Culture medium composition and culturing method

Abstract

To provide a culture medium composition capable of being used for culturing mammalian embryonic stem cells, the composition being free from serum and pituitary extract and also free from animal-derived proteins including albumin derived therefrom, and also to provide a culturing method using the culture medium composition.SOLUTION: A culture medium composition for three-dimensional culture of mammalian pluripotent stem cells, the composition containing (A1) activin, (A2) a TGFβ subfamily protein, (A3) a GSK3 inhibitor, (A4) a Wnt inhibitor, and (A5) polyvinyl alcohol (PVA), and containing none of (B) serum and pituitary extract and albumin derived therefrom and other animal-derived proteins. A culturing method including a step of culturing mammalian pluripotent stem cells in the culture medium composition under feeder-cell-free three-dimensional culture conditions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a medium composition for culturing mammalian pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells, etc.), and a method for culturing mammalian pluripotent stem cells using the same. [Background technology]

[0002] In recent years, the demand for meat has increased dramatically due to global population and economic growth, and research and development of cultured meat is underway as one way to meet this demand. Cultured meat can be produced by artificially culturing muscle cells and fat cells contained in meat from cows, pigs, etc., and giving them a three-dimensional structure resembling muscle tissue and / or fat tissue. The muscle cells and fat cells required for this can be produced from stem cells that have the ability to differentiate into these cells, such as embryonic stem cells (ES cells) derived from cows, etc.

[0003] Methods for producing and culturing pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, derived from bovine or other mammalian animals, and medium compositions used for these methods are known, for example, from the following patent documents:

[0004] Patent Document 1 describes a method for producing embryonic stem cells from ungulates (such as cattle), which involves culturing ungulate blastocysts and pluripotent stem cells established therefrom in a medium (e.g., serum-free medium) containing inactivated feeder cells, FGF2, and a Wnt inhibitor, and which may further contain a ROCK inhibitor, activin A (a SMAD activator), and the like. Patent Document 1 also describes that this method produces stable ungulate embryonic stem cells that proliferate for long periods while maintaining stable cell morphology and a normal karyotype, possess a transcriptome and epigenome indicative of pluripotency, and are capable of forming teratomas containing three germ layers. The medium "CTFR" used in the examples of Patent Document 1 (see page 40, "Derivation and culture of CTFR-bESCs") is similar to the medium "MTeSR" (but does not contain FGF2 or TGFβ), but contains bovine serum albumin (BSA).

[0005] Patent Document 2 describes a method for producing expanded potential stem cells (EPSCs), which involves culturing pluripotent cells in a medium containing two or more inhibitors selected from a Ras-ERK inhibitor, an SKF (Src Kinase family) inhibitor, a GSK3 inhibitor, and a Wnt inhibitor, and may further contain a JNK (Jun N-Terminal Kinase) inhibitor, a p38 inhibitor, a ROCK inhibitor, LIF, activin (a SMAD activator), or the like. EPSCs are said to resemble naive embryonic stem cells and are capable of differentiating into three germ layers, including the placenta. Patent Document 2 describes the production of EPSCs derived from various mammals, including humans and non-humans. However, the examples disclosed only show EPSCs produced from mouse-, human-, or porcine-derived embryonic stem cells or induced pluripotent stem cells, and does not disclose bovine EPSCs. The serum-free medium "N2B27" used in the examples of Patent Document 2 (see page 38, etc.) (a 1:1 mixture of N2 medium, which is DMEM / F12 medium with N2 supplements added, and B27 medium, which is neurobasal medium with B27 supplements added) is a product that generally contains biological components such as albumin.

[0006] Patent Document 3 describes a method for producing aggregates of non-genetically modified, non-human animal-derived pluripotent stem cells (PSCs), the method comprising: (a) seeding at least one PSC in a growth medium to form a seeded suspension culture, the growth medium being a serum-free liquid medium containing a combination of the growth factor bFGF and (i) at least one additional growth factor and (ii) at least one small molecule selected from the group consisting of an inhibitor of the Wnt-β-catenin signaling pathway, CHIR 99021 (C22H18Cl2N8), PD 0325901 (C16H14F3IN2O4), and A 83-01 (C25H19N5S); and (b) growing the suspension culture under conditions that allow aggregate formation and aggregate growth, thereby forming homogeneous aggregates of the PSCs. In Patent Document 3, proteins of the transforming growth factor beta (TGF-β) superfamily (TGF-β-1, TGFβ-3, activin-A, and any combination thereof) are exemplified as the "(i) at least one additional growth factor," but the examples do not disclose any embodiments using such proteins of the TGF-β superfamily (especially a specific combination of multiple types). Furthermore, the serum-free medium "mTeSR1 (registered trademark) (STEMCELL Technologies Inc. Canada)" used in the examples of Patent Document 3 (see paragraph 0277) is a product that may contain components processed or derived from blood, plasma, or serum, such as albumin, or biological components other than serum, plasma, or hemolymph (such as bovine pituitary extract).

