Composition to be used for differentiation inhibition of myoblast, composition to be used for proliferation promotion of myoblast, differentiation inhibition method of myoblast, and proliferation promotion method of myoblast

Abstract

To provide a composition or the like to be used for differentiation inhibition of a myoblast or proliferation promotion of a myoblast.SOLUTION: A composition to be used for differentiation inhibition of a myoblast contains proteoglycan.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to a composition for use in inhibiting myoblast differentiation, a composition for use in promoting myoblast proliferation, a method for inhibiting myoblast differentiation, a method for promoting myoblast proliferation, and the like. [Background technology]

[0002] In recent years, cultured meat has been attracting attention as a way to solve problems such as global warming caused by methane gas contained in livestock breath and meat supply shortages due to population growth.

[0003] Because meat such as loin and thigh that we normally eat is animal muscle tissue (skeletal muscle), there is a need for the development of cultured meat derived from muscle tissue. Such muscle tissue can be obtained by proliferating myoblasts and inducing differentiation after the proliferation. Currently, serum medium is used to proliferate the myoblasts, but due to the high cost of serum medium, the production of cultured meat derived from muscle tissue is not practical. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Guan, X et al., (2021). Trends and ideas in technology, regulation and public acceptance of cultured meat. Future Foods, 3, 100032. [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2020-089381 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an object of the present disclosure is to provide a composition for use in inhibiting myoblast differentiation or promoting myoblast proliferation. [Means for solving the problem]

[0007] To achieve the above object, the composition for use in inhibiting myoblast differentiation of the present disclosure contains a proteoglycan.

[0008] The medium for use in inhibiting myoblast differentiation of the present disclosure comprises the composition for use in inhibiting myoblast differentiation of the present disclosure.

[0009] The medium kit for use in inhibiting myoblast differentiation of the present disclosure comprises the composition for use in inhibiting myoblast differentiation of the present disclosure and a medium.

[0010] The compositions of the present disclosure for use in promoting myoblast proliferation include proteoglycans.

[0011] The medium for use in promoting the proliferation of myoblasts of the present disclosure comprises the composition for use in promoting the proliferation of myoblasts of the present disclosure.

[0012] A kit for use in promoting the proliferation of myoblasts according to the present disclosure comprises a composition for use in promoting the proliferation of myoblasts according to the present disclosure and a medium.

[0013] The method of inhibiting myoblast differentiation of the present disclosure comprises the step of culturing myoblasts in the presence of proteoglycan.

[0014] The method for promoting myoblast proliferation of the present disclosure comprises the step of culturing myoblasts in the presence of proteoglycan.

[0015] The cell population of the present disclosure comprises two or more myoblasts, the two or more myoblasts being adhered via an extracellular matrix, the extracellular matrix comprising a proteoglycan heterologous to the species of the myoblasts.

[0016] The method for producing myotubes disclosed herein includes a step of culturing myoblasts and a step of inducing myotubes from the cultured myoblasts, and the culturing is carried out using the differentiation-inhibiting method disclosed herein or the proliferation-promoting method disclosed herein.

[0017] The cell population of the present disclosure comprises two or more myotube cells, the two or more myotube cells being adhered via an extracellular matrix, the extracellular matrix comprising a heterologous proteoglycan relative to the species of the myotube cells.

[0018] The method of producing the cultured meat, processed cultured meat, processed meat, or meat product of the present disclosure includes producing the cultured meat, processed cultured meat, processed meat, or meat product from a cell population comprising the myotubes of the present disclosure.

[0019] The cultured meat, cultured meat derivative, or meat product of the present disclosure comprises a cell population comprising the myotubes of the present disclosure.

[0020] According to the present disclosure, for example, it is possible to provide a composition for use in inhibiting myoblast differentiation or promoting myoblast proliferation. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the main disaccharide structures of each chondroitin sulfate. [Figure 2] FIG. 2 is a photograph and graph showing the results of inhibiting differentiation and promoting proliferation of myoblasts when cultured for three days in a medium containing proteoglycan. [Figure 3] FIG. 3 shows photographs and graphs showing the results of inhibiting differentiation and promoting proliferation of myoblasts when cultured for 3 or 5 days using a medium containing proteoglycan. [Figure 4] FIG. 4 is a photograph and a graph showing the results of suspension culture for 5 days using a medium containing proteoglycan. [Figure 5] FIG. 5 is a photograph and a graph showing the results of culturing for 3 days using a medium containing proteoglycan. [Figure 6] FIG. 6 shows photographs and a graph showing the results of culturing chicken embryo-derived myoblasts in a proteoglycan-containing medium for 6 hours. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0023] <Definition> As used herein, "proteoglycan" refers to a molecule (glycoprotein) in which a protein (core protein) and a glycosaminoglycan (GAG, also called a "polysaccharide" or "sugar chain") are covalently bonded. The proteoglycan exists as an extracellular matrix in, for example, skin, organs, cartilage, and the like. The glycosaminoglycan is generally known as a sugar chain having a long chain structure without a branched structure. Examples of the proteoglycan include the following: Aggrecan family (also called lectican family or hyalectan family): Aggrecan, versican, neurocan, brevican, etc. Small leucine rich proteoglycans (SLRPs) family: biglycan, decorin, fibromodulin, lumican, epiphycan (PG-Lb), keratocan, mimecan, etc. Basement membrane proteoglycans: perlecan, agrin, permacan, etc. Others: Testican, Biglycan, Serglycin, Syndecan, Dystroglycan, Claustrin, Glypican, Keratocan, etc. The proteoglycans can also be classified into, for example, chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, and keratan sulfate proteoglycans depending on the type of GAG bound to the protein.

[0024] Examples of the GAG include chondroitin, chondroitin sulfate (CS), dermatan sulfate (DS, chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. Examples of the chondroitin include O-type sugar chains whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine, and iO-type sugar chains whose main structure is a disaccharide structure of iduronic acid and acetylgalactosamine (hereinafter referred to as "chondroitin sulfate O" and "chondroitin sulfate iO," respectively). The chondroitin sulfate (CS) has a structure in which a sulfate group is added to a sugar chain in which the disaccharide structure of glucuronic acid and acetylgalactosamine is repeated. Examples of the chondroitin sulfate include chondroitin sulfate A (A-type), whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine tetrasulfate, and chondroitin sulfate C (C-type), whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine hexasulfate. Dermatan sulfate (DS) has a structure in which a sulfate group is added to a sugar chain in which the disaccharide structure of iduronic acid and acetylgalactosamine is repeated. Examples of the dermatan sulfate include chondroitin sulfate iA (iA-type), whose main disaccharide structure is a disaccharide structure of iduronic acid and acetylgalactosamine tetrasulfate, and chondroitin sulfate iC (iC-type), whose main disaccharide structure is a disaccharide structure of iduronic acid and acetylgalactosamine hexasulfate. Each chondroitin sulfate has, for example, the disaccharide structure shown in FIG. 1 as its main disaccharide structure. In Figure 1, the sulfate group (sulfo group) is bonded to a hydrogen atom, but the present disclosure is not limited to this. The sulfate group of the GAG may, for example, be ionized by elimination of the hydrogen atom, or may form a salt.

[0025] As used herein, the term "myoblast" refers to a mononuclear cell in the process of myogenesis, which is in an undifferentiated state. The myoblast forms a multinucleated myotube through cell fusion, and then matures into a myocyte (muscle cell). That is, the myoblast has the ability to form muscle fibers. The myoblast can be identified, for example, by a marker. Examples of the marker include MyoD, Myf5, Pax7, etc. The myoblast is, for example, a MyoD-, Myf5-, and / or Pax7-positive cell.

