Mass culture of pluripotent stem cells

Abstract

To improve proliferation properties of pluripotent stem cells, while producing a large quantity of pluripotent stem cells maintaining their undifferentiated state.SOLUTION: Provided is a method for producing pluripotent stem cells, comprising the steps of (a) and (b) below: (a) filling a culture vessel with a liquid medium containing FGF2 and subsequently raising the temperature of the liquid medium in the culture vessel to a temperature at which pluripotent stem cells can grow; and (b) seeding pluripotent stem cells in the liquid medium in the culture vessel and culturing them while suspending them in the liquid medium, the volume of the liquid medium used being 1 L or more per culture vessel, and in step (a), the temperature of the liquid medium in the culture vessel before raising the temperature being -20°C or higher and 18°C or lower, and in step (a), the temperature at which the pluripotent stem cells can grow being 30°C or higher and 40°C or lower.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for mass culturing pluripotent stem cells on a commercial scale, and a method for producing somatic cells from pluripotent stem cells obtained by this method. This application claims priority based on Japanese Patent Application No. 2019-42797, filed on March 8, 2019, the contents of which are incorporated herein by reference. [Background technology]

[0002] Pluripotent stem cells, such as ES cells and iPS cells, have the ability to proliferate indefinitely and the pluripotency to differentiate into various cell types. Recent research results have made it increasingly possible to apply the cellular properties of pluripotent stem cells to fundamental treatments for intractable diseases and lifestyle-related diseases. For example, it is already possible to induce differentiation of pluripotent stem cells into cardiac muscle cells, skeletal muscle cells, neurons, megakaryocytes, hematopoietic stem cells, airway epithelial cells, germ cells, dendritic cells, eosinophils, mast cells, chondrocytes, T cells, erythropoietin-producing cells, intestinal epithelium, pancreatic cells, liver cells, and alveolar epithelial cells. On the other hand, regenerative medicine using pluripotent stem cells faces challenges before it can be put to practical use, one of which is the productivity of pluripotent stem cells. Generally, regenerative medicine requires a large amount of pluripotent stem cells; for example, treating the liver requires 20 billion pluripotent stem cells. Therefore, pluripotent stem cells must be produced efficiently and in large quantities, but general plate cultures can produce only 10 6 cm 2 This requires a culture area of ​​20,000 10cm dishes, which is a common culture vessel. As such, it is extremely difficult to scale up culture using flat plate culture, and it is not realistic because it requires securing a huge amount of space for the manufacturing facility. To solve this problem, suspension culture, in which cells are cultured three-dimensionally while suspended in a medium, has been reported in recent years. For example, Patent Document 1 discloses a technique for culturing cells in a medium while rotating the cells to produce cell aggregates. Furthermore, Patent Document 2 discloses a technique for preventing adhesion of cell clusters to each other by adding a water-soluble polymer to the culture medium to increase viscosity, and then culturing the cells in suspension so that the average diameter of the cell clusters is 200 μm or more and 300 μm or less. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-304866 [Patent Document 2] International Publication No. 2013 / 077423 Summary of the Invention [Problem to be solved by the invention]

[0004] To perform mass culture of pluripotent stem cells, the inventors heated commercially available cell culture medium to approximately 37°C and repeatedly poured the medium into a culture vessel heated to approximately 37°C until a volume suitable for suspension culture was reached. Pluripotent stem cells were then seeded into the culture vessel and cultured in suspension with stirring. As a result, they discovered a problem: the proliferation rate of pluripotent stem cells was reduced compared to that of pluripotent stem cells cultured on a plate. Furthermore, when the undifferentiated state of pluripotent stem cells obtained by the suspension culture was analyzed, they also discovered a problem: an increase in pluripotent stem cells that had deviated from the undifferentiated state. [Means for solving the problem]

[0005] The present inventors conducted extensive research to solve the above-mentioned problems and found that repeated injection of heated medium into a heated culture vessel destabilizes the components of the medium injected into the culture vessel from the early to mid-stage, thereby reducing the proliferation rate of pluripotent stem cells. Furthermore, they found that this phenomenon induces the deviation of pluripotent stem cells from their undifferentiated state. Therefore, the present inventors injected liquid medium into a culture vessel until the volume reached a level suitable for suspension culture, raised the medium temperature in the culture vessel to approximately 37°C, and then seeded pluripotent stem cells into the culture vessel for suspension culture. As a result, the present inventors made the surprising discovery that the above-mentioned culture method improves the proliferation rate of pluripotent stem cells and maintains their undifferentiated state, leading to the completion of the present invention. Specifically, the present invention includes the following inventions. (1) A method for producing pluripotent stem cells, comprising the steps of: (a) and (b) (a) filling a culture vessel with a liquid medium containing FGF2, and then increasing the temperature of the liquid medium in the culture vessel to a temperature at which pluripotent stem cells can grow; and (b) seeding pluripotent stem cells in the liquid medium in the culture vessel and culturing them in suspension; Including, The volume of liquid medium used is 1 L or more per culture vessel. In the step (a), the temperature of the liquid medium in the culture vessel before increasing the temperature is -20°C or higher and 18°C ​​or lower; In the step (a), the temperature at which the pluripotent stem cells can grow is 30°C or higher and 40°C or lower. Manufacturing method. (2) The method according to (1), wherein in step (a), the temperature of the liquid medium is increased by at least 20°C. (3) The production method according to (1) or (2), wherein in step (a), the temperature of the liquid medium in the culture vessel is increased while stirring the liquid medium. (4) The method according to any one of (1) to (3), wherein no growth factor is added in the step (a). (5) The method according to any one of (1) to (4), wherein a growth factor is added in the step (b). (6) The method according to any one of (1) to (5), wherein the liquid medium contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate. (7) The method according to any one of (1) to (6), wherein the pluripotent stem cells are embryonic stem cells or iPS cells (induced pluripotent stem cells). (8) The method according to (4) or (5), wherein the growth factor is at least one selected from the group consisting of FGF2 and TGF-β1. (9) A method for producing somatic cells, comprising the steps of: (a) to (c) (a) filling a culture vessel with a liquid medium containing FGF2, and then increasing the temperature of the liquid medium in the culture vessel to a temperature at which pluripotent stem cells can grow; (b) seeding pluripotent stem cells in the liquid medium in the culture vessel and culturing them in suspension; and (c) culturing the pluripotent stem cells obtained in step (b) in the presence of a differentiation-inducing factor to induce differentiation; Including, The volume of liquid medium used is 1 L or more per culture vessel. In the step (a), the temperature of the liquid medium in the culture vessel before increasing the temperature is -20°C or higher and 18°C ​​or lower; In the step (a), the temperature at which the pluripotent stem cells can grow is 30°C or higher and 40°C or lower. Manufacturing method. (10) The method according to (9), wherein in step (a), the temperature of the liquid medium is increased by at least 20°C. (11) The production method according to (9) or (10), wherein in the step (a), the temperature of the liquid medium in the culture vessel is increased while stirring the liquid medium. (12) The method according to any one of (9) to (11), wherein no growth factor is added in the step (a). (13) The method according to any one of (9) to (12), wherein a growth factor is added in the step (b). (14) The method according to any one of (9) to (13), wherein the somatic cells are at least one selected from the group consisting of cardiomyocytes, skeletal muscle cells, nerve cells, megakaryocytes, hematopoietic stem cells, airway epithelial cells, germ cells, dendritic cells, eosinophils, mast cells, chondrocytes, T cells, erythropoietin-producing cells, intestinal epithelium, pancreatic cells, liver cells, alveolar epithelial cells, and kidney cells. [Effects of the Invention]

[0006] According to the method for producing pluripotent stem cells of the present invention, it is possible to improve the proliferation of pluripotent stem cells and produce pluripotent stem cells in large quantities while maintaining their undifferentiated state. According to the method for producing somatic cells of the present invention, somatic cells can be produced efficiently and in large quantities. The pharmaceutical composition of the present invention can be used to treat diseases such as intractable diseases and lifestyle-related diseases. DETAILED DESCRIPTION OF THE INVENTION

[0007] The following describes in detail the embodiments of the present invention. However, the following description is intended to facilitate understanding of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the embodiments described below are also included in the scope of the present invention.

[0008] In the present invention, the term "cell" refers to a cell having adhesive properties (adherent cell). The adhesive cell may be an animal-derived cell, preferably a mammal-derived cell, more preferably a biological tissue-derived cell or a cell derived from a biological tissue-derived cell, particularly preferably an epithelial tissue-derived cell or a cell derived from an epithelial tissue cell, or a connective tissue-derived cell or a cell derived from a connective tissue-derived cell, or a muscle tissue-derived cell or a neural tissue-derived cell or a cell derived from a neural tissue-derived cell, even more preferably an animal-derived stem cell or a cell differentiated from an animal-derived stem cell, most preferably an animal-derived pluripotent stem cell or a cell differentiated from an animal-derived pluripotent stem cell, even more preferably a mammal-derived pluripotent stem cell or a cell differentiated from a mammal-derived pluripotent stem cell, and most preferably a human-derived pluripotent stem cell or a cell differentiated from a human-derived pluripotent stem cell.

[0009] In the present invention, a "stem cell" refers to a cell that can differentiate into other cells and has the ability to self-replicate. Among "stem cells," cells that have the multipotency (pluripotency) to differentiate into all types of cells that constitute the body and can continue to proliferate indefinitely while maintaining pluripotency when cultured in vitro under appropriate conditions are referred to as "pluripotent stem cells." Specific examples of pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), EG cells, which are pluripotent stem cells derived from fetal primordial germ cells (Shamblott MJ et al., Proc. Natl. Acad. Sci. USA. (1998) 95, pp. 13726-13731), GS cells, which are pluripotent stem cells derived from testes (Conrad S., Nature (2008) 456, pp. 344-349), and iPS cells (induced pluripotent stem cells), which are induced pluripotent stem cells derived from somatic cells.