[0007] Non-Patent Document 1 describes that "ON2 / AscleStem PSC medium" (ON2) is a suitable xeno-free medium for human induced pluripotent stem cells (iPS cells). ON2 is a DMEM / F-12 mixed medium containing various amino acids, vitamins, and inorganic salts, and is supplemented with L-ascorbic acid, selenate, insulin, and transferrin, as well as FGF2, albumin, activin A, LIF (leukemia inhibitory factor), and chondroitin sulfate. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2019 / 140260 [Patent Document 2] WO2016 / 079146 [Patent Document 3] Patent Publication No. 2022-535192 (US2022 / 0220439) [Non-patent literature]

[0009] [Non-Patent Document 1] Hua et al. Stem Cell Research & Therapy (2022) 13:223https: / / doi.org / 10.1186 / s13287-022-02879-z Summary of the Invention [Problem to be solved by the invention]

[0010] Conventional methods for culturing pluripotent stem cells, such as embryonic stem cells derived from bovine or other sources, have used medium compositions supplemented with, or potentially containing, animal-derived proteins such as serum, pituitary extract, or albumin contained therein (e.g., bovine serum albumin (BSA)). However, for reasons of safety and consumer preference, it is desirable that pluripotent stem cells used to produce cultured meat for consumption be recovered from a medium composition that is substantially free of such animal-derived components.

[0011] The present invention addresses the need to provide a medium composition that can be used to culture mammalian embryonic stem cells, preferably to produce mammalian pluripotent stem cells (embryonic stem cells, etc.) useful for producing cultured meat from cattle, pigs, etc., specifically to establish and maintain cell lines, and that does not contain serum, pituitary extract, or animal-derived proteins such as albumin derived therefrom, as well as a culture method using such a medium composition. [Means for solving the problem]

[0012] The present inventors discovered that a medium composition containing at least activin, a TGFβ subfamily protein, a GSK3 inhibitor, a Wnt inhibitor, and polyvinyl alcohol (PVA) in effective amounts, and optionally further containing an Src inhibitor, a STAT3 activator, and a ROCK inhibitor, can culture bovine and other embryonic stem cells for long periods (e.g., 10 or more passages) even without the addition of serum, pituitary extract, or animal-derived proteins contained therein, such as albumin, to the medium composition, thereby completing the present invention. Note that the conventional media prepared using mTeSR1 or N2 / B27 in the inventions described in the above-mentioned patent documents contain animal-derived proteins such as albumin, but do not disclose that the inclusion of animal-derived proteins is unnecessary by combining specific components, as in the present invention. Activin and TGFβ subfamily proteins are both proteins belonging to the TGFβ superfamily, known as "SMAD activators." One of the features of the present invention is the use of a combination of activin and a TGFβ subfamily protein as essential components.

[0013] That is, in one aspect, the present invention provides the following medium composition and culture method. [1] (A1) activin, (A2) TGFβ subfamily proteins, (A3) GSK3 inhibitor, (A4) Wnt inhibitors, and (A5) Polyvinyl alcohol (PVA) Contains (B) Serum and pituitary extracts, and albumin and other animal-derived proteins derived therefrom. Does not contain A medium composition for three-dimensional culture of mammalian pluripotent stem cells. [2] (A6) The medium composition according to Item 1, further containing an Src inhibitor. [3] (A7) The medium composition according to Item 1, further comprising a STAT3 activator. [4] (A8) The medium composition according to Item 1, further containing a ROCK inhibitor. [5] Item 1. The medium composition according to Item 1, wherein the mammal is an artiodactyl. [6] Item 6. The medium composition according to Item 5, wherein the artiodactyla is cattle or sheep. [7] Item 1. The medium composition according to Item 1, wherein the pluripotent stem cells are stem cells derived from an embryo, including embryonic stem cells. [8] A culture method comprising the step of culturing mammalian pluripotent stem cells in the medium composition according to any one of Items 1 to 7 under feeder cell-free three-dimensional culture conditions. [9] Item 9. The culture method according to Item 8, wherein the culture step is carried out on a plate-shaped culture substrate having a plurality of depressions forming compartments in which the cultured organisms are cultured and banks interposed between adjacent depressions on the upper surface thereof, and the adjacent banks and depressions are continuous curved surfaces. [Effects of the Invention]

[0014] In the present invention, by incorporating the specific combination of ingredients described above into a medium composition, it is possible to obtain a medium composition that is capable of efficiently culturing embryonic stem cells from bovine, ovine, and other mammals, even without containing animal-derived proteins such as albumin. The pluripotent stem cells obtained using such a medium composition of the present invention are desirable as pluripotent stem cells for producing cultured meat for consumption. [Brief explanation of the drawings]

[0015] [Figure 1] Optical microscope images of bovine embryonic stem cells obtained by step [1] of the Example. [A] When medium 1 was used, [B] When medium 2 was used. [Figure 2] Immunostained images showing desmin expression in skeletal muscle cells obtained by step [2] of the Example. [A] DAPI-stained image, [B] desmin-stained image, [C] DAPI / desmin-stained image. DETAILED DESCRIPTION OF THE INVENTION

[0016] In this specification (including the disclosure in the Examples), when multiple numerical ranges are exemplified for a certain item (for example, the concentration of a specific component), it is possible to define a new numerical range by combining the upper limit of one numerical range with the lower limit of another numerical range, and embodiments resulting from the definition of such new numerical ranges are also encompassed by the present invention.

[0017] -Culture composition- The medium composition of the present invention comprises: (A1) activin, (A2) TGFβ family protein, (A3) GSK3 inhibitor, (A4) Wnt inhibitor, and (A5) polyvinyl alcohol (PVA), and optionally (A6) Src inhibitor, (A7) ROCK inhibitor, and (A8) STAT3 activator, (B) free of serum and pituitary extracts, and albumin and other animal-derived proteins derived therefrom; A medium composition for three-dimensional culture of mammalian pluripotent stem cells.

[0018] That is, the medium composition of the present invention contains the above-mentioned essential components (A1) to (A5) in effective amounts, and may further contain the above-mentioned optional components (A6) to (A8) in effective amounts, but does not contain the above-mentioned component (B) (it is not intentionally added, or a medium in which component (B) is pre-blended is not used).