[0026] As used herein, "myotube cells" refer to multinucleated cells that differentiate from myoblast cells. The myotube cells can be identified, for example, by a marker. Examples of the marker include myosin heavy chain (MHC) and myogenin. That is, the myotube cells are MHC- and myogenin-positive cells. In the myotube cells, for example, the expression level of the MHC marker is increased by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more compared to the myoblast cells.

[0027] As used herein, "muscle cells" refers to cells that constitute muscle fibers. Examples of muscle cells include cardiac muscle cells, smooth muscle cells, and skeletal muscle cells (muscle fibers). Muscle cells can be identified, for example, by markers. Examples of markers include MHC and myogenin. Muscle cells are, for example, MHC and / or myogenin-positive cells.

[0028] As used herein, "positive" (+) means that a higher signal is detected by an analytical method such as flow cytometry, which utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. As used herein, "negative" (-) means that a signal equivalent to or lower than a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected.

[0029] As used herein, "inhibition of myoblast differentiation" means that myoblast differentiation is significantly inhibited. The inhibition can also be referred to as, for example, alleviation, reduction, or decrease.

[0030] As used herein, "promoting myoblast proliferation" means that the proliferation of myoblasts is significantly promoted. The promotion can also be referred to as, for example, enhancement, improvement, or increase.

[0031] As used herein, "adhesion culture" refers to culturing in a state in which cells are fixed by adhesion or the like to a solid surface such as a culture vessel.

[0032] As used herein, "suspension culture" refers to culturing cells in a state where the cells are not fixed to a solid surface such as a culture vessel by adhesion or the like, i.e., in a suspended state. The suspension culture can be performed, for example, by subjecting the culture vessel containing the cells to agitation culture, shaking culture, or rotation culture.

[0033] As used herein, the term "cell population" refers to a collection of cells that includes a desired cell and is composed of one or more cells. In the cell population, the proportion of the desired cells among all cells (also referred to as "purity") can be quantified, for example, as the proportion of cells expressing one or more markers expressed by the desired cells. The purity is, for example, the proportion among live cells. The purity can be measured by methods such as flow cytometry, immunohistochemistry, and in situ hybridization. The purity of the desired cells in the cell population is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% 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 cell population can also be referred to as, for example, a cell preparation.

[0034] As used herein, the term "cell mass" (spheroid) refers to a mass-shaped cell population formed by cell aggregation. The cell mass can be formed, for example, by three-dimensional suspension culture of cells.

[0035] As used herein, "cultured meat" refers to meat made by cultivating animal cells. in In vitro Cultured meat refers to meat produced using cell masses or tissues formed by culturing in a laboratory. Cultured meat is also known as lab-grown meat, test tube meat, cell-cultured meat, artificial meat, or synthetic meat.

[0036] As used herein, "meat" refers to meat for consumption, including, for example, livestock meat, rabbit meat, poultry meat, cultured meat, and mixtures thereof.

[0037] In this specification, "processed meat" refers to meat products obtained by processing meat. Examples of processed meat include shaped meats obtained by adding additives to raw meat, such as ham, sausage, bacon, and the like, and binding the resulting meat, fat-injected processed meat obtained by injecting fat into raw meat, tenderized meat, and tumbling meat.

[0038] In this specification, "meat products" refers to dried meat products, uncooked meat products, specially cooked meat products, cooked meat products, etc., as defined by the Food Sanitation Act. Examples of the meat products include ham, sausage, bacon, beef jerky, roast chicken, roast beef, corned beef, hamburger steak, etc.

[0039] As used herein, the term "kit" generally refers to a unit in which the components to be provided (e.g., reagents, culture media, other components such as additives, instructions, etc.) are provided separately in two or more compartments. The kit can be suitably used to provide a composition that is not provided in a mixed state, but is preferably mixed immediately before use, for reasons of stability, etc. The kit preferably includes, for example, instructions or instructions on how to use the components to be provided (e.g., reagents, culture media, other components such as additives, etc.), or instructions or instructions describing how to treat the components. As used herein, when the kit is used as a culture kit, the kit may include instructions, etc., describing how to use the culture media and other components, etc.

[0040] As used herein, "instructions" or "manuals" refer to written instructions to a technician or other user on how to use the present disclosure. The instructions may, for example, describe instructions on how to use the presently disclosed differentiation-inhibiting method, proliferation-promoting method, manufacturing method, or kit. The instructions may be a package insert and are typically provided in paper form, but are not limited thereto and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, e-mail, etc.).

[0041] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure and each embodiment are mutually applicable unless otherwise specified. In this specification, the expression "to" is used to mean the numerical or physical values before and after it. In this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0042] <Composition for use in inhibiting myoblast differentiation> In one aspect, the present specification provides a composition for use in inhibiting myoblast differentiation. The composition for use in inhibiting myoblast differentiation of the present disclosure (hereinafter also referred to as the "first composition") contains proteoglycan, as described above.

[0043] As a result of extensive research, the present researchers have found that proteoglycans can inhibit the differentiation of myoblasts into myotubes. According to the present disclosure, even when myoblasts are cultured under conditions for inducing myotubes, such as in a medium containing a reduced amount of serum, the myoblasts can be cultured while their differentiation into myotubes is inhibited. Therefore, the first composition of the present disclosure allows the myoblasts to be cultured while their differentiation into myotubes is inhibited. Furthermore, the first composition of the present disclosure allows, for example, the amount of serum added during the culture of the myoblasts to be reduced, thereby enabling the maintenance culture of the myoblasts to be performed at lower cost.

[0044] The proteoglycan is not particularly limited, and is preferably aggrecan, for example.

[0045] The proteoglycan may be, for example, an animal proteoglycan. The animal is not particularly limited, and examples thereof include mammals (mammals) such as pigs, cows, and whales; avian animals (birds) such as chickens; fish such as flatfish (e.g., flounder), salmonids (e.g., chum salmon, Atlantic salmon), and rays (including skunkfish); and mollusks such as squid. The animal is preferably salmon, pig, chicken, flounder, or ray. The animal species from which the proteoglycan is derived is preferably different from the animal species from which serum, described below, is derived.

[0046] The animal species from which the proteoglycan is derived may be the same as or different from the animal species from which the myoblasts described below are derived. Examples of combinations of the origin of the proteoglycan and the myoblasts include salmon and mouse, salmon and chicken, salmon and pig, salmon and cow, cow and pig, and cow and chicken.

[0047] The proteoglycan may be derived from a tissue containing proteoglycan. Examples of the tissue include epithelial tissue such as skin, cartilaginous tissue such as cartilage, digestive organs, circulatory organs, respiratory organs, and placenta. Specific examples of the tissue include cartilage, fins, digestive organs, circulatory organs, respiratory organs, and ears.

[0048] Examples of the sugar chain of the proteoglycan include chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. The sugar chain is preferably chondroitin sulfate.

[0049] The peak top molecular weight of the proteoglycan is, for example, preferably 300,000 to 1,300,000, or 400,000 to 1,200,000. The peak top molecular weight can be measured, for example, by GPC (Gel Permeation Chromatography). The GPC method is performed, for example, under the following conditions, and the standard samples (molecular weight markers, pullulan) are individually injected into an HPLC system to obtain a molecular weight calibration curve, thereby allowing calculation.