[0010] The pluripotent stem cells used in the present invention are particularly preferably ES cells or iPS cells. ES cells are pluripotent stem cells derived from early embryos. iPS cells are cultured cells that have been conferred pluripotency by introducing reprogramming factors into somatic cells, thereby reprogramming them to an undifferentiated state. Examples of reprogramming factors that can be used include OCT3 / 4, KLF4, SOX2, and c-Myc (Takahashi K, et al. Cell. 2007;131:861-72). Examples of factors that can be used include OCT3 / 4, SOX2, LIN28, and Nanog (Yu J, et al. Science. 2007;318:1917-20). The manner in which these factors are introduced into cells is not particularly limited, and examples include gene transfer using a plasmid, synthetic RNA, and direct protein introduction. iPS cells generated using methods such as microRNA, RNA, and low-molecular-weight compounds may also be used. Pluripotent stem cells, including ES cells and iPS cells, may be commercially available, provided, or newly prepared. Examples of iPS cells that can be used include the 253G1, 201B6, 201B7, 409B2, 454E2, HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, Nips-B2, TkDN4-M, TkDA3-1, TkDA3-2, TkDA3-4, TkDA3-5, TkDA3-9, TkDA3-20, hiPSC 38-2, MSC-iPSC1, and BJ-iPSC1 strains. Examples of ES cells that can be used include KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SEES-1, SEES-2, SEES-3, SEES-4, SEES-5, SEES-6, SEES-7, HUES8, CyT49, H1, H9, HS-181, and RPChiPS771-2 strains. Newly prepared clinical-grade iPS cells or ES cells may also be used. The origin of the cells used to generate iPS cells is not particularly limited, and examples include fibroblasts and lymphocytes.

[0011] The present invention can provide a cell aggregate. A cell aggregate is a mass-like cell group formed by three-dimensional aggregation of multiple cells, and is also called a spheroid. The cell aggregate is usually approximately spherical. The cells constituting the cell aggregate are not particularly limited as long as they are one or more types of 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. Examples of pluripotent stem cell markers include alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1.

[0012] The pluripotent stem cell markers of the present invention can be detected by any detection method known in the art. Methods for detecting expression markers include, but are not limited to, flow cytometry. When cells are detected in flow cytometry using a fluorescently labeled antibody that emits stronger fluorescence than a negative control (isotype control), the cells are determined to be "positive" for the marker. The proportion of cells that are positive for a fluorescently labeled antibody analyzed by flow cytometry is sometimes referred to as the positive rate. Furthermore, any fluorescently labeled antibody known in the art can be used, including, but not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc.

[0013] When the cells constituting a cell aggregate or cell population are pluripotent stem cells, the percentage (proportion) of cells expressing and / or positively testing for a pluripotent stem cell marker can be, 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, or 99% or more. The upper limit of the percentage of cells is not particularly limited and can be 100% or less, or even 100%. A cell aggregate or cell population in which the percentage of cells expressing and / or positively testing for a pluripotent stem cell marker falls within the above range is highly undifferentiated and is a more homogeneous cell population. Note that the term "pluripotent stem cell marker" is synonymous with "undifferentiation marker," and the two terms can be used interchangeably.

[0014] The dimensions of the cell aggregates produced by one or more embodiments of the present invention are not particularly limited, but when observed under a microscope, the upper limit of the dimension of the widest part in the observed image is, for example, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, or 200 μm or less. The lower limit is, for example, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, or 100 μm or more. Cell aggregates within such a size range are preferable as a cell growth environment because oxygen and nutrients can be easily supplied to the cells inside.

[0015] In a population of cell aggregates produced by one or more embodiments of the present invention, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the cell aggregates constituting the population may have dimensions within the above ranges, on a weight basis. In a population of cell aggregates containing 20% ​​or more cell aggregates with dimensions within the above ranges, oxygen and nutrients are easily supplied to the cells within each individual cell aggregate, making it a preferable environment for cell growth. In a cell population produced by one or more embodiments of the present invention, the proportion of live cells (viability) among the cells constituting the population is preferably, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. A cell population with a viability within the above range is in a preferable state for cell growth.

[0016] The somatic cells of the present invention are not particularly limited as long as they can be induced to differentiate from pluripotent stem cells and can exist in a living body. Examples of the somatic cells include somatic stem cells (mesenchymal stem cells and neural stem cells derived from bone marrow, adipose tissue, dental pulp, placenta, fetal membrane, umbilical cord blood, amnion, chorion, etc.), neurons, glial cells, oligodendrocytes, Schwann cells, cardiomyocytes, cardiac progenitor cells, hepatocytes, liver progenitor cells, α cells, β cells, fibroblasts, chondrocytes, corneal cells, vascular endothelial cells, vascular endothelial progenitor cells, pericytes, skeletal muscle cells, megakaryocytes, hematopoietic stem cells, airway epithelial cells, germ cells, dendritic cells, eosinophils, mast cells, T cells, erythropoietin-producing cells, intestinal epithelium, alveolar epithelial cells, kidney cells, pancreatic cells, and liver cells. These cells may be transfected with genes or may have a target gene knocked down in the genome.

[0017] The cells used in the present invention may be derived from any animal, for example, mammals such as rodents such as mice, rats, and hamsters; primates such as humans, gorillas, and chimpanzees; and livestock or pet animals such as dogs, cats, rabbits, cows, horses, sheep, and goats, although cells derived from humans are particularly preferred.

[0018] According to the production method of the present invention, a cell population of any of three germ layers, such as endodermal cells, ectodermal cells, and mesodermal cells, and somatic cells obtained therefrom, are produced from pluripotent stem cells. The three germ layers include endodermal cells, mesodermal cells, and ectodermal cells. Examples of somatic cells include those already described.

[0019] Endodermal cells in the present invention have the ability to differentiate into tissues of organs such as the digestive tract, lung, thyroid gland, pancreas, and liver, cells of secretory glands opening into the digestive tract, peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most part of the urethra, and part of the ureter), and are sometimes generally referred to as definitive endoderm (DE). Differentiation of pluripotent stem cells into endodermal cells can be confirmed by measuring the expression levels of genes specific to endodermal cells. Examples of genes specific to endodermal cells include SOX17, FOXA2, CXCR4, AFP, GATA4, and EOMES. Note that, throughout this specification, endodermal cells may be referred to as definitive endoderm.

[0020] Mesodermal cells in the present invention differentiate into body cavities and the mesothelium lining them, muscles, skeletons, skin dermis, connective tissue, heart, blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidneys, ureters, gonads (testes, uterus, and gonadal epithelium), etc. Examples of genes specific to mesodermal cells include MESP1, MESP2, FOXF1, BRACHYURY, HAND1, EVX1, IRX3, CDX2, TBX6, MIXL1, ISL1, SNAI2, FOXC1, and PDGFRα.

[0021] Ectodermal cells in the present invention form the epidermis of 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), and lenses. During development, some ectodermal cells invaginate into a groove to form neural tubes, which also serve as the source of neurons and melanocytes in the central nervous system, such as the brain and spinal cord. They also form the peripheral nervous system. Examples of genes specific to ectodermal cells include FGF5, OTX2, SOX1, and PAX6.

[0022] According to the production method of the present invention, somatic cells can be produced by further inducing differentiation into the three germ layers obtained by inducing differentiation of the above-mentioned pluripotent stem cells. Somatic cells obtained from endodermal cells are described below.

[0023] In the present invention, primitive gut cells (PGT) form the foregut, midgut, and hindgut. The midgut is connected to the yolk sac, and the extraembryonic allantois branches from the hindgut. The pharynx of the respiratory system also forms from the foregut. Some organs differentiate directly from the gut, such as the stomach and intestines, while others form by budding from the gut, such as the liver, gallbladder, pancreas, and spleen (lymphatic organ). Differentiation of endodermal cells into primitive gut cells can be confirmed by measuring the expression level of genes specific to primitive gut cells. Examples of genes specific to primitive gut cells include HNF-1β and HNF-4α.

[0024] In the present invention, posterior foregut cells (PFG) are cells differentiated from primitive intestinal cells and can be identified by measuring the expression levels of genes specific to posterior foregut cells, such as PDX1 and HNF6.

[0025] In the present invention, pancreatic progenitor cells (PP) are cells differentiated from posterior foregut cells and can differentiate into exocrine and endocrine cells of the pancreas. Differentiation of posterior foregut cells into pancreatic progenitor cells can be confirmed by measuring the expression level of genes specific to pancreatic progenitor cells. Examples of genes specific to pancreatic progenitor cells include PDX1 and NKX6.1.

[0026] Pancreatic endocrine precursor cells (EP) of the present invention are cells differentiated from pancreatic precursor cells and can differentiate into pancreatic endocrine cells (α cells, β cells, δ cells, ε cells, PP cells, etc.). Differentiation into pancreatic precursor cells can be confirmed by measuring the expression level of genes specific to pancreatic precursor cells. Examples of genes specific to pancreatic precursor cells include PDX1, NKX6.1, NeuroG3, and NeuroD1.

[0027] Pancreatic β cells in the present invention are cells differentiated from pancreatic endocrine precursor cells and secrete insulin. Differentiation of pancreatic endocrine precursor cells into pancreatic β cells can be confirmed by measuring the expression levels of genes specific to pancreatic β cells. Examples of genes specific to pancreatic β cells include insulin, NKX6.1, MAFA, and PDX1.

[0028] According to the production method of the present invention, somatic cells that can be used for treating digestive systems such as the pancreas, liver, stomach, and intestines can be obtained using an endodermal cell population induced to differentiate from pluripotent stem cells. Examples of embodiments for treating each digestive system are described below, but the present invention is not limited to these.

[0029] In the intestinal system, if intestinal progenitor cells such as crypt cells are obtained, they can be transplanted using a catheter or the like to be used to treat ulcerative colitis, Crohn's disease, short bowel disease, and the like.

[0030] In the pancreatic system, if pancreatic β cells (sometimes referred to as insulin-producing cells) are obtained, they can be used to treat diabetes by transplanting them via a catheter or enclosed in an immunoisolation device.

[0031] In the liver system, for example, if albumin-producing cells are obtained, they can be transplanted via a catheter or enclosed in an immune isolation device, and used to treat trauma accompanied by massive bleeding.

[0032] In addition, therapeutic tissues from the digestive system, such as the pancreas, liver, stomach, and intestines, can be obtained by culturing them using polymeric support carriers. For example, liver tissue obtained by induction can be used to treat liver metabolic disorders such as liver cancer, cirrhosis, acute liver failure, and hemochromatosis. Lung cell tissue can be transplanted into affected areas to treat pulmonary and respiratory diseases such as cystic fibrosis and asthma. Renal tissue containing mesangial cells, tubular epithelial cells, and glomerular cells can be directly transplanted to treat renal failure, nephritis, and dialysis. Metabolic cells from the liver can be generated to produce albumin-producing cells, blood coagulation factor-producing cells, and metabolic enzyme-producing cells such as α-1 antitrypsin. These metabolic enzymes can be directly injected or administered intravenously to treat deficiencies of these proteins. For example, pancreatic cells capable of metabolism, such as pancreatic β cells, can be obtained, and insulin produced by the pancreatic β cells can be directly injected to treat type 1 diabetes.