[0019] "Mammals" include humans and non-human mammals. Non-human mammals include, for example, Primates such as chimpanzees, gorillas (Hominidae), orangutans (Pongoidae), and monkeys (Cercopithecidae); Rodents such as mice and rats (Muridae); Artiodactyla such as pigs (Suidae), cattle, sheep, and goats (Bovidae); Perissodactyla such as horses (Equidae); and Carnivora such as dogs (Canidae) and cats (Felidae). In the present invention, "mammals" are preferably mammals of the Order Artiodactyla, such as pigs (Suidae), cattle, sheep, and goats (Bovidae), more preferably cattle and sheep, and particularly preferably cattle.

[0020] As a term well known to those skilled in the art, "pluripotent stem cells" refers to cells that have the ability to self-proliferate and pluripotency (the ability to differentiate into all three germ layers: endoderm, mesoderm, and ectoderm). Pluripotent stem cells also include cells that can be harvested from other living organisms, such as embryonic stem cells (ES cells, etc.), induced pluripotent stem cells (iPS cells, etc.), and embryonic germ stem cells (EG cells). Pluripotent stem cells may be in a naive state or in a primed state (such as common ES cells and iPS cells). Whether a cell is a "pluripotent stem cell" can be confirmed by known techniques, for example, by determining whether the cell has at least one of the characteristics of a "pluripotent stem cell," such as the ability to self-proliferate (colony-forming ability) or the ability to differentiate into multiple germ layers.

[0021] (A1) Activin Activin itself is a component known in the technical field related to cell culture (also known as a type of SMAD activator), and a similar component can be used in the present invention. Activin includes activin A, activin AB, and activin B, and for example, activin A is a preferred activin in the present invention.

[0022] The type and concentration of activin can be appropriately determined depending on the desired level of the effects of the present invention, taking into consideration the types and concentrations of other components contained in the medium composition of the present invention. In one embodiment of the present invention, the medium composition of the present invention contains activin A as the activin, and its concentration in the medium composition is usually 0.1 to 200 ng / mL, preferably 1 to 100 ng / mL. When another type of activin is used, its concentration range can be similar to the above-mentioned concentration range for activin A.

[0023] (A2) TGFβ subfamily proteins TGFβ subfamily proteins are known in the art of cell culture (and are also known as a type of SMAD activator), and similar components can be used in the present invention. TGFβ subfamily proteins include TGFβ1, TGFβ2, and TGFβ3, with TGFβ3 being a preferred TGFβ subfamily protein in the present invention.

[0024] The type and concentration of the TGFβ subfamily protein can be appropriately determined depending on the desired level of the effects of the present invention, taking into consideration the types and concentrations of other components contained in the medium composition of the present invention. In one embodiment of the present invention, the medium composition of the present invention contains TGFβ3 as the TGFβ subfamily protein, and its concentration in the medium composition is usually 0.1 to 50 ng / mL, preferably 1 to 10 ng / mL. When other types of TGFβ subfamily proteins are used, their concentration range can also be set to the same as the above-mentioned concentration range for TGFβ3.

[0025] (A3) GSK3 inhibitor "GSK (Glycogen synthase kinase) 3 inhibitors" are known in the art related to cell culture, and similar compounds can be used in the present invention. GSK3 inhibitors include agents that inhibit the function of GSK-3α and GSK-3β (GSK-3α / β inhibitors) and agents that selectively inhibit the function of GSK-3β (GSK-3β inhibitors), either of which may be used in the present invention. Specific examples of GSK3 inhibitors include, but are not limited to, CHIR99021 (CAS No. 252917-06-9), CHIR98014 (CAS No. 252935-94-7), BIO (CAS No. 667463-62-9), SB216763 (CAS No. 280744-09-4), LY2090314 (CAS No. 603288-22-8), and LiCl (lithium chloride).

[0026] The type and concentration of the GSK3 inhibitor can be appropriately determined depending on the desired level of the effect of the present invention, taking into consideration the types and concentrations of other components contained in the medium composition of the present invention. In one embodiment of the present invention, the medium composition of the present invention contains CHIR99021 as the GSK3 inhibitor, and its concentration in the medium composition is usually 0.1 to 20 μM, preferably 0.5 to 10 μM. When other types of GSK3 inhibitors are used, their concentration range can also be set to be similar to the above-mentioned concentration range for CHIR99021.

[0027] (A4) Wnt inhibitor "Wnt inhibitors" are known compounds in the art related to cell culture, and similar compounds can be used in the present invention. Wnt inhibitors may be drugs known as "tankyrase inhibitors," "axin activators," "axin-β-catenin complex stabilizers," etc. Specific examples of Wnt inhibitors include, but are not limited to, XAV939 (CAS No. 284028-89-3), IWR-1 (CAS No. 1127442-82-3), and IWP-2 (CAS No. 686770-61-6).

[0028] The type and concentration of the Wnt inhibitor can be appropriately determined depending on the desired level of the effect of the present invention, taking into consideration the types and concentrations of other components contained in the medium composition of the present invention. In one embodiment of the present invention, the medium composition of the present invention contains XAV939 as a Wnt inhibitor, and its concentration in the medium composition is usually 0.1 to 50 μM, preferably 0.5 to 20 μM. When other types of Wnt inhibitors are used, their concentration range can also be set to the same as the concentration range described above for XAV939.

[0029] (A5) Polyvinyl alcohol (PVA) Polyvinyl alcohol (PVA) is a known component in the field of cell culture, and similar components can be used in the present invention. For example, the product "P8136" (Sigma-Aldrich, 87-90% hydrolyzed, average molecular weight 30,000-70,000) can be used as PVA.

