent culture method.

[0258] (Evaluation Example 6) After thawing each of the cell stocks prepared in Examples 1, 2, and 3 and the cell stock prepared in Comparative Example 1, the cell viability was measured using NC-200. The results are shown in Table 8 and FIG. 4.

[0259]

[0260] As shown in Table 8 and Figure 4, the stocks prepared by the method of the present invention in Examples 1, 2, and 3 had higher survival rates after thawing than the stock prepared by the conventional method in Comparative Example 1, demonstrating that high-quality cell stocks can be prepared by the method of the present invention.

[0261] (Evaluation Example 7: Adhesion rate of cell stock to adherent culture) The cell stocks prepared in Examples 1, 2, and 3 and the cell stock prepared in Comparative Example 1 were thawed, and then iMatrix-511 (Matrixome) was added at 0.5 μg / cm ２ Coated 25cm ２ In a culture flask, 6000 cells / cm ２ and inoculated at 37°C and 5% CO ２Adhesion culture was performed under a 5 mL atmosphere. The medium used was StemFit® AK02N (Ajinomoto Co.) supplemented with Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 10 μM. The day the cells were seeded was designated culture day 0. On culture day 1, the culture flask was observed, the number of adherent cells in the field of view was counted, and converted to the number of adherent cells in the entire culture vessel. The adhesion rate (the ratio of cells that had adhered at 24 hours of culture to the number of seeded cells) was then calculated. The results are shown in Table 9 and Figure 5.

[0262]

[0263] As shown in Table 9 and Figure 5, the cell stocks of Examples 1, 2, and 3 prepared by the method of the present invention were found to have significantly higher adhesion rates and higher quality than the cell stock of Comparative Example 1 prepared by the conventional method. Furthermore, surprisingly, the adhesion rates exceeded 100%, suggesting that the seeded cells had already begun to proliferate immediately after seeding. By preparing cell stocks using the method of the present invention, it is possible to shorten the number of culture days and improve the efficiency when using the stock to produce differentiated cells.

[0264] (Production Example 6: Adherent culture of human iPS cell line Ff-I14s04) Frozen human iPS cell line Ff-I14s04 (Institute for iPS Cell Research and Application, Kyoto University) was thawed, and then iMatrix-511MG (Matrixome) was added at 0.5 μg / cm ２ Coated 150cm ２ In a culture flask, 1000 cells / cm ２ and inoculated at 37°C and 5% CO ２ Adherent culture was performed under a 50% RH atmosphere. StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) was used as the medium, and the day the cells were seeded was designated as culture day 0. The entire medium was replaced on culture days 1, 4, 6, 8, 9, and 10. The medium volume was 30 mL. Only at the time of cell seeding, Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 10 μM. On culture day 11, TrypLE was added for subculturing. ＴＭThe cells were treated with Select Enzyme (Life Technologies Japan, Inc.) for 5 minutes, detached from the culture surface with a cell scraper, and dispersed into single cells. These cells were suspended and collected in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM.

[0265] (Evaluation Example 8: Cell yield in adherent culture using starting cells) The viable cell counts of the cells recovered from the culture in Production Example 2 and Production Example 6 were measured using NC-200. The results are shown in Table 10.

[0266]

[0267] As shown in Table 10, Production Example 2, in which starting cells were seeded and cultured in small-scale adherent culture at a high seeding density, then passaged to a medium-scale culture for a total of two passages (10 days), and the cells were recovered, yielded a larger number of cells than Production Example 6, in which starting cells were seeded and cultured in medium-scale adherent culture at a low seeding density, then cultured for a total of one passage (11 days). The cell coverage of the culture surface when the cells were recovered was approximately 60% in both Production Examples 2 and 6. In other words, when using starting cells with a small number of cells, dense seeding in small containers and stepwise culture tends to be more efficient than thin seeding in large containers and long-term culture, and tends to yield a sufficiently large number of cells that can be used for subsequent suspension culture.

[0268] (Evaluation Example 9: Undifferentiated state of cells in adhesion culture using starting cells) The cells recovered from the culture in Production Example 2 and Production Example 6 were analyzed by flow cytometry in the same manner as in Evaluation Example 2. The results are shown in Table 11.

[0269]

[0270] As shown in Table 11, the positive rate of undifferentiation markers was slightly reduced in the cells of Production Example 6, suggesting the possibility that the quality of the cells may have deteriorated before the suspension culture step. This is thought to be due to the state of the raw material cells becoming unstable due to the low seeding density, and the fact that the cells became denser within each cell colony due to the extended culture period in order to sufficiently proliferate the cells, resulting in cell damage. This demonstrates the usefulness of seeding cells at a high seeding density and culturing them in adhesion culture, as in the more preferred method of the present invention.