[0033] Furthermore, any of the cell populations of the three germ layers obtained by the production method of the present invention, as well as the somatic cells obtained therefrom, can be used to evaluate the efficacy / toxicity of test substances, to elucidate their mechanisms of action, or to analyze the mechanisms of biological phenomena.

[0034] In the present invention, cells isolated after culturing in an adherent or suspended state can be used. Here, "isolated cells" refer to cells in a state in which a plurality of cells have been detached and dispersed from a group of cells adhering to the culture vessel or culture carrier, or a group of cells adhering to each other, to form single cells by detaching and dispersing them. The cell group to be isolated may be in a suspended state in a liquid medium. The isolation method is not particularly limited, but a detachment agent (a cell detachment enzyme such as trypsin or collagenase), a chelating agent such as EDTA (ethylenediaminetetraacetic acid), or a mixture of a detachment agent and a chelating agent can be suitably used. The detachment agent is not particularly limited, but trypsin, Accutase (registered trademark), TrypLE, etc. TM Express Enzyme (Life Technologies), TrypLE TM Examples include Select Enzyme (Life Technologies), Dispase (registered trademark), collagenase, etc. The cells that have been cryopreserved after isolation can also be suitably used in the present invention.

[0035] In the present invention, "culturing in suspension" refers to growing cells in a suspended state in a liquid medium, and can be abbreviated as "suspension culture." Cells in suspension culture exist as aggregated cell masses in a liquid medium. Although not particularly limited, in the present invention, this method is used as a technique for culturing cells three-dimensionally to mass-produce them. Note that the culture method used for the maintenance culture of pluripotent stem cells is not limited to this, and cells may be cultured in either an adherent or suspended state.

[0036] Suspension culture may be static culture or culture under conditions in which the liquid medium is flowing, but culture under conditions in which the liquid medium is flowing is preferred. Culture under conditions in which the liquid medium is flowing is preferred as it promotes cell aggregation. Examples of culture under conditions in which the liquid medium is flowing to promote cell aggregation include culture under conditions in which the liquid medium is flowing so that cells gather at a single point due to stress (centrifugal force, centripetal force) caused by flow such as swirling flow or oscillating flow, and culture under conditions in which the liquid medium is flowing due to linear reciprocating motion, and culture using swirling flow and / or oscillating flow is particularly preferred.

[0037] In the present invention, the term "rotation culture" (including shaking culture) refers to a culture method in which a liquid medium flows so that cells gather at a single point due to stress (centrifugal force, centripetal force) caused by a swirling flow. Specifically, this method is carried out by rotating a culture vessel containing a liquid medium containing cells along a generally horizontal plane in a closed orbit such as a circle, ellipse, flattened circle, or flattened ellipse, or by rotating the liquid medium in the vessel using a stirrer bar or agitator blade while the culture vessel remains stationary. The latter method can be achieved, for example, by using a spinner flask-type culture vessel equipped with agitator blades. Such culture vessels are commercially available or made to order, and can also be used. In this case, the amount of liquid medium or culture solution used may be within the range recommended by the culture vessel manufacturer or within the range conceivable by one skilled in the art.

[0038] The rotation speed in the rotary culture method is not particularly limited, but the upper limit can be, for example, 200 rpm or less, 150 rpm or less, 120 rpm or less, 115 rpm or less, 110 rpm or less, 105 rpm or less, 100 rpm or less, 95 rpm or less, or 90 rpm or less. The lower limit can be, for example, 1 rpm or more, 10 rpm or more, 50 rpm or more, 60 rpm or more, 70 rpm or more, 80 rpm or more, or 90 rpm or more. The rotation width during rotary culture is not particularly limited, but the lower limit can be, for example, 1 mm or more, 10 mm or more, 20 mm or more, or 25 mm or more. The upper limit of the rotation width can be, for example, 200 mm or less, 100 mm or less, 50 mm or less, 30 mm or less, or 25 mm or less. The rotation radius during rotary culture is also not particularly limited, but is set so that the rotation width is within the above-mentioned range. The lower limit of the turning radius may be, for example, 5 mm or more, or 10 mm or more, and the upper limit may be, for example, 100 mm or more, or 50 mm or more.

[0039] In the present invention, "rocking culture" refers to a culture method in which a rocking flow is imparted to a liquid medium by linear reciprocating motion, such as rocking agitation. Specifically, rocking is performed by rocking a culture vessel containing a liquid medium containing cells in a plane perpendicular to a generally horizontal plane. The rocking speed is not particularly limited. For example, assuming one reciprocating motion is one cycle, the lower limit is 2 or more, 4 or more, 6 or more, 8 or more, or 10 or more times per minute, while the upper limit is 15 or fewer, 20 or fewer, 25 or fewer, or 50 or fewer times per minute. During rocking, it is preferable to provide the culture vessel with a slight angle, i.e., an induction angle, relative to the vertical plane. The rocking angle is not particularly limited. For example, the lower limit can be 0° or more, 1° or more, 2° or more, 4° or more, 6° or more, or 8° or more, while the upper limit can be 10° or less, 12° or less, 15° or less, 18° or less, or 20° or less. Setting the rocking culture conditions within this range is preferable because it makes it possible to produce cell aggregates of appropriate dimensions.

[0040] The seeding density of cells in suspension culture in a liquid medium (cell density at the start of suspension culture) can be adjusted as appropriate, but the lower limit of the seeding density is, for example, 0.01 × 10 5 individual cells / mL or more, 0.1×10 5 cells / mL or more, or 1 x 10 5 The upper limit of the seeding density is, for example, 10 × 10 6 Individual cells / mL or less, 20×10 5 cells / mL or less, or 10 x 10 5 When the seeding density is in this range, cell aggregates of appropriate size are likely to be formed. The seeding density is, for example, 0.1 × 10 5 Individual cells / mL, 0.2×10 5 Individual cells / mL, 0.3×10 5 Individual cells / mL, 0.4×10 5 Individual cells / mL, 0.5×10 5 Individual cells / mL, 0.6×10 5 Individual cells / mL, 0.7×10 5 Individual cells / mL, 0.8×10 5 Individual cells / mL, 0.9×10 5 Individual cells / mL, 1×10 5 Individual cells / mL, 1.5×10 5 Individual cells / mL, 2×10 5 Individual cells / mL, 3×10 5 Individual cells / mL, 4×10 5 Individual cells / mL, 5×10 5 Individual cells / mL, 6×10 5 Individual cells / mL, 7×10 5 individual cells / mL, 8×10 5 Individual cells / mL, 9×10 5 cells / mL, or 10 x 10 6 Individual cells / mL is also acceptable.

[0041] The extent to which cells are grown in suspension culture, or how the morphology and state of the cells are adjusted, can be determined appropriately depending on the type and properties of the cells to be cultured, the purpose of the culture, the type of liquid medium, and the culture conditions. The cells used in suspension culture are preferably cells that have been previously cultured in a maintenance culture step, recovered in a recovery step, and, if necessary, dissociated into single cells. After suspension culture, the culture medium is discarded and the cells are recovered by a conventional method. At this time, the cells are preferably recovered as single cells by detachment or dispersion treatment. The recovered cells may be subjected to the next step either directly or, if necessary, after washing with a buffer, physiological saline, or liquid medium.

[0042] The liquid medium used in the present invention is a liquid substance prepared for cell culture. In principle, it contains at least the minimum amount of components essential for cell growth and / or maintenance. For example, it can be prepared by using any animal cell culture medium as a basal medium and appropriately adding other components, such as culture additives, as needed. The liquid medium used in the present invention is preferably one suitable for cell suspension culture. Note that solid media that are partially or completely gelled tend to remain in the liquid delivery tube during delivery, and temperature changes can induce morphological changes and changes in the concentration of major components. Therefore, the medium used in the present invention is a liquid substance. Note that, in this specification, liquid medium may also be referred to simply as medium.

[0043] The viscosity of the liquid medium of the present invention is not particularly limited as long as it is within a range that can be recognized as liquid by a person skilled in the art. Specifically, when the temperature of the liquid medium is 25°C, the viscosity is preferably 100 mPa·s or less, 90 mPa·s or less, 80 mPa·s or less, 70 mPa·s or less, 60 mPa·s or less, 50 mPa·s or less, 40 mPa·s or less, or 30 mPa·s or less, and is preferably greater than 0 mPa·s, 1 mPa·s or more, 2 mPa·s or more, 3 mPa·s or more, 4 mPa·s or more, or 5 mPa·s or more.

[0044] The viscosity of the liquid medium is not particularly limited as long as it is a viscometer capable of measuring the viscosity of a liquid medium, and can be measured, for example, with a B-type viscometer (Tokimec Co., Ltd.) Specifically, a measurement sample is placed in a glass container with an inner diameter of 60 mm, and measurements are taken three times under the following conditions: liquid temperature 25°C, rotor No. 2, rotation speed 60 rpm, and retention time 30 seconds, and the average value can be used as the measured value (viscosity).

[0045] In the present invention, the term "not added" refers to the absence of exogenous addition of factors such as proteins, peptides, and compounds identified as not being added to the culture or conditioned medium. When factors such as proteins, peptides, and compounds identified as not being added to the culture or conditioned medium are introduced through continuous culturing operations, the concentration is adjusted to less than 1% (volume / volume), less than 0.5% (volume / volume), less than 0.1% (volume / volume), less than 0.05% (volume / volume), less than 0.01% (volume / volume), or less than 0.001% (volume / volume).

[0046] Examples of basal media that can be used include, but are not limited to, 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 / Nutrient Mixture F-12 Ham)). As the DMEM / F12 medium, a medium in which DMEM medium and Ham's F12 medium are mixed in a weight ratio of, for example, 60 / 40 or more and 40 / 60 or less, a weight ratio of 55 / 45 or more and 45 / 55 or less, or equal amounts (50 / 50 weight ratio) can be used.

[0047] In the present invention, a "culture additive" refers to a substance other than serum that is added to a culture medium for the purpose of culture. Specific examples of culture additives include, but are not limited to, L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, growth factors, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and antibiotics. Insulin, transferrin, and cytokines may be naturally derived substances isolated from animal tissues or serum (preferably human, mouse, rat, bovine, horse, goat, etc.), or may be recombinant proteins produced by genetic engineering. Growth factors that can be used include, but are not limited to, basic fibroblast growth factor-2 (FGF2), transforming growth factor-β1 (TGF-β1), activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF, and IGFBP-7. Antibiotics that can be used include, but are not limited to, penicillin, streptomycin, amphotericin B, and the like.