[0030] The type (average molecular weight, etc.) and concentration of PVA can be appropriately determined according to the desired level of the effects of the present invention, taking into consideration the types and concentrations of other components contained in the medium composition of the present invention. In one embodiment of the present invention, the concentration of PVA in the medium composition of the present invention is usually 0.01 to 10 wt%, and preferably 0.1 to 5 wt%.

[0031] The medium composition of the present invention may further contain components other than the essential components (A1) to (A5) and the optional components (A6) to (A7) (excluding component (B)) as necessary. The type of additional component and its concentration in the medium composition can be appropriately selected and set, taking into consideration the type and concentration of other components in the medium composition of the present invention, as long as the effects of the present invention are not lost. Specific examples of additional components include, but are not limited to, the components contained in "ON2" described in Non-Patent Document 1, i.e., L-ascorbic acid, selenate, insulin, transferrin, FGF2 (bFGF), chondroitin sulfate, other amino acids necessary for cells, vitamins, and inorganic salts. The medium composition of the present invention may be a medium modified from "ON2" described in Non-Patent Document 1, particularly a medium obtained by removing albumin from "ON2."

[0032] (A6) Src inhibitor The Src inhibitor is a component that is preferably added when the medium composition of the present invention is used to culture EPSCs (expanded potential stem cells, see Patent Document 2) or naive pluripotent stem cells, for example.

[0033] "Src inhibitors" are drugs known in the art related to cell culture, and similar drugs can be used in the present invention. Src inhibitors may be drugs known as "SFK (Src Family Kinase) inhibitors," "Lck / Src inhibitors," etc. Specific examples of Src inhibitors include, but are not limited to, WH-4-023 (CAS No. 837422-57-8), bosutinib (also known as SKI-606, CAS No. 380843-75-4), dasatinib (also known as BMS-354825, CAS No. 302962-49-8), and saracatinib (also known as AZD0530, CAS No. 379231-04-6).

[0034] The type and concentration of the Src inhibitor can be appropriately selected and set depending on the desired level of the effect of the present invention and the intended use of the medium composition, taking into consideration the types and concentrations of other components contained in the medium composition of the present invention. In one embodiment of the present invention, the medium composition of the present invention contains WH-4-023 as the Src inhibitor, and its concentration in the medium composition is usually 0.1 to 20 μM, preferably 0.1 to 5 μM. When other types of Src inhibitors are used, their concentration range can also be set in accordance with the above-mentioned concentration range for WH-4-023.

[0035] When the medium composition of the present invention does not contain an Src inhibitor, the activin concentration in the medium composition can be made relatively low. For example, an embodiment in which the medium composition of the present invention contains an Src inhibitor and activin at a concentration of 2 to 200 ng / mL can be modified to an embodiment in which the medium composition does not contain an Src inhibitor and contains activin at a concentration of 5 to 120 ng / mL.

[0036] (A7)STAT3 activator "STAT (Signal Transducer and Activator of Transcription) 3 activators" are drugs known in the art related to cell culture, and similar drugs can be used in the present invention. Examples of STAT 3 activators include, but are not limited to, interferon (IFN), epidermal growth factor (EGF), interleukin 5 (IL5), interleukin 6 (IL6), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), and bone morphogenetic protein 2 (BMP2).

[0037] The type and concentration of the STAT3 activator can be appropriately selected and set depending on the desired level of the effect of the present invention and the intended use of the medium composition, taking into consideration the types and concentrations of other components contained in the medium composition of the present invention. In one embodiment of the present invention, the medium composition of the present invention contains LIF as a STAT3 inhibitor, and its concentration in the medium composition is usually 0.1 to 200 ng / mL, preferably 0.2 to 50 ng / mL. When other types of STAT3 inhibitors are used, their concentration range can also be set in accordance with the above-mentioned concentration range for LIF.

[0038] (A8) ROCK inhibitor A ROCK inhibitor is a component that is preferably added when the medium composition of the present invention is used, for example, for passaging pluripotent stem cells.

[0039] "ROCK (Rho-associated protein kinase) inhibitors" are drugs known in the art related to cell culture, and similar drugs can be used in the present invention. Specific examples of ROCK inhibitors include, but are not limited to, Y-27632 (CAS No. 146986-50-7), thiazovivin (CAS No. 1226056-71-8), and GSK429286A (CAS No. 864082-47-3).

[0040] The type and concentration of the ROCK inhibitor can be appropriately selected and set depending on the desired level of the effect of the present invention and the intended use of the medium composition, taking into consideration the types and concentrations of other components contained in the medium composition of the present invention. In one embodiment of the present invention, the medium composition of the present invention contains Y-27632 as a ROCK inhibitor, and its concentration in the medium composition is usually 0.1 to 100 μM, preferably 1 to 50 μM. When other types of ROCK inhibitors are used, their concentration range can be similar to the above-mentioned concentration range for Y-27632.

[0041] The medium composition of the present invention can be prepared by adding essential components (A1) to (A5) and optional components (A6) to (A8), as well as other additional components as necessary, to a basal medium that does not contain component (B), such as a serum-free medium, but does not contain serum, pituitary extract, or albumin and other animal-derived proteins derived therefrom.

[0042] Basal media such as serum-free media can be any of various basal media known in the art related to cell culture, and may be commercially available. Examples of basal media include DMEM (Dulbecco's Modified Eagle's Medium), αMEM (Eagle's Minimum Essential Medium α-modified), Ham's F-10 medium, Ham's F-12 medium, or mixtures thereof. In one embodiment of the present invention, the medium composition of the present invention contains a mixed medium of DMEM and Ham's F-12 as the basal medium.