[0271] (Production Example 7: Suspension culture of human iPS cell line Ff-I14s04) 150 cm ２ The culture flask used was 1000 cells / cm ２ and iMatix-511 was seeded at 0.25 μg / cm ２ Human iPS cell line Ff-I14s04 (Center for iPS Cell Research and Application, Kyoto University) was cultured in adherent culture in the same manner as in Production Example 1, except that the cells were seeded by mixing the medium at the time of seeding so that the concentration was 10 μM and the cells were collected on day 8 of culture. The cells were cultured at a concentration of 1 × 10 per mL using StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) containing 10 μM Y-27632 and 20 μM IWR-1-endo. ５ The cell suspension was prepared so that it contained 320 mL of cells. 320 mL of the cell suspension was inoculated into BioBlu 1c Single-Use Vessels (Eppendorf). The reactor inoculated with the cells was stirred at 75 rpm, maintained at 37°C, and vented under 5% CO2. ２ Suspension culture was performed under the same conditions. StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) was used as the medium. The day the cells were seeded was designated as culture day 0, and the entire medium was replaced on culture days 1 and 2. The medium replacement on culture day 1 used StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) containing a final concentration of 7 μM Y-27632, 20 μM IWR-1-endo, and 1 μM LY333531, and the medium replacement on culture day 2 used StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) containing a final concentration of 3 μM Y-27632, 20 μM IWR-1-endo, and 1 μM LY333531. On culture day 3, TrypLE was used for subculture. ＴＭThe cells were treated with Select Enzyme (Life Technologies Japan, Inc.) for 5 minutes, and cell clumps were dispersed into single cells by pipetting. The cells were suspended and collected in StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) containing Y-27632 at a final concentration of 10 μM.

[0272] (Example 4: Low-temperature preparation of stock solution) The cells collected from suspension culture in Production Example 7 were transferred to a STEM-CELLBANKER (Xenogen Pharma) that had been refrigerated at around 4°C in advance to a cell density of 1.0 × 10 ６ The cells were suspended to a concentration of 1000 cells / mL. The STEM-CELLBANKER (stock solution) containing the suspended cells was then kept cold on a cooling core (Corning Incorporated), and 1 mL of the solution was dispensed into NUNC cryotubes (Thermo Fisher Scientific) that were also kept cold on a cooling core. The cells were then kept cold on the cooling core, and slowly frozen at a cooling rate of 1°C / mL using a Mr. Frosty (Thermo Fisher Scientific) at 10, 30, 60, and 120 minutes after being suspended in the STEM-CELLBANKER to prepare cell stocks.

[0273] (Example 5: Preparation of stock solution at room temperature) A cell stock was prepared in the same manner as in Example 4, except that the cells cultured in suspension and collected in Production Example 7 were prepared as a stock solution and left to stand until freezing at room temperature (22°C).

[0274] (Evaluation Example 10: Confirmation of viable cell rate before and after preparation of cell stock) The cell stocks prepared in Examples 4 and 5 were thawed, and the number of viable cells was measured using an NC-200. From the measurement results, the ratio of the number of viable cells after stock preparation and thawing to the number of viable cells filled was calculated. The results are shown in Figure 6.

[0275] As shown in Figure 6, when cell stocks are prepared at room temperature using conventional methods, the number of viable cells contained in the resulting cell stock tends to decrease as the waiting time before freezing, i.e., the time required for the filling process to produce a large amount of cell stock, increases. However, when cell stocks are prepared under refrigerated conditions using the preferred method of the present invention, the number of viable cells does not decrease over time. This is thought to be because refrigeration prevents cell apoptosis and other cell declines. In other words, the preferred method of the present invention makes it possible to produce cell stocks using high-quality pluripotent stem cells expanded in the culture process without compromising their quality.

[0276] (Evaluation Example 11: Aggregate formation rate in suspension culture of cell stock) The cell stocks prepared in Examples 1, 2, and 3 and the cell stock prepared in Comparative Example 1 were thawed and then inoculated into a 30 mL reactor (ABLE) at 10,000 cells / mL. The cells were then incubated at 37°C and 5% CO with stirring at 100 rpm. ２ Suspension culture was performed under atmospheric conditions. The medium used was StemFit® AK02N (Ajinomoto Co.) supplemented with Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 10 μM. The day the cells were seeded was designated culture day 0, and cell aggregates formed on culture day 1 were collected, treated with Accutase (Innovative Cell Technologies, Inc.) for 10 minutes, and dissociated into single cells by pipetting. The cells were suspended in StemFit® AK02N (Ajinomoto Co.) containing Y-27632 at a final concentration of 10 μM, and the number of viable cells was measured using an NC-200. The aggregate formation rate (the ratio of cells forming cell aggregates at 24 hours of culture to the number of seeded cells) was calculated from the measured number of viable cells. The results are shown in Table 12 and Figure 7.

[0277]

[0278] As shown in Table 12 and Figure 7, the cell stocks of Examples 1, 2, and 3 prepared by the method of the present invention were found to have significantly higher aggregate formation rates and higher quality than the cell stock of Comparative Example 1 prepared by the conventional method. Furthermore, surprisingly, the aggregate formation rate exceeded 100%, suggesting that the seeded cells had already begun to proliferate immediately after seeding. By preparing a cell stock using the method of the present invention, it is possible to shorten the number of culture days and improve the efficiency when using the stock to produce differentiated cells.