[0048] The liquid medium used in the present invention preferably contains at least one selected from L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate, and more preferably contains all of these. Furthermore, L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate can be added to the medium in the form of a solution, derivative, salt, mixed reagent, or the like. For example, L-ascorbic acid may be added to the medium in the form of a derivative such as magnesium ascorbate 2-phosphate. Selenium may be added to the medium in the form of a selenite (e.g., sodium selenite). Insulin and transferrin may be naturally derived and isolated from tissues or serum of animals (preferably humans, mice, rats, cows, horses, goats, etc.), or may be recombinant proteins produced by genetic engineering. Insulin, transferrin, and selenium may be added to the medium in the form of the reagent ITS (insulin-transferrin-selenium). ITS is an additive for promoting cell growth that contains insulin, transferrin, and sodium selenite. Commercially available media containing at least one selected from L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate can be used. Commercially available media containing insulin and transferrin include CHO-S-SFM II (Life Technologies), Hybridoma-SFM (Life Technologies), eRDF Dry Powdered Media (Life Technologies), UltraCULTURE™ (BioWhittaker), and UltraDOMA. TM (BioWhittaker), UltraCHO TM (BioWhittaker), UltraMDCK TM (BioWhittaker), etc. can be used. STEMPRO (registered trademark) hESC SFM (Life Technologies), mTeSR1 (Veritas), TeSR2 (Veritas), etc. can also be suitably used. In addition, media used for culturing human iPS cells and human ES cells can also be suitably used.

[0049] In the present invention, the "culture vessel" is not particularly limited as long as it has a shape, size, structure, and configuration that allows pluripotent stem cells to be cultured in suspension, but it is preferable that the shape, capacity, structure, and configuration reduce adhesion of cells to the inner surface of the vessel. Note that in this specification, the culture vessel may be referred to as a culture tank.

[0050] The shape of the culture vessel is not particularly limited, and examples thereof include tank-shaped, flow reactor-shaped, dish-shaped, flask-shaped, well-shaped, bag-shaped, etc. The capacity of the culture vessel used in the present invention can be appropriately selected and is not particularly limited, and examples thereof include a culture vessel having a bottom surface of, for example, 25 cm2 in plan view, which is the area of ​​the bottom surface of the portion containing the culture medium. 2 More than 50cm 2 More than 100cm 2 More than 200cm 2 More than 300cm 2 More than 400cm 2 More than 500cm 2 More than 600cm 2 More than 700cm 2 More than 800cm 2 More than 900cm 2 More than 1000cm 2 More than 2000cm 2 More than 3000cm 2 More than 4000cm 2 More than 5000cm 2 More than 6000cm 2 More than 7000cm 2 More than 8000cm 2 More than 9000cm 2 or more than 10,000 cm 2 It is preferable that the viscosity is 1,000,000 cm or more. 2 Below, 500000cm 2 Below, 100000cm 2 Below, 90000cm 2 Below, 80000cm 2 Below, 70000cm 2 Below, 60000cm 2 Below, 50000cm 2 Below, 40000cm 2Below, 30000cm 2 Below, 20000cm 2 Below, 10000cm 2 Below, 9000cm 2 Below, 8000cm 2 Below, 7000cm 2 Below, 6000cm 2 Below, 5000cm 2 Below, 4000cm 2 Below, 3000cm 2 Below, 2000cm 2 or less than 1000cm 2 It is preferable that:

[0051] The volume (L / culture vessel) of the liquid medium used in the present invention is not particularly limited as long as it allows pluripotent stem cells to be cultured in suspension and allows cell aggregates to be formed. For example, the volume is preferably 0.1 L / culture vessel or more, 0.5 L / culture vessel or more, 1 L / culture vessel or more, 2 L / culture vessel or more, 3 L / culture vessel or more, 4 L / culture vessel or more, 5 L / culture vessel or more, 6 L / culture vessel or more, 7 L / culture vessel or more, 8 L / culture vessel or more, 9 L / culture vessel or more, 10 L / culture vessel or more, 20 L / culture vessel or more, 30 L / culture vessel or more, 40 L / culture vessel or more, 50 L / culture vessel or more, 60 L / culture vessel or more, 70 L / culture vessel or more, 80 L / culture vessel or more, 90 L / culture vessel or more, or 100 L / culture vessel or more. 0000L / culture vessel or less, 10000L / culture vessel or less, 9000L / culture vessel or less, 8000L / culture vessel or less, 7000L / culture vessel or less, 6000L / culture vessel or less, 5000L / culture vessel or less, 4000L / culture vessel or less, 3000L / culture vessel or less, 2000L / culture vessel or less, 1000L / culture vessel or less, 900L / culture vessel or less, 800L / culture vessel or less, 700L / culture vessel or less, 600L / culture vessel or less, 500L / culture vessel or less, 400L / culture vessel or less, 300L / culture vessel or less, 200L / culture vessel or less, 150L / culture vessel or less, 100L / culture vessel or less, 90L / culture vessel or less, 80L / culture vessel or less, 70L / culture vessel or less, 60L / culture vessel or less, or 50L / culture vessel or less.

[0052] In the present invention, the "maintenance culture step" refers to a step of culturing a cell population before suspension culture, or a cell aggregate obtained after suspension culture or the subsequent recovery step, in order to proliferate the cells while maintaining their undifferentiated state. The maintenance culture may be adhesion culture, in which the cells are cultured while attached to a culture substrate such as a container or carrier, or suspension culture, in which the cells are cultured while suspended in a liquid medium.

[0053] In the maintenance culture step, the target cells may be cultured by any animal cell culture method known in the art, regardless of whether they are adherent culture or suspension culture.

[0054] Specific embodiments of the liquid medium, cells, cell seeding density, culture vessel, shape of the culture vessel, and culture conditions used in the maintenance culture step are as described above.

[0055] The state of flow of the liquid medium in the maintenance culture step is not particularly limited, and may be static culture or fluid culture.

[0056] "Static culture" refers to culturing in a liquid medium in a culture vessel while it is stationary. This static culture is usually used in adherent culture.

[0057] "Flow-through culture" refers to culture under conditions in which the liquid medium is flowing. Specific embodiments of flow-through culture are as described above.

[0058] The extent to which the cells are grown in the maintenance culture step or how the morphology and state of the cells are adjusted may be determined appropriately depending on the type and properties of the cells to be cultured, the purpose of the culture, the type of liquid medium, and the culture conditions.

[0059] In the maintenance culture step, it is preferable to change the liquid medium at an appropriate frequency. The frequency of liquid medium change varies depending on the cell type, but can include, for example, at least once every 5 days, at least once every 4 days, at least once every 3 days, at least once every 2 days, or at least once a day. This frequency of liquid medium change is particularly suitable for culturing cell aggregates of stem cells. While the method for changing the liquid medium is not particularly limited, it is preferable to collect the entire cell culture composition containing the cell aggregates in a centrifuge tube, centrifuge the mixture, or allow it to stand for about 5 minutes, remove the supernatant while leaving the settled cell aggregates, and then add fresh liquid medium. The cell aggregates can then be gently dispersed, and the cell aggregate dispersion medium can be returned to the culture vessel, such as a plate, to continue culturing the cell aggregates. The culture period for this embodiment of the maintenance culture step is not particularly limited, but is preferably between 3 and 7 days.

[0060] After the maintenance culture step, the culture medium is discarded and the cells are recovered by conventional methods. At this time, the cells are preferably recovered as single cells by detachment or dispersion treatment. The recovered cells can be used directly or, if necessary, washed with a buffer (including PBS buffer), physiological saline, or liquid medium (preferably the liquid medium or basal medium used in the next step) before being subjected to the next step.

[0061] The "recovery step" is a step of recovering cultured cells from the culture medium after suspension culture and / or after the maintenance culture step, and is a selection step in the method of the present invention.

[0062] In the present invention, "(cell) recovery" refers to separating the cells from the culture medium and obtaining the cells. The cell recovery method may be any conventional method used in cell culture methods in the field, and is not particularly limited. Cell culture methods can generally be broadly divided into suspension culture and adherent culture. Below, we will explain the cell recovery methods after each culture method.

[0063] (Recovery method after suspension culture) When cultured using suspension culture, cells exist in a suspended state in the culture medium. Therefore, cell recovery can be achieved by removing the liquid components of the supernatant either by statically standing or by centrifugation. Alternatively, cell recovery methods such as filters or hollow fiber separation membranes can be selected. To remove the liquid components in a static state, the container containing the culture medium can be left standing for approximately 5 minutes, and the supernatant can be removed, leaving behind the settled cells and cell aggregates. Centrifugation can be performed at a rotation speed and for a processing time that does not damage the cells due to centrifugal force. For example, the lower limit of the rotation speed is not particularly limited as long as it allows the cells to settle, but it can be, for example, 500 rpm or more, 800 rpm or more, or 1000 rpm or more. On the other hand, the upper limit can be any speed that does not or is unlikely to damage the cells due to centrifugal force, such as 1400 rpm or less, 1500 rpm or less, or 1600 rpm or less. The lower limit of the treatment time is not particularly limited as long as it allows the cells to settle at the rotation speed, 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 rotation, for example, 30 seconds, 6 minutes, 8 minutes, or 10 minutes. The recovered cells can be washed as needed. The washing method is not limited. For example, the washing method may be the same as that described in the "Post-process treatment" in the maintenance culture step above. The washing liquid may be a buffer (including PBS buffer), physiological saline, or liquid medium (basal medium is preferred).

[0064] (Recovery method after adherent culture method) When cells are cultured using the adherent culture method, many cells remain attached to the external matrix, such as the culture vessel or culture support. Therefore, to remove the culture medium from the culture vessel, simply tilt the vessel after culture to drain off the liquid components. Since the cells attached to the external matrix remain in the culture vessel, the culture medium and cells can be easily separated.

[0065] Thereafter, the surface of the cells adhered to the external matrix can be washed as needed. The washing solution can be, but is not limited to, a buffer (including PBS buffer), physiological saline, or liquid medium (basal medium is preferred). The washing solution after washing can be removed in the same manner as the culture medium. This washing step can be repeated multiple times.