[0043] -Culture method- The culture method of the present invention comprises a step of culturing mammalian pluripotent stem cells in the medium composition of the present invention under feeder cell-free three-dimensional culture conditions (sometimes referred to herein as the "culture step").

[0044] For details (specific examples, embodiments, etc.) of the medium composition used in the culture method of the present invention, please refer to the items described in this specification in relation to the medium composition of the present invention.

[0045] In the culture method of the present invention, the basic techniques for culturing mammalian pluripotent stem cells can be those used in known culture methods. The culture method of the present invention can be carried out essentially in accordance with such known culture methods, adjusting operations, conditions, etc. as necessary, except that the medium composition of the present invention is used.

[0046] For example, when embryonic stem cells are used as pluripotent stem cells, they can generally be produced by collecting the inner cell mass (ICM) from a late blastocyst of a donor mammal and culturing it in an appropriate medium. When induced pluripotent stem cells are used as pluripotent stem cells, they can generally be produced by introducing a specific gene (e.g., the four classical genes Oct3 / 4, Sox2, Klf4, and c-Myc, or an improved gene combination) into donor mammalian somatic cells (e.g., fibroblasts) using a retroviral vector or other vector or plasmid, or by introducing a specific compound, and then culturing the cells in an appropriate medium.

[0047] When preparing embryonic stem cells, inducible stem cells, or other pluripotent stem cells using the methods described above (before the first passage), the cells can be cultured, for example, for 3 to 10 days, using a two-dimensional (flat) culture method using a conventional medium composition and feeder cells.

[0048] Three-dimensional culture methods generally involve the use of scaffolds (culture supports) such as culture substrates with low cell adhesion, porous membranes, and hydrogels to allow cells to form aggregates (spheroids), thereby culturing cells in a three-dimensional state similar to that found in vivo. The present invention can utilize various culture methods known as three-dimensional culture methods for pluripotent stem cells (e.g., embryonic stem cells, induced pluripotent stem cells), as well as the culture equipment and culture conditions (e.g., temperature, atmosphere, etc.) used therefor. For example, the culture temperature can be set to 38.5°C, which is suitable for bovine cells.

[0049] In one embodiment of the present invention, the culturing step is preferably carried out on a plate-shaped culture substrate (sometimes referred to herein as a "specific culture substrate") having a plurality of depressions forming compartments in which the cultured organisms are cultured, and banks between adjacent depressions, located on the upper surface of the substrate, with adjacent banks and depressions forming a continuous curved surface. The specific culture substrate is the culture substrate described in WO2002 / 036011 (corresponding to Japanese Patent No. 5921437), and corresponds to a product available for purchase under the trademark "EZSPHERE (registered trademark)" (AGC Technoglass Co., Ltd.). The opening diameter of the "depression" is, for example, 200 to 1400 μm, and the opening depth is, for example, 100 to 600 μm. A culture substrate having depressions with an appropriate opening diameter and depth can be selected and used. For example, "EZSPHERE (registered trademark)" is available in dish types (35 mm, 60 mm, 100 mm) and well plate types (6 well, 24 well, 96 well) with various combinations of opening diameter and depth. 3 ~1×10 5 By seeding pluripotent stem cells using a cell suspension at a concentration of 1000 cells / mL, spheroids are formed in the wells.

[0050] In one embodiment of the present invention, the culture method of the present invention comprises culturing embryonic stem cells (typically obtained by the above-described embodiment) in the medium composition of the present invention for a desired number of passages. In this embodiment, the medium composition of the present invention can be replaced at appropriate times and intervals (e.g., daily). The culture period in the culture method of this embodiment can be, for example, the period from the appearance of embryonic stem cell colonies and establishment of embryonic stem cells to proliferation to a desired cell number.

[0051] In another embodiment of the present invention, the culture method of the present invention comprises temporarily maintaining the blastocyst or the like in the medium composition of the present invention while treating the blastocyst with a protease (e.g., pronase) to remove the zona pellucida in order to collect the ICM.

[0052] -Reference culture method and reference medium composition- In a further embodiment of the present invention, the culture method of the present invention is carried out when culturing mammalian pluripotent stem cells at a stage after they have been established by the "Reference Invention" described below, i.e., a mammalian pluripotent stem cell culture method (herein referred to as the "Reference Culture Method") carried out using a specific medium composition (herein referred to as the "Reference Medium Composition") described below. Hereinafter, the Reference Culture Method and Reference Medium Composition that can be used for the present invention are collectively referred to as the "Reference Invention."

[0053] The reference culture method includes culturing mammalian pluripotent stem cells in a serum-free medium (reference medium composition) supplemented with at least (1) a CDK8 / 19 inhibitor, (2) a ROCK inhibitor, (3) a STAT3 activator, and (4) a SMAD activator.

[0054] Serum-free medium As the "serum-free medium" in the reference culture method, various serum-free media known in the technical fields related to cell culture can be used. Such serum-free media may be commercially available or may be prepared by adding appropriate components to a basal medium.

[0055] Examples of the basal medium include DMEM (Dulbecco's modified Eagle's medium), αMEM (α-modified Eagle's minimal essential medium), Ham's F-10 medium, Ham's F-12 medium, and mixtures thereof.

[0056] In one embodiment of the reference invention, the serum-free medium is "mTeSR™1" (STEMCELL Technologies, Basal Medium + 5X Supplement) or "ON2 / AscleStem PSC medium" (see Non-Patent Document 1 cited above).