[0050] (Measurement conditions for peak top molecular weight (Mp)) HPLC system: LC-20AD (Shimadzu Corporation) Column: TSKgel G5000-PWXL φ7.8mm x 300mm (Tosoh Corporation) Eluent: pH 6.8 phosphate buffer Flow rate: 0.5mL / min Column temperature: 40℃ Detector: Differential refractive index detector (RID-20A, Shimadzu Corporation) Injection volume: 50μL Molecular weight marker: Shodex STANDARD P-82 (pullulan) The peak top molecular weight (Mp) and weight average molecular weight (Mw) / number average molecular weight (Mn) of the molecular weight markers are as follows: STD P-800:Mp:739,000, Mw / Mn:1.24 STD P-400:Mp:348,000, Mw / Mn:1.33 STD P-200:Mp:216,000, Mw / Mn:1.22 STD P-100:Mp:107,000, Mw / Mn:1.12 STD P-50: Mp:49,400, Mw / Mn:1.08 STD P-20:Mp:22,000, Mw / Mn:1.08 STD P-10: Mp:9,800, Mw / Mn:1.07 STD P-5: Mp: 6,300, Mw / Mn: 1.09

[0051] The origin of the myoblasts is not particularly limited, and examples thereof include humans and non-human animals. Examples of the non-human animals include animals raised for livestock farming, such as rabbits, sheep, horses, cattle, pigs, goats, and chickens; birds; primates, such as monkeys, gorillas, chimpanzees, and marmosets; fish; mice, rats, dogs, and cats. When muscle cells derived from the myoblasts are used for food, such as cultured meat, the animals are preferably animals raised for livestock farming.

[0052] The "inhibition of myoblast differentiation" can be evaluated, for example, by determining whether the differentiation of myoblasts into myotubes is inhibited in the presence of a target substance during myoblast culture, compared to the absence of the target substance. Specifically, the inhibitory effect of the target substance on myoblast differentiation can be evaluated, for example, by determining the difference between the myoblast differentiation rate into myotubes in the absence of the target substance and the myoblast differentiation rate in the presence of the target substance, when the culture period is the same. The differentiation rate can be, for example, the rate of myoblast differentiation into myotubes when myoblasts are cultured for 1 to 5 days. In this evaluation, the differentiation rate can be calculated, for example, from the proportion of cells identified by a myotube marker to the total number of myoblasts after culture. The evaluation can be carried out by comparing the differentiation rate of myoblasts into myotubes in the presence of the target substance with the differentiation rate of myoblasts into myotubes in the absence of the target substance, and if the differentiation rate is, for example, 5% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, or 99% or less, the target substance can be evaluated as having an inhibitory effect on myoblast differentiation.

[0053] <Culture medium for inhibiting myoblast differentiation> In another aspect, the present disclosure provides a medium for use in inhibiting myoblast differentiation. The medium for use in inhibiting myoblast differentiation of the present disclosure (hereinafter also referred to as "first medium") contains the composition for use in inhibiting myoblast differentiation of the present disclosure. The first medium of the present disclosure allows myoblasts to be cultured while their differentiation into myotubes is inhibited.

[0054] The first medium of the present disclosure can be composed of, for example, a basal medium and, optionally, other components, such as additives, added to the basal medium. In the first medium of the present disclosure, the first composition is, for example, contained in the basal medium. The basal medium can be, for example, a known medium suitable for culturing myoblasts or a medium equivalent thereto. The basal medium can be, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, or a mixture thereof.

[0055] The first medium of the present disclosure may contain blood-derived components such as serum or plasma, or may be a serum-free medium that does not contain these components. Examples of the serum include serum derived from animals. Examples of the animals include animals raised for livestock purposes, such as fetal cattle, horses, sheep, goats, pigs, rabbit serum, and chicken serum, as well as mules, donkeys, guinea pigs, hamsters, rats, and mice. When the myoblast-derived muscle cells are intended for consumption, the serum is preferably derived from animals raised for livestock purposes. When the first medium of the present disclosure contains serum, the serum concentration in the medium is, for example, more than 0 (v / v)% and not more than 10 (v / v), 0.1 to 7.5 (v / v)%, 0.5 to 5 (v / v)%, or 1 to 3 (v / v)%.

[0056] In the first medium of the present disclosure, the concentration of the proteoglycan is, for example, 100 to 5000 μg / ml, 150 to 4500 μg / ml, 200 to 3000 μg / ml, 250 to 2500 μg / ml, or 500 to 2000 μg / ml. The concentration of the proteoglycan is preferably the concentration of a proteoglycan not derived from the serum, i.e., added.

[0057] The first medium of the present disclosure may contain, for example, other components. The other components are not particularly limited, and examples thereof include albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, and the like. The other components may be, for example, one type or two or more types. The concentrations of the other components are not particularly limited.

[0058] <Culture medium kit for inhibiting myoblast differentiation> In another aspect, the present disclosure provides a culture medium kit for use in inhibiting myoblast differentiation. The culture medium kit for use in inhibiting myoblast differentiation of the present disclosure (hereinafter also referred to as a "first culture medium kit") comprises the composition for use in inhibiting myoblast differentiation of the present disclosure and a culture medium. The first culture medium kit of the present disclosure allows myoblasts to be cultured while their differentiation into myotubes is inhibited.

[0059] In the first culture medium kit of the present disclosure, the description of the basal medium in the first culture medium can be applied to the medium.

[0060] The first culture medium kit of the present disclosure may contain, for example, the other components (constituents). When the first culture medium kit of the present disclosure contains the additive, the additive may be an additive to be added to the medium before culture, or an additive to be added during cell culture. The addition may be continuous or intermittent.

[0061] The first culture medium kit of the present disclosure may further include, for example, a container for storing the composition for use in inhibiting myoblast differentiation of the present disclosure and the culture medium.

[0062] The first culture medium kit of the present disclosure may include, for example, myoblasts for use in culture. The first culture medium kit of the present disclosure may also include, for example, instructions or manuals.

[0063] In the first culture medium kit of the present disclosure, for example, each component may be contained separately, or some or all of the components may be contained in a mixed or unmixed state. In the first culture medium kit of the present disclosure, when all components are contained in a mixed or unmixed state in a single container, the kit of the present disclosure can also be called, for example, a culture medium.

[0064] The first culture medium kit of the present disclosure can also be suitably used as a test kit or research kit for use in inhibiting myoblast differentiation, for example.

[0065] <Composition for use in promoting proliferation of myoblasts> In another aspect, the present disclosure provides a composition for use in promoting myoblast proliferation. The composition for use in promoting myoblast proliferation of the present disclosure (hereinafter also referred to as the "second composition") contains proteoglycan as described above.

[0066] As a result of extensive research, the present researchers have found that proteoglycans can promote the proliferation of myoblasts. According to the present disclosure, even when myoblasts are cultured under conditions for inducing myoblasts into myotubes, such as in a medium containing a reduced amount of serum, the proliferation of the myoblasts can be promoted while suppressing their differentiation into myotubes. Therefore, the second composition of the present disclosure allows the proliferation of myoblasts while suppressing their differentiation into myotubes. Furthermore, the first composition of the present disclosure allows, for example, the amount of serum added during the culture of the myoblasts to be reduced, thereby enabling the proliferation and culture of the myoblasts to be performed at lower cost.

[0067] The proliferation can be identified, for example, by a marker, such as Ki67.