[0066] Next, the cell population adhered to the external matrix is ​​detached from the external matrix by any method known in the art. Typically, scraping, a detaching agent containing a protease as an active ingredient, a chelating agent such as EDTA, or a mixture of a detaching agent and a chelating agent is used.

[0067] Scraping is a method of mechanically removing cells attached to an external matrix using a scraper or the like. However, because cells are easily damaged by mechanical manipulation, when the recovered cells are to be further cultured, a detachment method in which the scaffolding portion of the cells that is fixed to the external matrix is ​​chemically destroyed or decomposed to release the adhesion between the cells and the external matrix is ​​preferred.

[0068] The peeling method uses a peeling agent and / or a chelating agent. Examples of peeling agents include, but are not limited to, trypsin, collagenase, pronase, hyaluronidase, elastase, as well as commercially available enzymes such as Accutase (registered trademark) and TrypLE. TM Express Enzyme (Life Technologies, Inc.), TrypLE TM Select Enzyme (Life Technologies, Inc.), Dispase (registered trademark), etc. can be used. The concentration and treatment time of each detachment agent may be within the ranges commonly used for cell detachment or dispersion. For example, in the case of trypsin, the lower limit of the concentration in the solution is not particularly limited as long as it is a concentration that can detach cells, but may be, for example, 0.01% or more, 0.02% or more, 0.03% or more, 0.04% or more, 0.05% or more, 0.08% or more, or 0.10% or more.

[0069] 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 cell lysis or other effects due to the action of trypsin, and may be, for example, 0.15% or less, 0.20% or less, 0.25% or less, or 0.30% or less. The treatment time, while dependent on the trypsin concentration, is not particularly limited as long as it is a time that allows cells to be sufficiently detached from the external matrix by the action of trypsin, and may be, for example, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, or 5 minutes or more. On the other hand, the upper limit of the treatment time is not particularly limited as long as it does not cause cell lysis or other effects due to the action of trypsin, and may be, for example, 8 minutes or less, 10 minutes or less, 12 minutes or less, 15 minutes or less, 18 minutes or less, or 20 minutes or less. Similar procedures can be used with other detachment agents or chelating agents. When using commercially available detachment agents, the concentration and treatment time described in the attached protocol can be used. The cells recovered after this recovery process can also be disaggregated into single cells, if necessary.

[0070] In the present invention, "reducing to single cells" refers to dispersing a cell aggregate in which multiple cells adhere or aggregate to each other, such as a monolayer cell fragment or a cell aggregate, into a single, free cell state.

[0071] Single-cell dissociation can be achieved by increasing the concentration of the detachment agent and / or chelating agent used in the above-mentioned detachment method and / or by increasing the treatment time with the detachment agent and / or chelating agent. For example, in the case of trypsin, the lower limit of the trypsin concentration in the solution is not particularly limited as long as it is a concentration that can disperse cell aggregates, but it may be, for example, 0.15% or more, 0.18% or more, 0.20% or more, or 0.24% or more. On the other hand, the upper limit of the trypsin 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 lysing them, but it may be 0.25% or less, 0.28% or less, or 0.30% or less. Furthermore, although the treatment time depends on the trypsin concentration, the lower limit is not particularly limited as long as it is a time that allows the cell aggregates to be sufficiently dispersed by the action of trypsin, and it may be, for example, 5 minutes or more, 8 minutes or more, 10 minutes or more, 12 minutes or more, or 15 minutes or more. On the other hand, the upper limit of the treatment time is not particularly limited as long as it is a time that does not cause the cells themselves to be affected by the action of trypsin, such as by being lysed, and may be, for example, 18 minutes or less, 20 minutes or less, 22 minutes or less, 25 minutes or less, 28 minutes or less, or 30 minutes or less.

[0072] When using a commercially available detachment agent, it should be used at a concentration that can disperse the cells into a single cell state within the range specified in the attached protocol. After treatment with the detachment agent and / or chelating agent, light physical treatment can be performed to promote the formation of single cells. This physical treatment is not limited, but examples include pipetting the cells together with the solution multiple times. Furthermore, if necessary, the cells may be passed through a strainer or mesh.

[0073] The single-cells can be recovered by removing the supernatant containing the release agent by standing or centrifugation. The recovered cells may be washed as needed. The centrifugation conditions and washing method may be as described above.

[0074] Specific embodiments of the present invention will be described below, but the present invention is not limited thereto, and the description already given will be omitted.

[0075] The production method of the present invention comprises the following steps (a) and (b): (a) filling a culture vessel with a liquid medium and then increasing the temperature of the liquid medium in the culture vessel to a temperature at which pluripotent stem cells can grow; and (b) seeding pluripotent stem cells in the liquid medium in the culture vessel and culturing them in suspension; The manufacturing method includes:

[0076] Step (a): After filling a culture vessel with liquid medium, the step of raising the temperature of the liquid medium in the culture vessel to a temperature at which pluripotent stem cells can proliferate corresponds to a preliminary step before the step of culturing pluripotent stem cells in suspension, and it is preferable that this step and step (b) are performed consecutively.

[0077] In step (a), when filling the culture vessel with unheated liquid medium (liquid medium maintained at the temperature at which it was stored), the operator may fill the culture vessel directly with the liquid medium, or, if the culture vessel and the container in which the liquid medium is stored are connected by a liquid transfer tube such as a pipe, the liquid may be transferred from container to container in a sterile environment using air pressure, etc. The temperature of the liquid medium when filling the culture vessel is not particularly limited as long as it is a temperature at which the components contained in the liquid medium can remain stable for at least 6 months. For example, the temperature is preferably −80° C. or higher, −70° C. or higher, −60° C. or higher, −50° C. or higher, −40° C. or higher, −30° C. or higher, −20° C. or higher, −10° C. or higher, −5° C. or higher, 0° C. or higher, or 4° C. or higher, and is also preferably 18° C. or lower, 15° C. or lower, 10° C. or lower, 9° C. or lower, 8° C. or lower, 7° C. or lower, 6° C. or lower, or 5° C. or lower. In another embodiment, in order to make the filling operation more efficient and stabilize the composition of the liquid medium, for example, after filling with a liquid medium at 4° C., the temperature inside the culture vessel may be lowered to −20° C., so that the temperature of the liquid medium is −20° C. In other words, it is preferable to appropriately adjust the temperatures of the liquid medium at the time of filling and the liquid medium before heating within a temperature range at which the components contained in the liquid medium described above are stabilized.

[0078] Next, in step (a), the temperature of the liquid medium in the culture vessel is raised to a temperature at which pluripotent stem cells can proliferate. The temperature of the liquid medium in the culture vessel before the temperature increase may be the same as the temperature of the liquid medium when the culture vessel is filled, or may be changed within a temperature range at which the components contained in the liquid medium can be stabilized. Specifically, the temperature is preferably −80°C or higher, −70°C or higher, −60°C or higher, −50°C or higher, −40°C or higher, −30°C or higher, −20°C or higher, −10°C or higher, −5°C or higher, 0°C or higher, or 4°C or higher, and is also preferably 18°C ​​or lower, 15°C or lower, 10°C or lower, 9°C or lower, 8°C or lower, 7°C or lower, 6°C or lower, or 5°C or lower. In particular, a temperature of −20°C or higher or 10°C or lower in the liquid medium in the culture vessel before the temperature increase is preferred, as this significantly improves the proliferation of pluripotent stem cells.

[0079] The temperature increase of the liquid medium in step (a) (the range of increase in the temperature of the liquid medium) can be selected appropriately and is not particularly limited, and may be, for example, 15°C increase, 16°C increase, 17°C increase, 18°C ​​increase, 19°C increase, 20°C increase, 21°C increase, 22°C increase, 23°C increase, 24°C increase, 25°C increase, 26°C increase, 27°C increase, 28°C increase, 29°C increase, 30°C increase, 31°C increase, 32°C increase, 33°C increase, 34°C increase, 35°C increase, 36°C increase, 37°C increase, 38°C increase, 39°C increase, 40°C increase, or above. Preferably, the temperature is raised by 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., or 60° C. In particular, the range in which the temperature of the liquid medium is raised is preferably 20° C. or higher and 57° C. or lower, more preferably 22° C. or higher and 57° C. or lower, and particularly preferably 37° C. or higher and 57° C. or lower.

[0080] The temperature at which the pluripotent stem cells can grow in step (a) is not particularly limited as long as it is a temperature at which the pluripotent stem cells can survive and grow, but is preferably, for example, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, or 35°C or higher, and is preferably 42°C or lower, 41°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, or 37°C or lower.

[0081] In step (a), it is preferable to increase the temperature of the liquid medium in the culture vessel while stirring the liquid medium. Examples of embodiments for stirring the liquid medium in the culture vessel include stirring under conditions in which the liquid medium flows due to stress (centrifugal force, centripetal force) caused by flow such as swirling flow or oscillating flow, or stirring under conditions in which the liquid medium flows due to linear reciprocating motion. Stirring utilizing swirling flow and / or oscillating flow is preferred. More specifically, the temperature of the liquid medium in the culture vessel can be efficiently increased by swirling the liquid medium in the vessel using a stirrer such as a stirring blade.

[0082] In step (a), it is preferable not to add a growth factor. It has been discovered by the present invention that the temperature change in the liquid medium in this step induces destabilization of growth factors. Examples of growth factors include FGF2, TGF-β1, Activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF, and IGFBP-7, with FGF2 and / or TGF-β1 being particularly preferred.

[0083] Step (b): The step of seeding pluripotent stem cells in the liquid medium in the culture vessel and culturing them in suspension corresponds to a step subsequent to the step of increasing the temperature of the liquid medium in the culture vessel, and it is preferable that step (a) and this step are performed consecutively.

[0084] In step (b), the method for culturing pluripotent stem cells in suspension can be the suspension culture method described above, or the method described in Japanese Patent No. 6238265.

[0085] In step (b), it is preferable to add a growth factor. It has been discovered by the present invention that adding a growth factor in this step efficiently proliferates pluripotent stem cells. Examples of growth factors include FGF2, TGF-β1, Activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF, and IGFBP-7, with FGF2 and / or TGF-β1 being particularly preferred. The concentration of the growth factor added is not particularly limited as long as it is within a concentration range that allows pluripotent stem cells to proliferate, but is preferably 1 μg / L or more, 3 μg / L or more, 5 μg / L or more, 7 μg / L or more, or 10 μg / L or more, and is preferably 500 μg / L or less, 400 μg / L or less, 300 μg / L or less, 200 μg / L or less, or 100 μg / L or less.