[0279] (Evaluation Example 12: Cell Cycle Analysis of Cell Stock) After thawing, the cell stocks prepared in Example 1 and Comparative Example 1 were stained using the Cell Meter Fluorimetric Fixed Cell Cycle Assay Kit (AAT Bioquest). Then, analysis was performed using a Guava easyCyte 8HT (Lumix). Cell cycle analysis was performed using the Watson (Pragmatic) method on cell populations extracted by FSC / SSC dot plot using Flowjo (Becton Dickinson). The results are shown in Table 13 and Figure 8. The DNA amounts corresponding to the peaks in the overlapping shaded areas in Figure 8 were used as the DNA amounts at the boundaries between the G2 / M phase and the S phase, and between the G0 / G1 phase and the S phase cell populations.

[0280]

[0281] As shown in Table 13, the cell stock of Example 1 prepared by the method of the present invention had a higher proportion of G2 / M phase cells and a higher ratio of G2 / M phase cells to G0 / G1 phase cells compared to the cell stock of Comparative Example 1 prepared by the conventional method. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. (a) thawing frozen cells that will be used as the source of the cell stock to be prepared; and (b) culturing the thawed starting cells in an adherent culture; and (c) culturing the adherently cultured cells in suspension; and (d) dispensing the suspension-cultured cells into containers for stock storage; and (e) freezing the cells dispensed into the container; A method for producing a pluripotent stem cell stock, comprising:

2. The number of raw material cells used in the step (b) is 1 × 10 ６ cells or less, and the number of cells at the end of the culture in the step (c) is 1 x 10 ８ The method of claim 1 , wherein the target cell is a cell or more.

3. The method of claim 1 , wherein the adhesion rate of the raw material cells is 70% or less.

4. In the adherent culture of the step (b), 3 × 10 ３ cells / cm ２ The method of claim 1 , wherein the cells are seeded at a density equal to or greater than 1000 μg / ml.

5. The method of claim 1 , wherein step (b) comprises passaging.

6. The method of claim 5, wherein the passage is into a container having a larger surface area.

7. The method according to claim 1, wherein the suspension culture in step (c) is carried out by perfusion of the medium.

8. The method of claim 7 , wherein the method of perfusing the medium includes increasing the amount of medium perfusion in accordance with cell growth.

9. The method according to claim 7, further comprising controlling the amount of medium perfusion so as to maintain the pH of the culture solution between 6.5 and 9.0 by perfusing the medium.

10. 2. The production method according to claim 1, wherein the suspension culture in the step (c) comprises varying the concentration of carbon dioxide gas supplied within a range of 10 to 0% as the culture progresses.

11. The method according to claim 1 , wherein the suspension culture in step (c) is a suspension agitation culture.

12. The method of claim 11 , wherein the stirring suspension culture comprises reducing the stirring speed during the culture period.

13. The method according to claim 1, wherein the amount of culture medium for the suspension culture in step (c) is 100 mL or more.

14. The specific growth rate of the cells at the end of the step (c) is 0.70 days －１ The manufacturing method according to claim 1 .

15. The method of claim 1 , wherein step (c) comprises breaking down the cell aggregate into single cells.

16. The method of claim 15 , wherein the dissociation into single cells comprises an enzyme treatment in the presence of a ROCK inhibitor.

17. The method of claim 1, wherein in step (d), at least one of the container and the cell suspension is maintained at 10°C or below.

18. The method of claim 1, wherein a stock of 100 or more pluripotent stem cells is produced.

19. The method according to claim 1 , wherein the dispensing in step (d) is carried out using a multiple-barrel pipette.

20. The method according to claim 1 , wherein the medium used for the culture in the step (c) contains a ROCK inhibitor.

21. The method according to claim 20, wherein the ROCK inhibitor is Y-27632.

22. 2. The production method according to claim 1, wherein the medium used for the culture in the steps (b) and (c) contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.

23. The method according to claim 1, wherein the culture medium used for the culture in the steps (b) and (c) contains FGF2 and / or TGF-β1.

24. The production method according to claim 1, wherein the proportion of cells that are positive for OCT4 is 90% or more and the proportion of cells that are positive for TRA-1-60 is 90% or more in the pluripotent stem cells that constitute the stock.

25. (f) thawing the cryopreserved pluripotent stem cells and then culturing them in an adherent manner; and (g) a step of subjecting the adherently cultured cells to suspension culture. A method for enhancing the quality of pluripotent stem cells, comprising:

26. A pluripotent stem cell stock having a cell survival rate of 90% or more after thawing, and in which, when subjected to adherent culture after thawing, the number of adherent cells after 24 hours of culture is 0.8 times or more the number of seeded cells.

27. A pluripotent stem cell stock having a cell viability of 90% or more after thawing, and having a viable cell count of 0.8 times or more the number of seeded cells after 24 hours of culture when subjected to suspension culture after thawing.

28. A pluripotent stem cell stock, in which the proportion of cells in the G2 / M phase of the cell cycle of the cells contained therein is 1.5 times or more the proportion of cells in the G0 / G1 phase.