[0068] The "myoblast proliferation-promoting effect" can be evaluated, for example, by determining whether the proliferation of myoblasts is promoted in the presence of a target substance during myoblast culture, compared to the absence of the target substance. Specifically, the myoblast proliferation-promoting effect of the target substance can be evaluated, for example, by determining the difference between the proliferation rate of myoblasts in the absence of the target substance and the proliferation rate of myoblasts in the presence of the target substance, for the same culture period. In this evaluation, the proliferation rate can be calculated as the proliferation rate (increase rate) of myoblast cell number when myoblasts are cultured for, for example, 1 to 5 days in the presence or absence of the target substance, based on the number of cells at the start of myoblast culture. The evaluation can be made such that the target substance is evaluated as having the effect of promoting myoblast proliferation when the rate of increase of myoblasts in the presence of the target substance is, for example, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more compared to the rate of increase of myoblasts in the absence of the target substance.

[0069] <Culture medium for promoting myoblast proliferation> In another aspect, the present disclosure provides a medium for use in promoting myoblast proliferation. The medium for use in promoting myoblast proliferation of the present disclosure (hereinafter also referred to as "second medium") contains the composition for use in promoting myoblast proliferation of the present disclosure. The second medium of the present disclosure can promote the proliferation of myoblasts while suppressing their differentiation into myotubes.

[0070] <Culture medium kit for promoting myoblast proliferation> In another aspect, the present disclosure provides a culture medium kit for use in promoting myoblast proliferation. The culture medium kit for use in promoting myoblast proliferation of the present disclosure (hereinafter also referred to as a "second culture medium kit") comprises the composition for use in promoting myoblast proliferation of the present disclosure and a culture medium. The second culture medium kit of the present disclosure can promote the proliferation of myoblasts while suppressing their differentiation into myotubes.

[0071] <Method for inhibiting myoblast differentiation> In another aspect, the present disclosure discloses a method capable of inhibiting myoblast differentiation. The method of inhibiting myoblast differentiation (hereinafter also referred to as "differentiation-inhibiting method") of the present disclosure includes a step of culturing myoblasts in the presence of proteoglycan. Since the differentiation-inhibiting method of the present disclosure cultures myoblasts in the presence of proteoglycan, it is possible to culture myoblasts in a state in which differentiation into myotubes is inhibited.

[0072] The culture is carried out, for example, using a medium containing the proteoglycan. For example, the medium may be the same as the first medium described above.

[0073] In the culture, the concentration of the proteoglycan is, for example, 100 to 5000 μg / ml, 150 to 4500 μg / ml, 200 to 3000 μg / ml, 250 to 2500 μg / ml, or 500 to 2000 μg / ml. The concentration of the proteoglycan is, for example, the concentration in the medium.

[0074] The proteoglycan may be homologous to or heterologous to the species of the myoblasts. The heterologous proteoglycan may be derived from an animal other than cow. In the heterologous case, the combination of the proteoglycan and the myoblasts may be, for example, salmon and mouse, salmon and chicken, salmon and pig, salmon and cow, cow and pig, cow and chicken, etc.

[0075] The proteoglycan is heterologous to the serum described below. Examples of the combination of the proteoglycan and the serum include salmon and horse, salmon and cow, etc.

[0076] The culture may be performed in the presence or absence of serum. When the culture is performed in the presence of serum, the serum concentration is, for example, more than 0 (v / v)% and not more than 10 (v / v), 0.1 to 7.5 (v / v), 0.5 to 5 (v / v), or 1 to 3 (v / v). The serum concentration is, for example, the concentration in the medium.

[0077] In the culture, for example, the total number of cells after the culture is 4 to 6 times the total number of cells at the start of the culture.

[0078] The culture temperature is, for example, about 30 to 40°C, preferably about 37°C. The culture can be carried out, for example, in an atmosphere of CO2-containing air. The CO2 concentration is, for example, about 1 to 10%, or about 2 to 5%. The culture is preferably carried out in a humid environment.

[0079] The culture may be, for example, adhesion culture, suspension culture, etc. The suspension method is not particularly limited as long as it allows cells to be cultured in a suspended state, and examples thereof include spinner culture, rotation culture, and shaking culture.

[0080] After the culture, the lower limit of the proportion (cell number) of myoblasts to all cells is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% 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. After the culture, the upper limit of the proportion (cell number) of myoblasts to all cells is, for example, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 80% or less. After the culture, the proportion of the myoblasts to the total cells (cell number) is, for example, 50 to 99%, 50 to 90%, 50 to 80%, 50 to 70%, 50 to 60%, 60 to 99%, 60 to 90%, 60 to 85%, 60 to 80%, 60 to 75%, 60 to 70%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 85%, 75 to 99%, 75 to 90%, 80 to 99%, 80 to 95%, 80 to 90%, 90 to 99%, 91% to 98%, 92% to 97%, or 93 to 95%.

[0081] After the culture, the lower limit of the proportion of myotubes to all cells (cell number) is, for example, more than 0%, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more. After the culture, the upper limit of the proportion of myotubes to all cells (cell number) is, for example, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. After the culture, the proportion (cell number) of the myotube cells to the total cells is, for example, more than 0% and not more than 50%, more than 0% and not more than 40%, more than 0% and not more than 30%, more than 0% and not more than 20%, more than 0% and not more than 10%, 5 to 50%, 5 to 40%, 5 to 30%, 5 to 20%, 5 to 10%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 50%, 20 to 40%, 20 to 30%, 30 to 50%, 30 to 40%, 40 to 50%, 1 to 10%, 3 to 9%, 4 to 8%, or 5 to 7%.

[0082] <Method for promoting myoblast proliferation> In another aspect, the present disclosure discloses a method capable of promoting myoblast proliferation. The method for promoting myoblast proliferation (hereinafter also referred to as the "proliferation promotion method") of the present disclosure includes a step of culturing myoblasts in the presence of proteoglycan. Because the proliferation promotion method of the present disclosure cultures myoblasts in the presence of proteoglycan, it is possible to promote myoblast proliferation while suppressing differentiation into myotubes.

[0083] <Cell population containing myoblasts> In another aspect, the present disclosure provides a cell population that can be used, for example, to induce myotubes. The cell population of the present disclosure (hereinafter also referred to as a "first cell population") contains two or more myoblasts, which are adhered via an extracellular matrix, and the extracellular matrix contains proteoglycans heterologous to the species of the myoblasts. The first cell population of the present disclosure can be suitably used, for example, to induce myotubes.

[0084] The first cell population may include, for example, a cell cluster containing the two or more myoblasts.

[0085] As described above, in the first cell population, the extracellular matrix contains proteoglycans heterologous to the species of the myoblasts. That is, the extracellular matrix contains exogenous proteoglycans not derived from the myoblasts. The exogenous proteoglycans are, for example, serum-derived proteoglycans in the culture medium for culturing the cell population and / or heterologous proteoglycans added to the culture medium for culturing the cell population. The heterologous proteoglycans are, for example, proteoglycans derived from animals other than cows. Examples of combinations of the proteoglycans and the myoblasts include salmon and mouse, salmon and chicken, salmon and pig, salmon and cow, cow and pig, and cow and chicken.

[0086] In the first cell population, the lower limit of the proportion (cell number) of the myoblasts to all cells is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% 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. In the first cell population, the upper limit of the proportion (cell number) of the myoblasts to all cells is, for example, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 80% or less. In the first cell population, the proportion (cell number) of the myoblasts to the total cells is, for example, 50 to 99%, 50 to 90%, 50 to 80%, 50 to 70%, 50 to 60%, 60 to 99%, 60 to 90%, 60 to 85%, 60 to 80%, 60 to 75%, 60 to 70%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 85%, 75 to 99%, 75 to 90%, 80 to 99%, 80 to 95%, 80 to 90%, 90 to 99%, 91% to 98%, 92% to 97%, or 93 to 95%.