[0086] Furthermore, the manufacturing method of the present invention allows somatic cells to be obtained by carrying out the following step (c) using pluripotent stem cells obtained after the above-mentioned steps (a) and (b). The method for producing somatic cells of the present invention comprises the following steps (a) to (c): (a) filling a culture vessel with a liquid medium and then increasing the temperature of the liquid medium in the culture vessel to a temperature at which pluripotent stem cells can grow; (b) seeding pluripotent stem cells in the liquid medium in the culture vessel and culturing them in suspension; and (c) culturing the pluripotent stem cells obtained in step (b) in the presence of a differentiation-inducing factor to induce differentiation; The manufacturing method includes: Step (c): The step of culturing the pluripotent stem cells obtained in step (b) in the presence of a differentiation-inducing factor and inducing differentiation corresponds to a subsequent step after the step of culturing the pluripotent stem cells in suspension, and it is preferable to carry out step (b) and this step consecutively.

[0087] Examples of differentiation-inducing factors used in step (c) include substances that act on TGFβ signaling, substances that act on Wnt signaling, substances that act on Hedgehog signaling, substances that act on BMP signaling, and substances that act on Nodal / Activin signaling. Specifically, the differentiation-inducing factors described in WO 2016 / 063986, WO 2012 / 020845, and WO 2016 / 060260 can be used.

[0088] The cell population containing the somatic cells according to the present invention can be used as a pharmaceutical composition. That is, the present invention provides a pharmaceutical composition containing the somatic cells according to the present invention and a cell population containing those somatic cells, and a pharmaceutically acceptable medium. The present invention also provides a pharmaceutical composition containing the somatic cells according to the present invention and a cell population containing those somatic cells, and other cells that can be administered.

[0089] The pharmaceutical composition of the present invention can be used as a cell therapy agent, for example, an agent for treating intractable diseases. According to the present invention, there are provided a method for transplanting cells into a patient or subject, and a method for treating a disease in a patient or subject, which comprise the step of administering to the patient or subject a therapeutically effective amount of a cell population containing the somatic cells of the present invention.

[0090] According to the present invention, there is provided use of a cell population comprising somatic cells according to the present invention for the manufacture of a pharmaceutical composition.

[0091] The present invention provides use of a cell population comprising somatic cells according to the present invention for the manufacture of a cell therapeutic agent.

[0092] The pharmaceutical composition of the present invention may be a cell population containing somatic cells diluted with a pharmaceutically acceptable medium. The pharmaceutically acceptable medium is not particularly limited, as long as it is a solution that can be administered to a patient or subject. The pharmaceutically acceptable medium may be an infusion preparation, and examples thereof include, but are not limited to, water for injection, physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution, amino acid solution, initiation solution (solution No. 1), dehydration rehydration solution (solution No. 2), maintenance infusion solution (solution No. 3), postoperative recovery solution (solution No. 4), and Plasma-Lyte A (registered trademark).

[0093] In the present invention, a "patient or subject" typically refers to a human, but may also refer to other animals. Examples of other animals include, but are not limited to, mammals such as dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, and Japanese macaques), ferrets, rabbits, and rodents (mice, rats, gerbils, guinea pigs, and hamsters); and birds such as chickens and quails.

[0094] In the present invention, "treatment" includes, but is not limited to, significantly improving at least one of the following in a patient or subject: vital prognosis, functional prognosis, survival rate, weight loss, anemia, diarrhea, bloody stool, abdominal pain, fever, loss of appetite, malnutrition, vomiting, fatigue, rash, inflammation, ulcer, erosion, fistula, stenosis, intestinal obstruction, internal bleeding, rectal bleeding, convulsions, pain, decreased liver function, or blood test results.

[0095] The pharmaceutical composition of the invention may contain any component used in treating a patient or subject, including, but not limited to, salts (e.g., saline, Ringer's solution, Bicanate infusion), polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxide (DMSO), amino acids, and medium components (e.g., components contained in RPMI 1640 medium, etc.).

[0096] The pharmaceutical composition of the present invention may contain various additives for increasing storage stability, isotonicity, absorbability, and / or viscosity, such as emulsifiers, dispersing agents, buffers, preservatives, humectants, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters, etc. Examples of thickeners include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, etc. The concentration of the thickener depends on the thickener selected, but can be set arbitrarily within a concentration range that is safe when administered to a patient or subject and achieves the desired viscosity.

[0097] The pharmaceutical composition of the present invention may contain one or more other pharmaceutical agents in addition to somatic cells. Examples of such other pharmaceutical agents include, but are not limited to, antibiotics, albumin preparations, vitamin preparations, and anti-inflammatory agents. Examples of such anti-inflammatory agents include, but are not limited to, 5-aminosalicylic acid preparations, steroid preparations, immunosuppressants, and biological agents. Examples of such 5-aminosalicylic acid preparations include, but are not limited to, salazosulfapyridine and mesalazine. Examples of such steroid preparations include, but are not limited to, cortisone, prednisolone, and methylprednisolone. Examples of the immunosuppressants include tacrolimus, cyclosporine, methotrexate, azathiprine, 6-mercaptopurine, etc., while examples of the biological agents include, but are not limited to, infliximab, adalimumab, ustekinumab, secukinumab, ixekizumab, brodalumab, tocilizumab, vedolizumab, filgotinib, golimumab, certolizumab pegol, abatacept, etanercept, etc. In addition, the other pharmaceutical agents may be other types of cells.

[0098] The pH of the pharmaceutical composition of the present invention can be near neutral, for example, pH 5.5 or higher, pH 6.0 or higher, pH 6.5 or higher, or pH 7.0 or higher, and can be, but is not limited to, pH 10.5 or lower, pH 9.5 or lower, pH 8.5 or lower, or pH 8.0 or lower.

[0099] The cell concentration of the pharmaceutical composition of the present invention is a concentration of cells that, when administered to a patient or subject, can provide a therapeutic effect against a disease compared to a patient or subject not administered the composition. The specific cell concentration can be appropriately determined depending on the dosage form, administration method, purpose of use, and the age, weight, and symptoms of the patient or subject. The lower limit of the cell concentration of the pharmaceutical composition of the present invention is not particularly limited, but can be, for example, 1.0 × 10 5 pcs / mL or more, 1.0×10 6 pcs / mL or more, 1.2×10 6 pcs / mL or more, 1.4×10 6 pcs / mL or more, 1.6×10 6 pcs / mL or more, 1.8×10 6 pcs / mL or more, 2.0×10 6 pcs / mL or more, 3.0×10 6 pcs / mL or more, 4.0×10 6 pcs / mL or more, 5.0×10 6 pcs / mL or more, 6.0×10 6 pcs / mL or more, 7.0×10 6 pcs / mL or more, 8.0×10 6 pcs / mL or more, 9.0×10 6 pcs / mL or more, 9.5×10 6 cells / mL or more, or 1.0 x 10 7 The upper limit of the cell concentration in the pharmaceutical composition of the present invention is not particularly limited, but is, for example, 1.0 × 10 10 pcs / mL or less, 1.0×10 9 pcs / mL or less, 8.0×10 8 pcs / mL or less, 6.0×10 8 pcs / mL or less, 4.0×10 8 pcs / mL or less, 2.0×10 8 particles / mL or less, or 1.0 x 10 8 It is less than 1000 cells / mL.

[0100] One embodiment of the pharmaceutical composition of the present invention is a liquid preparation, preferably an injectable liquid preparation. As an injectable liquid preparation, for example, liquid preparations suitable for injection are known in International Publication WO2011 / 043136 and Japanese Patent Application Laid-Open No. 2013-256510.

[0101] The pharmaceutical composition of the present invention can also be in the form of an injectable liquid formulation as described in the above-mentioned literature. Furthermore, the liquid formulation may be a cell suspension or a liquid preparation in which cells are dispersed in the liquid. Furthermore, the form of the cells contained in the liquid formulation is not particularly limited, and may be, for example, a single cell or a cell aggregate.

[0102] When the pharmaceutical composition of the present invention is an injectable solution, the lower limit of the cell concentration in the injectable solution is 1.0 × 10 6 pcs / mL or more, 1.2×10 6 pcs / mL or more, 1.4×10 6 pcs / mL or more, 1.6×10 6 pcs / mL or more, 1.8×10 6 pcs / mL or more, 2.0×10 6 pcs / mL or more, 3.0×10 6 pcs / mL or more, 4.0×10 6 pcs / mL or more, 5.0×10 6 pcs / mL or more, 6.0×10 6 pcs / mL or more, 7.0×10 6 pcs / mL or more, 8.0×10 6 pcs / mL or more, 9.0×10 6 pcs / mL or more, 9.5×10 6 cells / mL or more, or 1.0 x 10 7 The upper limit of the cell concentration in the injectable solution is preferably 1.0 × 10 cells / mL or more, from the viewpoint of facilitating the preparation and administration of the injectable solution. 9 pcs / mL or less, 8.0×10 8 pcs / mL or less, 6.0×10 8 pcs / mL or less, 4.0×10 8 pcs / mL or less, 2.0×10 8 particles / mL or less, or 1.0 x 10 8It is preferable that the number is less than 1 / mL.

[0103] According to another embodiment of the present invention, the pharmaceutical composition of the present invention may be a transplant preparation having a cell mass or sheet-like structure. Examples of transplant preparations having a cell mass structure include a transplant preparation containing a cell mass obtained by adhering isolated cells with an adhesive (e.g., fibrinogen), as disclosed in International Publication WO 2017 / 126549. Examples of transplant preparations having a sheet-like structure include cell sheets obtained by culturing cells using a temperature-responsive culture dish (e.g., UpCell® (CellSeed)), laminates of sheet-like cell cultures and fibrin gels, and cell-coated sheets in which a cell suspension is applied to a sheet-like substrate, as disclosed in International Publication WO 2006 / 080434 and Japanese Patent Application Laid-Open No. 2016-52272. The pharmaceutical composition of the present invention can also be prepared into various transplant preparations having a cell mass or sheet-like structure, for example, by using the methods described in the above-mentioned documents.

[0104] According to another embodiment of the present invention, the pharmaceutical composition of the present invention may be a gel formulation in which cells are mixed with any gel. For example, a cell therapy agent containing a cell-hydrogel composition as an active ingredient is known in JP-A-2017-529362. The pharmaceutical composition of the present invention may also be a gel formulation described in the above document.