[0057] (1) CDK8 / 19 inhibitor "CDK (Cyclin-Dependent Kinase) 8 / 19 inhibitors" are drugs that selectively inhibit the function of CDK8 and its isoform, CDK19. These drugs are known in the art related to cell culture, and similar drugs can be used in the present invention. Examples of CDK8 / 19 inhibitors include, but are not limited to, ETP-47799 (CAS No. 923596-52-5), AS2863619 (CAS No. 2241300-51-4), and SEL120 (SEL120-34A) hydrochloride (CAS No. 1609452-30-3). The type and concentration of the drug used as a CDK8 / 19 inhibitor can be appropriately selected and set, taking into account the effects of the present invention.

[0058] In one embodiment of the present invention, the reference medium composition contains ETP-47799 as a CDK8 / 19 inhibitor. The concentration of ETP-47799 in the reference medium composition can be adjusted as appropriate, but can be, for example, 0.01 to 10 μM, and preferably 0.1 to 2.0 μM.

[0059] (2) ROCK inhibitors The ROCK inhibitor in the Reference Invention is the same as the (A8) ROCK inhibitor described in relation to the culture method (medium composition) of the present invention. The concentration of the ROCK inhibitor in the Reference Invention can be appropriately set taking into consideration the type of ROCK inhibitor used and the effects of the Reference Invention. In one embodiment of the Reference Invention, the Reference Medium Composition contains Y-27632 as a ROCK inhibitor. The concentration of Y-27632 in the Reference Medium Composition can be adjusted as appropriate, but can be, for example, 0.1 to 100 μM, preferably 1 to 50 μM. When other types of ROCK inhibitors are used in the Reference Medium Composition, the concentration range can be the same as the above concentration range for Y-27632.

[0060] (3) STAT3 activators The STAT3 activator in the Reference Invention is the same as the STAT3 activator (A7) described in relation to the culture method (medium composition) of the present invention. The concentration of the STAT3 activator in the Reference Invention can be appropriately set taking into consideration the type of STAT3 activator used and the effects of the Reference Invention. In one embodiment of the Reference Invention, the Reference Medium Composition contains LIF as a STAT3 activator. The concentration of LIF in the Reference Medium Composition can be adjusted as appropriate, but can be, for example, 1 to 200 ng / mL, and preferably 3 to 50 ng / mL. When another type of STAT3 activator is used in the Reference Medium Composition, the concentration range can be the same as the above-mentioned concentration range for LIF.

[0061] (4) SMAD activators "SMAD activators" are drugs known in the technical field related to cell culture, and similar drugs can be used in the present invention. Examples of SMAD activators include, but are not limited to, activin A, TGFβ1, and bone morphogenetic protein 4 (BMP4). Note that (A1) activin and (A2) TGFβ subfamily proteins, which were explained in relation to the culture method (medium composition) of the present invention, fall under the category of "SMAD activators." The type and concentration of the drug used as the SMAD activator can be appropriately selected and set, taking into consideration the effects of the present invention, etc.

[0062] In one embodiment of the reference invention, the reference medium composition contains activin A as a SMAD activator. The concentration of activin A in the reference medium composition can be adjusted as appropriate, for example, to 0.1 to 200 ng / mL, and preferably 5 to 100 ng / mL. When other types of SMAD3 activators are used in the reference medium composition, the concentration range can be similar to the above-mentioned concentration range for activin A.

[0063] The reference medium composition may further contain components other than the components (1) to (4) in addition to the serum-free medium. For example, the serum-free medium may further contain components such as glutathione, L-glutamine, lipids, thiamine, β-mercaptoethanol, BSA, pipecolic acid, GABA, bFGF, and L-ascorbic acid. The types and concentrations of these additional components can be appropriately selected and set, taking into account the effects of the reference invention.

[0064] The serum-free medium product "mTeSR1" contains glutathione, L-glutamine, lipids, thiamine, trace elements B and C, β-mercaptoethanol, BSA, pipecolic acid, LiCl (a GSK3 inhibitor), GABA, bFGF, and TGFβ1 (a SMAD activator). One or more of these components may be added as needed. For example, β-mercaptoethanol may be added to "mTeSR1" to a concentration of 100 mM. Furthermore, "mTeSR1" itself contains LiCl and TGFβ1, but these components may be added to achieve the preferred concentration ranges described herein.

[0065] The reference medium composition contains the above-mentioned components (1) to (4), and thereby exhibits the desired effect even without containing the (5) Src inhibitor and the (6) GSK3 inhibitor. However, if necessary, in addition to the above-mentioned components (1) to (4), the (5) SFK inhibitor may be further added, or the (5) Src inhibitor and the (6) GSK3 inhibitor may be further added.

[0066] (5) Src inhibitors The Src inhibitor in the Reference Invention is the same as the (A6) Src inhibitor described in relation to the culture method (medium composition) of the present invention. The concentration of the Src inhibitor in the Reference Invention can be appropriately determined taking into consideration the type of Src inhibitor used and the effects of the Reference Invention. In one embodiment of the Reference Invention, the Reference Medium Composition contains WH-4-023 as the Src inhibitor. The concentration of WH-4-023 in the Reference Medium Composition can be adjusted as appropriate, but can be, for example, 0.1 to 20 μM, preferably 0.1 to 5 μM. When other types of Src inhibitors are used in the Reference Medium Composition, the concentration range can be the same as the above concentration range for WH-4-023.