[0087] The first cell population may further include myotubes, for example. When the first cell population includes myotubes, the lower limit of the proportion (cell number) of the myotubes to all cells is, for example, more than 0%, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more. In the first cell population, the upper limit of the proportion (cell number) of myotube cells to all cells is, for example, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. In the first cell population, the proportion (cell number) of the myotube cells to the total cells is, for example, greater than 0% and less than 50%, greater than 0% and less than 40%, greater than 0% and less than 30%, greater than 0% and less than 20%, greater than 0% and less than 10%, 5 to 50%, 5 to 40%, 5 to 30%, 5 to 20%, 5 to 10%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 50%, 20 to 40%, 20 to 30%, 30 to 50%, 30 to 40%, or 40 to 50%.

[0088] The first cell population may be obtained, for example, by the differentiation-inhibiting method of the present disclosure or the proliferation-promoting method of the present disclosure.

[0089] <Method for producing myotube cells (muscle fibers)> In another aspect, the present disclosure discloses a method for producing myotubes (muscle fibers). The method for producing myotubes of the present disclosure (hereinafter also referred to as the "first production method") includes a step of culturing myoblasts and a step of inducing myotubes from the cultured myoblasts, and the culturing is carried out by the differentiation-inhibiting method of the present disclosure or the proliferation-promoting method of the present disclosure. The first production method of the present disclosure induces myoblasts proliferated under a differentiation-inhibited state into myotubes, thereby enabling efficient production of myotubes.

[0090] The induction can be achieved, for example, by lowering the serum concentration and / or the proteoglycan concentration in myoblast culture. Specifically, the induction can be achieved, for example, by lowering the proteoglycan concentration and / or the serum concentration in the differentiation-inhibiting method of the present disclosure.

[0091] In the first production method of the present disclosure, the culture can be carried out in the same manner as the culture in the differentiation-inhibiting method or proliferation-promoting method of the present disclosure.

[0092] The induction can be carried out using a medium, for example, in the same manner as in the culture. The induction can be carried out in the presence or absence of serum. When carried out in the presence of serum, the concentration of the serum is not particularly limited as long as it can induce the differentiation of the myoblasts into myotubes, and is, for example, more than 0% to 5% or less, or more than 0% to 2% or less. The concentration of the serum is, for example, the concentration in the medium.

[0093] The induction may be carried out in the presence or absence of proteoglycan. When the induction is carried out in the presence of proteoglycan, the concentration of the proteoglycan is not particularly limited as long as it can induce differentiation of the myoblasts into myotubes, and is, for example, more than 0 to 2000 μg / ml or less, or 500 to 1500 μg / ml. The concentration of the proteoglycan is, for example, the concentration in the medium.

[0094] The number of days of culture for the induction can be set depending on the period during which the myoblasts are induced to differentiate into myotubes. The lower limit of the number of days of culture can be, for example, one day or more, two days or more, or more. The upper limit of the number of days of culture can be, for example, 60 days or less, 30 days or less, or 1 day or less.

[0095] After the induction, the lower limit of the proportion (cell number) of myotubes to all cells is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% 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. After the induction, the upper limit of the proportion (cell number) of myotubes to all cells is, for example, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 80% or less. After the induction, the proportion (cell number) of the myotube cells to the total cells is, for example, 50 to 99%, 50 to 90%, 50 to 80%, 50 to 70%, 50 to 60%, 60 to 99%, 60 to 90%, 60 to 85%, 60 to 80%, 60 to 75%, 60 to 70%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 85%, 75 to 99%, 75 to 90%, 80 to 99%, 80 to 95%, 80 to 90%, 90 to 99%, 91% to 98%, 92% to 97%, or 93 to 95%.

[0096] After the induction, the lower limit of the proportion of myoblasts to all cells (cell number) is, for example, more than 0%, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more. After the induction, the upper limit of the proportion of myoblasts to all cells (cell number) is, for example, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. After the induction, the proportion of the myoblasts to all cells (cell number) is, for example, greater than 0% and less than 50%, greater than 0% and less than 40%, greater than 0% and less than 30%, greater than 0% and less than 20%, greater than 0% and less than 10%, 5 to 50%, 5 to 40%, 5 to 30%, 5 to 20%, 5 to 10%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 50%, 20 to 40%, 20 to 30%, 30 to 50%, 30 to 40%, 40 to 50%, 1 to 10%, 3 to 9%, 4 to 8%, or 5 to 7%.

[0097] After the induction, the myotube cells differentiated by the induction may be further cultured. The culture can be carried out, for example, by referring to Reference 1 below. Reference 1: Shimizu Kazunori. “Study on functional expression and evaluation of cultured skeletal muscle cells using microfabricated devices.” Journal of Bioengineering 99.3 (2021): 122-128.

[0098] <Cell population containing myotubes> In another aspect, the present disclosure provides a cell population that can be used, for example, in the production of cultured meat. The cell population of the present disclosure (hereinafter also referred to as a "second cell population") comprises two or more myotube cells, the two or more myotube cells being adhered via an extracellular matrix, and the extracellular matrix comprising a proteoglycan heterologous to the species of the myotube cells. The second cell population of the present disclosure can be suitably used, for example, in the production of cultured meat, as described below.

[0099] The second cell population may include, for example, a cell mass containing the two or more myotubes.

[0100] As described above, in the second cell population, the extracellular matrix contains proteoglycans heterologous to the species of the myotube cells. That is, the extracellular matrix contains exogenous proteoglycans not derived from the myoblasts. The exogenous proteoglycans are, for example, serum-derived proteoglycans in the culture medium for culturing the cell population and / or heterologous proteoglycans added to the culture medium for culturing the cell population. The heterologous proteoglycans are, for example, proteoglycans derived from animals other than cows. Examples of combinations of the proteoglycans and the myotube cells origin include salmon and mouse, salmon and chicken, salmon and pig, salmon and cow, cow and pig, and cow and chicken.

[0101] In the second cell population, the lower limit of the proportion (cell number) of the myotubes to all cells is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more. In the second cell population, the upper limit of the proportion (cell number) of the myoblasts to all cells is, for example, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50% or less. In the cell population, the proportion (cell number) of the myoblasts to all cells is 50 to 99%, 50 to 90%, 50 to 80%, 50 to 70%, 50 to 60%, 60 to 99%, 60 to 90%, 60 to 85%, 60 to 80%, 60 to 75%, 60 to 70%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 85%, 75 to 99%, 75 to 90%, 80 to 99%, 80 to 95%, 80 to 90%, 90 to 99%, 91% to 98%, 92% to 97%, or 93 to 95%.

[0102] The second cell population may further include myoblasts, for example. When the second cell population includes myoblasts, the lower limit of the proportion (cell number) of myoblasts to all cells is, for example, more than 0%, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 45% or more, or 49% or more. The upper limit of the proportion (cell number) of myoblasts to all cells in the second cell population is, for example, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In the second cell population, the proportion (cell number) of the myoblasts to the total cells is greater than 0% and less than 50%, greater than 0% and less than 40%, greater than 0% and less than 30%, greater than 0% and less than 20%, greater than 0% and less than 10%, 5-50%, 5-40%, 5-30%, 5-20%, 5-10%, 10-50%, 10-40%, 10-30%, 10-20%, 20-50%, 20-40%, 20-30%, 30-50%, 30-40%, 40-50%, 1-10%, 3-9%, 4-8%, or 5-7%.