[0105] The method of administering the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include subcutaneous injection, intradermal injection, intramuscular injection, intralymph node injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, direct injection into a local area, direct application, or direct implantation into a local area. According to one aspect of the present invention, the injectable solution is filled into a syringe and administered via an injection needle or catheter intravenously, intraarterially, intramyocardially, intraarterially, intrahepatic artery, intramuscularly, epidurally, gingivally, intraventricularly, subcutaneously, intradermally, intraperitoneally, or into the portal vein, but is not limited thereto. Methods for administering pharmaceutical compositions include intravenous injection, intravenous drip injection, local direct injection, and local direct implantation, as described in, for example, JP 2015-61520 A, Onken JE, et al. American College of Gastroenterology Conference 2006 Las Vegas, NV, Abstract 121, and Garcia-Olmo D, et al. Dis Colon Rectum 2005;48:1416-23. The pharmaceutical composition of the present invention can also be administered by the various methods described in the above documents.

[0106] The administration frequency of the pharmaceutical composition of the present invention is a frequency that can provide a therapeutic effect against a disease when administered to a patient or subject. The specific administration frequency can be appropriately determined depending on the dosage form, administration method, purpose of use, and the age, weight, and symptoms of the patient or subject, and is, for example, once every 4 weeks, once every 3 weeks, once every 2 weeks, once every week, twice every week, three times every week, four times every week, five times every week, six times every week, or seven times every week.

[0107] The administration period of the pharmaceutical composition of the present invention is a period that can provide a therapeutic effect against a disease when administered to a patient or subject. The specific administration period can be appropriately determined depending on the dosage form, administration method, purpose of use, and the age, weight, and symptoms of the patient or subject, and is, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0108] The timing of administering the pharmaceutical composition of the present invention to a patient or subject is not particularly limited, and examples include immediately after onset, within n days (n is an integer of 1 or more) after onset, immediately after diagnosis, within n days (n is an integer of 1 or more) after diagnosis, before remission, during remission, after remission, before relapse, during relapse, and after relapse. [Example]

[0109] The present invention will be described in more detail by the following examples, but these are merely illustrative and do not limit the present invention in any way.

[0110] Example 1 <Maintenance culture of pluripotent stem cells> The iPS cell line RPChiPS771 (ReproCell) was maintained in undifferentiated state by culturing on SNL feeder cells treated with mitomycin-C (WAKO) in iPS cell medium (DMEM / HAM'S F12 (WAKO) containing 20% ​​Knockout Serum Replacement (KSR; GIBCO), 1X Non-Essential Amino Acids (NEAA; WAKO), 55 μmol / L 2-Mercaptethanol (2-ME; GIBCO), 7.5 ng / mL recombinant human Fibroblast Growth Factor (FGF2; PEPROTECH), and 0.5X Penicillin and Streptomycin (PS; WAKO). Alternatively, the iPS cell line was cultured on vitronectin (GIBCO)-coated plates in ESSENTIAL 8 (WAKO) containing 1X Penicillin, Streptomycin, and Amphotericin B (WAKO). TM The cells were cultured in a medium (E8; GIBCO) to maintain undifferentiated state. Only at the time of seeding, Y27632 (WAKO) was added to a final concentration of 10 μM.

[0111] <Step of raising the temperature of the medium to a temperature at which pluripotent stem cells can grow> ESSENTIAL8 (500mL) stored at 4℃TM The medium was poured into a 5 L culture tank until the volume reached 3.5 L. Next, the temperature of the medium was increased while controlling the temperature with a temperature sensor attached to the tank so that the temperature of the medium in the 5 L culture tank was around 37°C.

[0112] <Suspension culture of pluripotent stem cells> The iPS cells cultured in the maintenance culture were treated with Accutase (Thermo Fisher Scientific) for about 5 minutes to detach and disperse into single cells. The dispersed cells were placed in ESSENTIAL 8 containing BSA (WAKO) at a final concentration of 5 mg / mL. TM After suspending in the medium, a portion was taken and stained with trypan blue to measure the cell count. Based on the measured cell count, 2 × 10 cells per mL were obtained. 5 Essentially, the cell suspension should contain 8 cells. TM After confirming that the temperature of the medium in the 5 L culture tank had reached approximately 37°C, the cell suspension was seeded in the culture tank and cultured under stirring for 5 days in an environment of 5% CO2 and 37°C. After the culture, TrypLE TM The cell aggregates were suspended in Select (Life Technologies) and treated for 10 minutes in an environment of 5% CO2 and 37°C. After treatment, the cell aggregates were dispersed into single cells using a micropipette, and the number of cells obtained after culture was measured by trypan blue staining. As a result of measuring the cell number, 7 × 10 9 We were able to obtain pluripotent stem cells (cells / lot).

[0113] (Comparative Example 1) <The process of repeatedly injecting the culture medium with the temperature increased into the culture tank> ESSENTIAL8 (500mL) stored at 4℃ TM The medium was immersed in warm water at 42°C, and the temperature of the medium in the medium container was raised to around 37°C, after which 500 mL of the medium was poured into a 5 L culture tank that had been separately heated to around 37°C. This pouring operation was repeated six times until the amount of medium in the 5 L culture tank reached 3.5 L.

[0114] <Suspension culture of pluripotent stem cells> As in Example 1, a cell suspension (2 × 10 5 After confirming that the temperature of the medium in the 5 L culture tank was maintained at around 37°C, the cell suspension was seeded in the culture tank and agitated culture was carried out for 5 days while maintaining an environment of 5% CO2 and 37°C. The number of cells after culture was measured in the same manner as in Example 1, and was found to be 1 x 10 9 Although pluripotent stem cells (cells / lot) could be obtained, it was revealed that the proliferation rate of the pluripotent stem cells was significantly reduced compared to Example 1.

[0115] <Analysis of medium components> To investigate the cause of the decreased proliferation of the pluripotent stem cells, a portion of the medium in the 5 L culture tank of Example 1 and Comparative Example 1 was collected, and the total protein content in each medium was measured using a protein assay bicinchoninic acid kit (Nacalai Tesque, Inc.). As a result, it was found that the total protein content in the medium of Comparative Example 1 was lower than that of Example 1. Furthermore, to identify the protein that was decreased in Comparative Example 1, a portion of the medium in the 5 L culture tank of Example 1 and Comparative Example 1 was collected, and the FGF2 content in each medium was measured using FGF2 ELISA kits (Biolegend, Inc.). As a result, it was found that FGF2 was detected in the medium of Example 1, but that FGF2 was below the detection limit in the medium of Comparative Example 1. From this, it was found that the decreased proliferation of pluripotent stem cells in Comparative Example 1 was caused by instability of the medium components, specifically, a significant decrease in growth factors (especially FGF2). Therefore, it was confirmed that pluripotent stem cells can be cultured in large quantities by adding a step of raising the temperature of the medium in the culture tank to a temperature at which pluripotent stem cells can proliferate before culture of iPS cells in suspension, as described in Example 1.

[0116] <Undifferentiated state of pluripotent stem cells> To examine the undifferentiated state of the pluripotent stem cells, a portion of the medium in the 5 L culture tanks of Example 1 and Comparative Example 1 was collected. Each cell suspension was centrifuged at 300 g for 5 minutes, the supernatant removed, and the cells were washed with phosphate-buffered saline (PBS). The cells were then fixed with 4% PFA (paraformaldehyde) at room temperature for 20 minutes, washed three times with PBS, and permeabilized overnight with cold methanol at -20°C. After washing three times with PBS, the cells were blocked with 3% FBS (fetal bovine serum) / PBS and stained with fluorescently labeled anti-OCT4 (Cat. No. 653703, Biolegend) for 1 hour at 4°C. After washing once with 3% FBS / PBS, the pluripotent stem cells were passed through a cell strainer and analyzed using a FACS Verse. Analysis revealed that the OCT4 positivity rate of the pluripotent stem cells obtained by the method of Example 1 was 99%. In contrast, the OCT4 positivity rate of the pluripotent stem cells obtained by the method of Comparative Example 1 was 55%. This confirmed that the undifferentiated state of pluripotent stem cells can be efficiently maintained by increasing the temperature of the medium in the culture tank to a temperature at which the pluripotent stem cells can proliferate before culturing the iPS cells in suspension, as described in Example 1.

[0117] (Comparative Example 2) We analyzed the effect on the proliferation of pluripotent stem cells in suspension culture when the step of raising the temperature of the medium in the culture tank to a temperature at which pluripotent stem cells can proliferate, as described in Example 1, is not performed.

[0118] <Suspension culture of pluripotent stem cells> Maintenance culture and suspension culture of pluripotent stem cells were carried out in the same manner as in Example 1, except that the step of raising the temperature of the medium in the culture tank to a temperature at which pluripotent stem cells can proliferate was not carried out. Specifically, 500 mL of ESSENTIAL 8, stored at a medium temperature of 4°C, was used. TM The medium was poured into a 5 L culture tank until the volume reached 3.5 L. Next, a cell suspension of iPS cells was seeded into the culture tank, and then agitation culture was carried out for 5 days in a 5% CO2 environment while the temperature of the medium in the culture tank was heated to around 37°C. As a result, 9 x 10 8Only pluripotent stem cells (cells / lot) could be obtained.

[0119] Example 2 <Consideration of culture scale> Suspension culture of iPS cells was carried out in the same manner as in Example 1, except that the culture volume in the culture tank was changed to 5 L, 10 L, 20 L, 30 L, 40 L, or 50 L. The number of iPS cells after the completion of culture was investigated in each case, and the results are shown in Table 1. As a control test, the effect of culture scale under the production conditions described in Comparative Example 1 was also tested.

[0120] [Table 1]

[0121] As shown in Table 1, it was confirmed that pluripotent stem cells can be cultured in large quantities by increasing the temperature of the culture medium in the culture tank to a temperature at which pluripotent stem cells can proliferate before suspension culturing iPS cells. In particular, the culture became more efficient as the amount of medium increased, and the proliferation rate of pluripotent stem cells improved 5 to 20 times compared to the control test. Furthermore, the use of the present invention revealed a correlation between the amount of medium and the proliferation rate of pluripotent stem cells.

[0122] Example 3 <Effect of medium temperature before heating> The initial medium temperature in the culture tank was changed to -20°C, -15°C, -10°C, 0°C, 10°C, and 15°C, and the volume of medium in the culture tank was changed to 10 L, but suspension culture of iPS cells was performed in the same manner as in Example 1. The cell count of iPS cells after the end of culture in each case was investigated, and the results are shown in Table 2.

[0123] As a control test, the effect of the initial medium temperature under the production conditions described in Comparative Example 2 was also tested.