[0067] (6) GSK3 inhibitors The GSK3 inhibitor in the Reference Invention is the same as the (A3) GSK3 inhibitor explained in relation to the culture method (medium composition) of the present invention. The concentration of the GSK3 inhibitor in the Reference Invention can be appropriately set taking into consideration the type of GSK3 inhibitor used, the effects of the Reference Invention, etc. In one embodiment of the Reference Invention, the Reference Medium Composition contains CHIR99021 as the GSK3 inhibitor. The concentration of CHIR99021 in the Reference Medium Composition can be adjusted as appropriate, but can be, for example, 0.1 to 20 μM, and preferably 0.3 to 10 μM. When another type of GSK3 inhibitor is used in the Reference Medium Composition, the concentration range can also be the same as the concentration range for CHIR99021 described above.

[0068] By containing the above-mentioned components (1) to (4), the reference medium composition can achieve the desired effect without containing (7) a Wnt inhibitor. In one embodiment of the reference invention, the reference medium composition does not contain (7) a Wnt inhibitor, which is not necessary.

[0069] (7) Wnt inhibitors The Wnt inhibitor in the Reference Invention is the same as the (A4) Wnt inhibitor explained in relation to the culture method (medium composition) of the present invention.

[0070] In one embodiment of the reference invention, the reference culture method comprises seeding the recovered inner cell mass into the medium composition of the present invention, followed by culturing the inner cell mass and proliferated embryonic stem cells in the reference medium composition until the first passage. In this embodiment, the reference medium composition can be replaced at appropriate times and intervals (e.g., every other day after 3 days). Furthermore, in the culture method of this embodiment, the reference medium composition can be modified to an embodiment that does not contain a ROCK inhibitor. The culture period of the reference culture method of this embodiment can be, for example, 3 to 10 days.

[0071] In one embodiment of the reference invention, the reference culture method comprises culturing embryonic stem cells (typically obtained by the above-described embodiment of the reference invention) in a reference medium composition for a desired number of passages. In this embodiment, the reference medium composition can be replaced at appropriate times and intervals (e.g., daily). The culture period in the reference culture method of this embodiment can be, for example, the period from the appearance of embryonic stem cell colonies and establishment of embryonic stem cells to proliferation to a desired cell number. The reference culture method may be performed by appropriate means (e.g., see the Examples and Reference Examples below) using plate culture, or may be performed by three-dimensional culture, as in the culture method of the present invention.

[0072] In another embodiment of the reference invention, the reference culture method comprises temporarily holding the blastocyst in the medium composition of the present invention while treating the blastocyst with a protease (e.g., pronase) to remove the zona pellucida in order to recover the ICM.

[0073] Mammalian pluripotent stem cells established by the reference invention can be cryopreserved and thawed by appropriate means (e.g., see the Examples and Reference Examples below). In one embodiment of the present invention, the culture method of the present invention can be carried out when establishing mammalian (e.g., bovine) embryonic stem cells by the reference culture method, cryopreserving the embryonic stem cells, and then culturing the thawed mammalian embryonic stem cells.

[0074] The uses of pluripotent stem cells established and maintained according to the present invention are not particularly limited and can be used for a variety of purposes, but in a preferred embodiment of the present invention, they are used to obtain cells useful for the production of cultured meat. Specifically, pluripotent stem cells established and maintained according to the present invention can be used to obtain cells that make up tissues contained in meat, such as skeletal muscle, smooth muscle, cardiac muscle, fat, blood vessels, and blood, i.e., muscle cells, adipocytes, vascular endothelial cells, vascular wall cells, blood cells, etc., or stem cells or precursor cells that can induce these, in order to produce cultured meat. Methods for inducing the above-mentioned muscle cells, adipocytes, etc. from pluripotent stem cells are known, and known methods can also be applied to the present invention. [Example]

[0075] In this example, "Medium 1" and "Medium 2" shown in the table below were prepared and used as the medium compositions of the present invention.

[0076] [Table 1]

[0077] [1] Cultivation of bovine embryonic stem cells Cryopreserved bovine embryonic stem cells were thawed and seeded onto a three-dimensional culture substrate (EZSPHERE® Dush 100mm, AGC Technoglass Corporation). Culture was performed for 3–6 days using the bovine embryonic stem cell medium (Medium 1 or Medium 2) shown in the table above until cell clumps reached 200–500 μm in size. After single cell collection using TrypLE select (Gibco), the cells were seeded onto bovine embryonic stem cell medium (Medium 1 or Medium 2) shown in the table above supplemented with the ROCK inhibitor Y-27632 (Wako) at a concentration of 1–10 μM. The medium was changed daily, and the cells were passaged every 3–6 days at a ratio of 1:5–1:10.

[0078] An optical microscope image of bovine embryonic stem cells after the above-described culture is shown in Figure 1. Whether medium 1 or medium 2 was used, aggregates formed on the three-dimensional culture substrate, demonstrating that bovine embryonic stem cells were successfully cultured.

[0079] [2] Differentiation of skeletal muscle cells According to the following procedure, the bovine embryonic stem cells prepared in step 2 of [1] above were induced to differentiate into skeletal muscle cells. (1) Bovine embryonic stem cells with aggregates of 200-500 μm were prepared. (2) Treatment with the cell detachment enzyme "Accutase" (Innovative Cell Technologies, Inc.) was carried out at 38.5°C for 5 minutes. (3) The cells were collected in skeletal muscle induction medium, centrifuged at 300 g for 4 minutes, and then the number of cells was counted. (4) The cells collected in (3) above were diluted to 5 × 10 3 cells / cm 2 The cells were seeded in the above skeletal muscle induction medium to a density of 1000 x g. (5) The cells were cultured for 6 to 10 days in the skeletal muscle induction medium used in (3) above, with the medium replaced every two days. (6) Treatment with 0.05% trypsin was carried out at 38.5°C for 5 minutes. (7) The cells were collected in a medium containing 10% serum and centrifuged at 300 g for 4 minutes. (8) The cells collected in (7) above were diluted to 5 × 10 3 cells / cm 2 The cells were seeded onto type I collagen-coated plates at a density of 1000 x g. (9) The cells were cultured for 6 to 8 days in the serum-containing medium used in (7) above, with the medium replaced every two days. (10) The medium was switched to “Myotube Medium” or “Myotube Fusion Medium” (Myocea, Inc.), and the medium was changed every 3 to 4 days.