[0103] <Method of manufacturing cultured meat, cultured meat products, processed meat, or meat products> In another aspect, the present disclosure provides a method for producing cultured meat, a processed cultured meat product, a processed meat, or a meat product. The method for producing cultured meat, a processed cultured meat product, a processed meat, or a meat product of the present disclosure (hereinafter also referred to as the "second production method") includes a step (production step) of producing cultured meat, a processed cultured meat product, a processed meat, or a meat product.

[0104] The second production method of the present disclosure may include, prior to the production step, a step of culturing myoblasts and a step of inducing myotubes from the cultured myoblasts. The culturing may be performed by the differentiation-inhibiting method of the present disclosure or the proliferation-promoting method of the present disclosure.

[0105] The processed meat or meat product may be, for example, a meat product formed by combining the cultured meat with existing meat. When the processed meat and meat product are produced from the cultured meat, the processed meat and meat product can be produced in the same manner as when the processed meat and meat product are produced from meat derived from animals raised for livestock farming.

[0106] In the production process, the cultured meat may be used in combination with meat derived from animals raised for livestock farming, for example.

[0107] The cultured meat, processed cultured meat, processed meat, or meat product may contain, for example, additives. The additives may be, for example, (food) additives used in the meat processing, such as coloring agents, antioxidants, thickeners, vitamins, etc.

[0108] <Cultured or processed meat> In another aspect, the present disclosure provides cultured meat or processed meat, which comprises a cell population comprising the myotube cells of the present disclosure. The cultured meat or processed meat of the present disclosure can incorporate the descriptions of the first composition and medium of the present disclosure, the second composition and medium of the present disclosure, the method of inhibiting differentiation of the present disclosure, and the method of producing cultured meat of the present disclosure.

[0109] The cultured meat or processed meat may be produced by the second production method of the present disclosure. [Example]

[0110] Next, examples of the present disclosure will be described. However, the present disclosure is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be abbreviated as "M."

[0111] [Example 1] It has been confirmed that the composition of the present disclosure inhibits differentiation of myoblasts and exhibits proliferation-promoting effects.

[0112] (1) Inhibition of myoblast differentiation and promotion of proliferation 3×10 4Mouse skeletal myoblast cell line (C2C12 cells) seeded at a density of 1000 cells / well were cultured in DMEM medium (Nacalai Tesque, Inc., 08458-45) containing 2% horse serum (Sigma-Aldrich, H1138) at 37°C and 5% CO2. The medium was supplemented with 500, 1000, or 2000 μg / ml of proteoglycan (salmon nasal cartilage-derived proteoglycan, Ichimaru Pharcos, Inc.). After 3 or 5 days of culture, the cells were fixed for 15 minutes at room temperature (approximately 25°C) using 4% paraformaldehyde in PBS. After fixation, the cells were permeabilized and blocked for 1 hour at room temperature using 1% Triton™-X, 1% BSA, and 10% FBS in PBS. After blocking, primary staining was performed using primary antibodies at 4°C overnight. The primary antibodies used were anti-myosin heavy chain antibody (MF20) (500-fold dilution, Cat No. MAB4470, R&D Systems), anti-myogenin antibody (500-fold dilution, Cat No. ab124800, Abcam), anti-p-Histone H3 antibody (C-2) (1000-fold dilution, Cat No. sc-374669, Santa Cruz), and anti-cleaved caspase-3 antibody (Asp175) (5A1E) (500-fold dilution, Cat No. #9664, Cell Signaling Technology). After primary staining, the cells were washed three times with PBS containing 0.1% Triton™-X. After washing, secondary staining was performed using secondary antibodies at room temperature for 2 hours. The secondary antibodies used were Goat anti-Mouse IgG H&L (Alexa Fluor (registered trademark) 488) (Cat No: ab150113, Abcam), Goat Anti-Mouse IgG H&L (Alexa Fluor (registered trademark) 594) (Cat No: ab150116, Abcam), and Goat Anti-Rabbit IgG H&L (Alexa Fluor (registered trademark) 594) (Cat No: ab150080).After the secondary staining, the cells were washed three times with PBS containing 0.1% Triton™-X. DAPI (Cat No: D9542, Sigma-Aldrich) was used to stain the nuclei. After the washing, the cells were observed using an optical microscope (product name: Keyence, BZ-X710). A control was performed in the same manner except that no proteoglycan was added to the medium. The cell count was measured by detaching the cells with trypsin and counting using a hemocytometer. These results are shown in Figures 2 and 3.

[0113] Figure 2 shows photographs and graphs showing the results of inhibiting differentiation and promoting proliferation of myoblasts when cultured for three days using a medium containing proteoglycan. In Figure 2(A), the scale bar indicates 100 μm. In Figure 2(B), the vertical axis indicates the number of cells (×10 5 The horizontal axis indicates the presence or absence of proteoglycan added to the medium. As shown in Figure 2(A), the number of cells expressing the differentiation marker MHC decreased in proportion to the amount of proteoglycan added. As shown in Figure 2(B), the number of cells after 3 days of culture was significantly increased when 1000 μg / ml of proteoglycan was added compared to the control. These results demonstrate that adding proteoglycan to the medium can inhibit the differentiation of myoblasts into myotubes and promote myoblast proliferation.

[0114] Figure 3 shows photographs and graphs showing the results of inhibiting myoblast differentiation and promoting proliferation when cultured for 3 or 5 days in a medium containing proteoglycan. In Figure 3(A), the scale bar indicates 200 μm. In Figure 3(A), the left column shows the results for MHC markers, the middle column shows the results for Ki67 markers, and the right column shows the results for cell nuclei stained with DAPI. In Figure 3(B), the vertical axis shows the percentage of MHC-positive cells normalized by the number of DAPI-positive cells, and the horizontal axis shows the presence or absence of proteoglycan in the medium. In Figure 3(C), the horizontal axis shows the percentage of Ki67-positive cells normalized by the number of DAPI-positive cells, and the horizontal axis shows the presence or absence of proteoglycan in the medium. As shown in Figure 3(A), when cultured for 5 days in a medium containing proteoglycan, the number of cells expressing MHC markers decreased compared to the control. Furthermore, when cells were cultured for 5 days in a medium supplemented with proteoglycan, the number of cells expressing the Ki67 marker increased compared to the control. Furthermore, as shown in Figure 3(B), when cells were cultured for 3 days in a medium supplemented with proteoglycan, the percentage of MHC-positive cells (normalized by the number of DAPI-positive cells) significantly decreased compared to the control. As shown in Figure 3(C), when cells were cultured for 5 days in a medium supplemented with proteoglycan, the percentage of Ki67-positive cells (normalized by the number of DAPI-positive cells) significantly increased compared to the control. These results demonstrate that the addition of proteoglycan to the medium can inhibit the differentiation of myoblasts into myotubes and promote myoblast proliferation.

[0115] (2) Inhibition of myoblast differentiation and promotion of proliferation in 3D culture Next, we investigated whether myoblast differentiation was inhibited and proliferation was promoted when myoblasts were cultured in three dimensions. 3Mouse skeletal myoblast cell line (C2C12 cells) seeded at a density of 100 cells / well were cultured in DMEM medium (Nacalai Tesque, Inc., 08458-45) containing 10% fetal bovine serum (Gibco, 10270-106) at 37°C and 5% CO2 for 24 hours to form cell clusters. After cell cluster formation, the medium was replaced with DMEM medium containing 2% horse serum, and suspension culture was performed. The 2% horse serum-containing DMEM medium was supplemented with 200, 400, 600, 800, or 1000 μg / ml of proteoglycan (Ichimaru Pharcos, Inc.). Controls were performed in the same manner except that no proteoglycan was added to the medium. These results are shown in Figure 4.