[0124] [Table 2]

[0125] As shown in Table 2, it was confirmed that pluripotent stem cells can be cultured in large quantities by including a step of raising the temperature of the culture medium in the culture tank to a temperature at which pluripotent stem cells can proliferate before suspension culturing iPS cells. In particular, the more the temperature of the liquid medium was raised, the more efficient the culture became, and the proliferation rate of pluripotent stem cells was improved by 2 to 20 times compared to the control test. Furthermore, the use of the present invention revealed a correlation between changes in the temperature of the liquid medium and the proliferation rate of pluripotent stem cells.

[0126] Example 4 <Consideration of growth factor addition conditions> DMEM / F12 medium, stored at 4°C, was supplemented with 64 mg / L magnesium ascorbate 2-phosphate and 543 mg / L sodium bicarbonate, and the medium was poured into a 5 L culture tank until the volume reached 3.5 L. The temperature of the medium in the 5 L culture tank was then increased while controlling the temperature using a temperature sensor attached to the tank to maintain the temperature at approximately 37°C. After confirming that the temperature of the medium in the 5 L tank was approximately 37°C, growth factors were added to the medium in the tank to achieve a concentration of 1% ITS (insulin-transferrin-selenium; Life Technologies), 100 μg / L FGF2, and 2 μg / L TGF-β1. iPS cells were cultured in the same manner as in Example 1 for maintaining undifferentiated cells at a concentration of 2 × 10 per mL. 5 The cells were seeded in the medium in the tank so that they contained 1.4 × 10 cells, and agitated culture was performed for 5 days while maintaining an environment of 5% CO2 and 37°C. After culture, the cell aggregates were suspended in TrypLE select and treated for 10 minutes in an environment of 5% CO2 and 37°C. After treatment, the cell aggregates were dispersed into single cells using a micropipette, and the number of cells obtained after culture was measured by trypan blue staining. The cell number measurement result was 1.4 × 10 10pluripotent stem cells (cells / lot) were obtained. These results demonstrate that by not adding growth factors during the process of raising the temperature of the medium in the culture tank to a temperature at which the pluripotent stem cells can proliferate, but adding growth factors during the process of suspension culture of the pluripotent stem cells, the proliferation rate of the pluripotent stem cells is significantly improved, nearly two-fold compared to the proliferation rate in Example 1.

[0127] Example 5 <Induction of differentiation into somatic cells (pancreatic β cells)> The iPS cell line RPChiPS771 (ReproCELL) is maintained in undifferentiated state by culturing on SNL feeder cells treated with Mitomycin-C (WAKO) in iPS cell medium (DMEM / HAM'S F12 (WAKO) containing 20% ​​Knockout Serum Replacement (KSR; GIBCO), 1X Non-Essential Amino Acids (NEAA; WAKO), 55 μmol / L 2-Mercaptethanol (2-ME; GIBCO), 7.5 ng / mL FGF2, and 0.5X Penicillin and Streptomycin (PS; WAKO). The medium is stored at 4°C in a 500 mL bottle of ESSENTIAL 8. TM The medium is poured into a 5L culture tank until the volume reaches 3.5L, and the temperature of the medium is raised while controlling the temperature using a temperature sensor attached to the tank so that the temperature in the 5L culture tank is around 37°C. The iPS cells cultured in the maintenance culture are treated with Accutase (Thermo Fisher Scientific) for about 5 minutes to detach them and disperse them into single cells. The dispersed cells are placed in ESSENTIAL 8 containing BSA (WAKO) at a final concentration of 5mg / mL. TM After suspending in the medium, a portion of the suspension is taken and stained with trypan blue to measure the cell count. Based on the measured cell count, 2 × 10 cells per mL were obtained. 5 Essentially, the cell suspension should contain 8 cells. TMThe iPS cells are prepared in a medium. After confirming that the temperature of the medium in the 5 L culture tank has reached approximately 37°C, the cell suspension is seeded into the culture tank and cultured under agitation for 5 days in a 5% CO2, 37°C environment. The iPS cell population obtained by the culture is cultured in a dish-like state for the first 2 days in RPMI 1640 containing 0.5% bovine serum albumin, 0.4x PS, 1 mmol / L sodium pyruvate, 1x NEAA, 80 ng / mL recombinant human activin A, 50 ng / mL FGF2, 20 ng / mL recombinant bone morphogenetic protein 4, and 3 μmol / L CHIR99021. On day 3, the CHIR99021 is removed from the medium and the cells are cultured in a dish-like state. On day 4, the cells are cultured in a dish-like state in a medium supplemented with 1% (vol / vol) KSR for an additional 1 day to induce differentiation of the pluripotent stem cells into endoderm cells. The cells were then cultured for 2 days in RPMI1640 containing 0.5% BSA, 1 mmol / L sodium pyruvate, 1x NEAA, 0.4x PS, 50 ng / mL FGF2, 50 ng / mL recombinant human FGF7 (PEPROTECH), 2% B27 supplement (GIBCO), 0.67 μmol / L EC23 (SANTA CRUZ), 1 μmol / L dorsomorphin (WAKO), 10 μmol / L SB431542 (WAKO), and 0.25 mol / L SANT1 (WAKO) to induce differentiation of endoderm cells into primitive gut cells (PGT). Next, the cells are cultured for 4 days in DMEM-high glucose (WAKO) containing 0.4x PS, 1x NEAA, 50ng / mL FGF2, 2% B27, 0.67μmol / L EC23, 1μmol / L dorsomorphin, 10μmol / L SB431542, and 0.25μmol / L SANT1 to induce differentiation of primitive gut cells (PGT) into posterior foregut cells (PFG: Pancreatic Progenitor).The cells were then cultured for 3 days in DMEM-high glucose containing 0.4x PS, 1x NEAA, 50ng / mL recombinant human FGF10 (Peprotech), 2% B27, 0.5μmol / L EC23, 1μmol / L dorsomorphin, 0.25μmol / L SANT1, 5μmol / L Alk5 inhibitor II (Biovision), and 0.3μmol / L indolactam V (ILV; Cayman) to induce differentiation of posterior foregut cells (PFG) into pancreatic progenitor cells (PP). Next, the cells are cultured for 3 days in Advanced-DMEM (GIBCO) containing 0.4xPS, 2mmol / L L-glutamine, 2% B27, 0.2μmol / L EC23, 1μmol / L dorsomorphin, 0.25μmol / L SANT1, 5μmol / L Alk5 inhibitor II, and 50ng / mL Exendin4 (SIGMA) to induce differentiation of pancreatic progenitor cells (PP) into pancreatic endocrine progenitor cells (EP). The cells were then cultured for 6 days in Advanced-DMEM containing 0.4xPS, 2 mmol / L L-glutamine, 2% B27, 10 ng / mL BMP4, 10 ng / mL FGF2, 50 ng / mL recombinant human hepatocyte growth factor (HGF; PEPROTECH), 50 ng / mL insulin-like growth factor 1 (IGF1; PEPROTECH), 5 μmol / L Alk5 inhibitor II, 50 ng / mL Exendin4, 5 mmol / L nicotinamide (SIGMA), and 5 μmol / L forskolin (WAKO). The differentiation of pancreatic endocrine progenitor cells (EPs) into pancreatic β cells was induced. While the above differentiation induction method is merely one embodiment, it is possible to induce differentiation of pluripotent stem cells into pancreatic β cells, ultimately resulting in the production of somatic cells.

Claims

1. A method for producing pluripotent stem cells, comprising the steps of: (a) and (b) (a) filling a culture vessel with a liquid medium containing FGF2, and then increasing the temperature of the liquid medium in the culture vessel to a temperature at which pluripotent stem cells can grow; and (b) seeding pluripotent stem cells in the liquid medium in the culture vessel and culturing them in suspension; Including, The volume of the liquid medium used is 1 L or more per culture vessel, In the step (a), the temperature of the liquid medium in the culture vessel before increasing the temperature is −20° C. or higher and 18° C. or lower; In the step (a), the temperature at which the pluripotent stem cells can grow is 30°C or higher and 40°C or lower. Manufacturing method.

2. The method according to claim 1, wherein the temperature of the liquid medium is increased by at least 20°C in step (a).

3. The method according to claim 1 or 2, wherein in the step (a), the temperature of the liquid medium in the culture vessel is increased while stirring the liquid medium.

4. The method according to any one of claims 1 to 3, wherein no growth factor is added in step (a).

5. The method according to any one of claims 1 to 4, wherein a growth factor is added in step (b).

6. The method according to any one of claims 1 to 5, wherein the liquid medium contains at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate.

7. The method according to any one of claims 1 to 6, wherein the pluripotent stem cells are embryonic stem cells or iPS cells (induced pluripotent stem cells).

8. The method according to claim 4 or 5, wherein the growth factor is at least one selected from the group consisting of FGF2 and TGF-β1.

9. A method for producing somatic cells, comprising the steps of: (a) producing a somatic cell; (a) filling a culture vessel with a liquid medium containing FGF2, and then increasing the temperature of the liquid medium in the culture vessel to a temperature at which pluripotent stem cells can grow; (b) seeding pluripotent stem cells in the liquid medium in the culture vessel and culturing them in suspension; and (c) culturing the pluripotent stem cells obtained in step (b) in the presence of a differentiation-inducing factor to induce differentiation; Including, The volume of the liquid medium used is 1 L or more per culture vessel, In the step (a), the temperature of the liquid medium in the culture vessel before increasing the temperature is −20° C. or higher and 18° C. or lower; In the step (a), the temperature at which the pluripotent stem cells can grow is 30°C or higher and 40°C or lower. Manufacturing method.

10. The method according to claim 9, wherein the temperature of the liquid medium is increased by at least 20°C in the step (a).

11. The method according to claim 9 or 10, wherein in the step (a), the temperature of the liquid medium in the culture vessel is increased while stirring the liquid medium.

12. The method according to any one of claims 9 to 11, wherein no growth factor is added in step (a).

13. The method according to any one of claims 9 to 12, wherein a growth factor is added in step (b).

14. The production method according to any one of claims 9 to 13, wherein the somatic cells are at least one selected from the group consisting of cardiomyocytes, skeletal muscle cells, nerve cells, megakaryocytes, hematopoietic stem cells, airway epithelial cells, germ cells, dendritic cells, eosinophils, mast cells, chondrocytes, T cells, erythropoietin-producing cells, intestinal epithelium, pancreatic cells, liver cells, alveolar epithelial cells, and kidney cells.

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