[0080] [3] Observation using a fluorescence microscope The cells cultured by the above procedure were immunostained according to the following procedure and observed under a fluorescence microscope. (1) The cultured cells were washed twice with PBS and then fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes. (2) The cells treated in (1) above were washed twice with PBS and then treated with 0.1% Triton-X100 (Sigma-Aldrich). (3) The cells treated in (2) above were washed twice with PBS and then subjected to a blocking treatment using 5% normal goat serum at room temperature for 30 minutes. (4) Immunostaining treatment using anti-desmin mouse monoclonal antibody (DAKO "IR606", undiluted) as the primary antibody was carried out at room temperature for 1 hour. (5) The cells treated in (4) above were washed three times with PBS (5 minutes each time), and then immunostained using Alexa488-labeled goat anti-mouse IgG antibody ("A11001", 1 / 500 dilution) as the secondary antibody at room temperature for 30 minutes. (6) The cells treated in (4) above were washed three times with PBS (5 minutes each time) and then mounted in the mounting medium "Mounting Medium With DAPI (AB104139)."

[0081] Fluorescence microscopic images of cells immunostained using the above procedure are shown in Figure 2. It can be seen that a sufficient number of myoblasts expressing Desmin were obtained using the above procedure.

[0082] <Reference Example> Establishment of bovine embryonic stem cells Bovine embryonic stem cells were established and maintained according to the following procedure (Reference Invention). Finally, the cryopreserved bovine embryonic stem cells were used in the above Example [1].

[0083] 1. Outgrowth (1.1) A 4-well plate previously prepared and seeded with mouse embryonic fibroblasts (MEF cells) or bovine-derived cells was removed from the incubator, and the inner cell mass recovered from bovine late stage blastocysts was seeded on it. (1.2) The ROCK inhibitor "Y-27632" (Wako) was added to the medium, and the medium was placed in a 38.5°C, 5% CO2 incubator.

[0084] 2. Establishment of bovine embryonic stem cells (2.1) The cultured cells were observed for 3 days without changing the medium, and then the medium was changed with the following medium for bovine embryonic stem cells (not containing the ROCK inhibitor "Y-27632").

[0085] [Table 2]

[0086] (2.2) The medium was replaced every other day with the medium for bovine embryonic stem cells, and the cells were cultured until they became confluent. (2.3) After about 10 days of growth, the cells reached confluence. Single cells were harvested using "TrypLE select" (Gibco) and then seeded onto the bovine embryonic stem cell medium containing the ROCK inhibitor. The cells were passaged 1:1 from one 4-well plate to another. (2.4) After two days, colonies appeared, confirming the establishment of bovine embryonic stem cells. (2.5) After the establishment of bovine embryonic stem cells, the medium was changed daily and the cells were passaged every 2 to 5 days at a ratio of 1:5 to 1:30.

[0087] 3. Cryopreservation (3.1) Bovine embryonic stem cells were cultured on a three-dimensional culture substrate until they reached aggregates of 500 μm or larger. (3.2) The aggregates on the three-dimensional culture substrate were collected in a centrifuge tube and left to stand for 1 minute or more to allow the aggregates to settle. (3.3) The supernatant was removed, and "TrypLE select" (Gibco) was added to the precipitated aggregates, followed by incubation at 38°C and 5% CO2 for 5 minutes. (3.4) After confirming that the aggregates were dispersed, the above-mentioned medium for pluripotent stem cells was added, and the mixture was centrifuged at 1000 rpm for 5 minutes. (3.5) After centrifugation, 1 × 10 6 The cells were suspended in a freezing medium so that there were 1 cell per tube, and the cells were stored at -80°C overnight. (3.6) The next day, the mixture was transferred to a liquid nitrogen tank and stored at −196°C in liquid phase.

Claims

1. (A1) activin, (A2) TGFβ subfamily proteins, (A3) GSK3 inhibitors, (A4) a Wnt inhibitor, and (A5) Polyvinyl alcohol (PVA) Contains (B) Serum and pituitary extracts, and albumin and other animal-derived proteins derived therefrom. Does not contain A medium composition for three-dimensional culture of mammalian pluripotent stem cells.

2. (A6) The medium composition according to claim 1, further comprising an Src inhibitor.

3. (A7) The medium composition of claim 1, further comprising a STAT3 activator.

4. (A8) The medium composition according to claim 1, further comprising a ROCK inhibitor.

5. The medium composition of claim 1 , wherein the mammal is an artiodactyl.

6. The medium composition of claim 5, wherein the artiodactyl is a cattle or sheep.

7. The medium composition of claim 1 , wherein the pluripotent stem cells are stem cells derived from an embryo, including embryonic stem cells.

8. A culture method comprising the step of culturing mammalian pluripotent stem cells in the medium composition according to any one of claims 1 to 7 under feeder cell-free three-dimensional culture conditions.

9. 9. The culture method according to claim 8, wherein the culture step is carried out on a plate-shaped culture substrate having a plurality of depressions forming compartments in which the cultured substance is cultured and bank portions interposed between adjacent depressions on the upper surface thereof, and the adjacent bank portions and depression portions being continuous curved surfaces.

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