[0116] Figure 4 shows photographs and graphs showing the results of 5 days of suspension culture using a medium containing proteoglycan. In Figure 4(A), the scale bar represents 100 μm. In Figure 4(B), the vertical axis represents the number of cells per cell cluster, and the horizontal axis represents the amount of proteoglycan added to the medium. As shown in Figure 4, the number of cells per cell cluster increased in a concentration-dependent manner. These results demonstrate that adding proteoglycan to the medium can promote myoblast proliferation.

[0117] (3) Promotes myoblast proliferation in high-serum medium Next, we investigated whether myoblast proliferation was promoted when myoblasts were cultured in a high-serum medium containing proteoglycan. 4 Mouse skeletal myoblast cell line (C2C12 cells) seeded at a density of 1000 cells / well were cultured for 3 days at 37°C and 5% CO2 in DMEM medium (Nacalai Tesque, Inc., 08458-45) containing 10% fetal bovine serum (Gibco, 10270-106). 1000 μg / ml of proteoglycan (Ichimaru Pharcos, Inc.) was added to the 10% fetal bovine serum-containing DMEM medium. A control was performed in the same manner except that no proteoglycan was added to the medium. These results are shown in Figure 5.

[0118] Figure 5 shows photographs and graphs showing the results of three days of culture using a medium containing proteoglycan. In Figure 5(A), the scale bar indicates 100 μm. In Figure 5(B), the vertical axis indicates the number of cells after three days of culture, and the horizontal axis indicates the amount of proteoglycan added to the medium. As shown in Figure 5, the number of cells after three days of culture increased with the addition of proteoglycan. These results demonstrate that adding proteoglycan to the medium can promote myoblast proliferation.

[0119] (4) Promotes myoblast proliferation in high-serum medium Next, we investigated whether myoblast proliferation was promoted when chicken-derived myoblasts were cultured in a high-serum medium. Specifically, chicken embryo-derived skeletal myoblasts were obtained by sorting from the thighs of 10-day-old chicken embryos. 5 × 10 5 The chick embryo-derived skeletal myoblasts were seeded at a density of 100 cells / well and cultured in DMEM medium (Nacalai Tesque, Inc., 08458-45) containing 10% fetal bovine serum (Gibco, 10270-106) at 37°C and 5% CO for 24 hours to confirm adhesion to the culture dish. After this confirmation, the medium was replaced with DMEM medium containing 2% horse serum, and the cells were cultured for 6 hours. The 2% horse serum-containing DMEM medium was supplemented with 500 or 1000 μg / ml of proteoglycan (Ichimaru Pharcos Co., Ltd.). After the culture, nuclear staining was performed using DAPI (Cat No. D9542, Sigma-Aldrich) and the cells were observed using an optical microscope (Keyence, BZ-X710). A control was performed in the same manner except that proteoglycan was not added to the medium. These results are shown in Figure 6.

[0120] Figure 6 shows photographs and graphs showing the results of culturing chicken embryo-derived myoblasts in a proteoglycan-containing medium for 6 hours. In Figure 6(A), the scale bar represents 100 μm. In Figure 6(B), the vertical axis represents the number of cells, and the horizontal axis represents the amount of proteoglycan added to the medium. As shown in Figure 6, when a medium containing proteoglycan was used, an increase in cell number was observed compared to the control. These results demonstrate that adding proteoglycan to the medium can promote the proliferation of chicken-derived myoblasts.

[0121] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0122] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Composition for use in inhibiting myoblast differentiation> (Appendix 1) A composition for use in inhibiting myoblast differentiation, comprising a proteoglycan. (Appendix 2) 2. The composition of claim 1, wherein the proteoglycan comprises aggrecan. (Appendix 3) 3. The composition of claim 1 or 2, wherein the proteoglycan comprises an animal proteoglycan. (Appendix 4) 4. The composition of claim 3, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray. (Appendix 5) 5. The composition according to any one of claims 1 to 4, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000. (Appendix 6) 6. The composition according to any one of claims 1 to 5, wherein the sugar chain of the proteoglycan is chondroitin sulfate. <Culture medium for inhibiting myoblast differentiation> (Appendix 7) A medium for use in inhibiting myoblast differentiation, comprising a composition for use in inhibiting myoblast differentiation described in any one of Appendices 1 to 6. <Culture medium kit for inhibiting myoblast differentiation> (Appendix 8) A culture medium kit for use in inhibiting myoblast differentiation, comprising a composition for use in inhibiting myoblast differentiation according to any one of Appendices 1 to 6, and a culture medium. <Composition for use in promoting proliferation of myoblasts> (Appendix 9) A composition for use in promoting myoblast proliferation, comprising a proteoglycan. (Appendix 10) 10. The composition of claim 9, wherein the proteoglycan comprises aggrecan. (Appendix 11) 11. The composition of claim 9 or 10, wherein the proteoglycan comprises an animal proteoglycan. (Appendix 12) 12. The composition of claim 11, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray. (Appendix 13) 13. The composition according to any one of claims 9 to 12, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000. (Appendix 14) 14. The composition according to any one of claims 9 to 13, wherein the sugar chain of the proteoglycan is chondroitin sulfate. <Culture medium for promoting myoblast proliferation> (Appendix 15) A medium for use in promoting the proliferation of myoblasts, comprising a composition for use in promoting the proliferation of myoblasts described in any one of Appendices 9 to 14. <Culture medium kit for promoting myoblast proliferation> (Appendix 16) A culture medium kit for use in promoting proliferation of myoblasts, comprising a composition for use in promoting proliferation of myoblasts described in any one of Appendices 9 to 14 and a culture medium. <Method for inhibiting myoblast differentiation> (Appendix 17) A method for inhibiting myoblast differentiation, comprising the step of culturing myoblasts in the presence of proteoglycan. (Appendix 18) 18. The differentiation-inhibiting method according to claim 17, wherein after the culture, the proportion of myoblasts among all cells is 90% or more. (Appendix 19) 19. The differentiation-inhibiting method according to claim 17 or 18, wherein after the culture, the proportion of myotube cells among all cells is 10% or less. (Appendix 20) 20. The differentiation-inhibiting method according to any one of appendix 17 to 19, wherein the concentration of the proteoglycan is 200 to 3000 μg / ml. (Appendix 21) 21. The differentiation-inhibiting method according to any one of claims 17 to 20, wherein the culturing is carried out until the total number of cells after the culturing is 4 to 6 times the total number of cells at the start of the culturing. (Appendix 22) 22. The differentiation-inhibiting method according to any one of claims 17 to 21, wherein the culture is carried out in the presence of serum. (Appendix 23) 23. The differentiation-inhibiting method described in Appendix 22, wherein the serum concentration is greater than 0%. (Appendix 24) 24. The differentiation-inhibiting method according to any one of claims 17 to 23, wherein the culture is a suspension culture. (Appendix 25) A differentiation-inhibiting method described in any one of Appendices 17 to 24, wherein the proteoglycan includes aggrecan. (Appendix 26) A differentiation-inhibiting method according to any one of Appendices 17 to 25, wherein the proteoglycan includes an animal proteoglycan. (Appendix 27) 27. The differentiation-inhibiting method according to any one of claims 17 to 26, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000. (Appendix 28) 28. The differentiation-inhibiting method according to any one of claims 17 to 27, wherein the sugar chain of the proteoglycan is chondroitin sulfate. (Appendix 29) A differentiation-inhibiting method according to any one of Appendices 17 to 28, wherein the proteoglycan is an animal proteoglycan. (Appendix 30) The differentiation-inhibiting method according to claim 29, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, and ray. (Appendix 31) 30. The differentiation-inhibiting method according to any one of claims 17 to 29, wherein the proteoglycan is heterologous to the species of the myoblast. <Method for promoting myoblast proliferation> (Appendix 32) A method for promoting myoblast proliferation, comprising the step of culturing myoblasts in the presence of proteoglycan. (Appendix 33) 33. The proliferation promoting method according to claim 32, wherein after the culture, the proportion of myoblasts among all cells is 90% or more. (Appendix 34) 34. The proliferation promotion method according to claim 32 or 33, wherein after the culture, the proportion of myotube cells among all cells is 10% or less. (Appendix 35) 35. The proliferation-promoting method according to any one of appendices 32 to 34, wherein the concentration of the proteoglycan is 200 to 3000 μg / ml. (Appendix 36) 36. The proliferation promoting method according to any one of Appendices 32 to 35, wherein the culturing is carried out until the total number of cells after the culturing is 4 to 6 times the total number of cells at the start of the culturing. (Appendix 37) 37. The method for promoting proliferation according to any one of claims 32 to 36, wherein the culture is carried out in the presence of serum. (Appendix 38) 38. The method for promoting proliferation described in Appendix 37, wherein the concentration of serum is greater than 0%. (Appendix 39) 39. The proliferation promotion method according to any one of Appendices 32 to 38, wherein the culture is a suspension culture. (Appendix 40) A proliferation-promoting method described in any of Appendices 32 to 39, wherein the proteoglycan includes aggrecan. (Appendix 41) A method for promoting proliferation according to any one of claims 32 to 40, wherein the proteoglycan comprises an animal proteoglycan. (Appendix 42) 42. The proliferation-promoting method according to any one of claims 32 to 41, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000. (Appendix 43) 43. The method for promoting proliferation according to any one of claims 32 to 42, wherein the sugar chain of the proteoglycan is chondroitin sulfate. (Appendix 44) A method for promoting proliferation according to any one of Appendices 32 to 43, wherein the proteoglycan is an animal proteoglycan. (Appendix 45) 45. The method for promoting proliferation described in Appendix 44, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, and ray. (Appendix 46) 46. The method of any of claims 32 to 45, wherein the proteoglycan is heterologous to the species of the myoblasts. <Cell population containing myoblasts> (Appendix 47) containing two or more myoblasts, the two or more myoblasts are adhered via an extracellular matrix; A cell population, wherein the extracellular matrix comprises proteoglycans heterologous to the species of the myoblasts. (Appendix 48) 48. The cell population of claim 47, comprising a cell mass containing two or more myoblasts. (Appendix 49) 49. The cell population according to claim 47 or 48, wherein the proportion of myoblasts among all cells in the cell population is 90% or more. (Appendix 50) Contains myotube cells, 50. The cell population according to any one of claims 47 to 49, wherein the proportion of myotube cells among all cells in the cell population is 10% or less. (Appendix 51) 51. The cell population of any of claims 47 to 50, wherein the proteoglycan comprises aggrecan. (Appendix 52) 52. The cell population of any of claims 47 to 51, wherein the proteoglycan comprises an animal proteoglycan. (Appendix 53) 53. The cell population of any of claim 52, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, and ray. (Appendix 54) 54. The cell population according to any one of Appendices 47 to 53, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000. (Appendix 55) 55. The cell population according to any one of Appendices 47 to 54, wherein the sugar chain of the proteoglycan is chondroitin sulfate. (Appendix 56) A cell population containing myoblasts, obtained by the method for inhibiting differentiation described in any one of Appendices 17 to 31 or the method for promoting proliferation described in any one of Appendices 32 to 46. <Method for producing myotube cells (muscle fibers)> (Appendix 57) Culturing myoblasts; and inducing myotubes from the cultured myoblasts, A method for producing myotubes, wherein the culturing is carried out by the differentiation-inhibiting method described in any one of Appendices 17 to 31 or the proliferation-promoting method described in any one of Appendices 32 to 46. <Cell population containing myotubes> (Appendix 58) comprising two or more myotubes, the two or more myotubes are adhered via an extracellular matrix; A cell population, wherein the extracellular matrix comprises proteoglycans heterologous to the species of the myotube cells. (Appendix 59) 59. The cell population of claim 58, comprising a cell mass containing the two or more myotube cells. (Appendix 60) 60. The cell population described in Appendix 58 or 59, wherein the proportion of myotube cells among all cells in the cell population is 50% or more. (Appendix 61) Contains myoblasts, 61. The cell population according to any one of claims 58 to 60, wherein the proportion of myoblasts among all cells in the cell population is 50% or less. (Appendix 62) 62. The cell population of any of claims 58 to 61, wherein the proteoglycan comprises aggrecan. (Appendix 63) 63. The cell population of any of claims 58 to 62, wherein the proteoglycan comprises an animal proteoglycan. (Appendix 64) 64. The cell population of any of claim 63, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, and ray. (Appendix 65) 65. The cell population according to any one of Appendices 58 to 64, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000. (Appendix 66) 66. The cell population described in any one of Appendices 58 to 65, wherein the sugar chain of the proteoglycan is chondroitin sulfate. (Appendix 67) A cell population containing myotubes obtained by the method for producing myotubes described in Appendix 57. <Method of manufacturing cultured meat, cultured meat products, processed meat, or meat products> (Appendix 68) 68. A method for producing cultured meat, a cultured meat product, a processed meat, or a meat product, comprising producing the cultured meat, a cultured meat product, a processed meat, or a meat product from a cell population comprising the myotube cells of any of Supplementary Notes 58 to 67. (Appendix 69) Culturing myoblasts; and inducing myotubes from the cultured myoblasts, A production method described in Appendix 68, wherein the culture is carried out using a differentiation-inhibiting method described in any one of Appendixes 17 to 31 or a proliferation-promoting method described in any one of Appendixes 32 to 46. <Cultured or processed meat> (Appendix 70) 68. Cultured meat, a cultured meat product, a processed meat, or a meat product comprising a cell population comprising myotubes of any of claims 58 to 67. [Industrial Applicability]

[0123] As described above, the present disclosure can provide, for example, a composition for use in inhibiting myoblast differentiation or promoting myoblast proliferation. Therefore, the present disclosure can be said to be extremely useful, for example, in the food industry.

Claims

1. A composition for use in inhibiting myoblast differentiation, comprising a proteoglycan.

2. The composition of claim 1 , wherein the proteoglycan comprises aggrecan.

3. The composition of claim 1 or 2, wherein the proteoglycan comprises an animal proteoglycan.

4. 4. The composition of claim 3, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray.

5. The composition according to any one of claims 1 to 4, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000.

6. The composition according to claim 1 , wherein the sugar chain of the proteoglycan is chondroitin sulfate.

7. A medium for use in inhibiting myoblast differentiation, comprising the composition for use in inhibiting myoblast differentiation according to any one of claims 1 to 6.

8. A culture medium kit for use in inhibiting myoblast differentiation, comprising the composition for use in inhibiting myoblast differentiation according to any one of claims 1 to 6 and a culture medium.

9. A composition for use in promoting myoblast proliferation, comprising a proteoglycan.

10. The composition of claim 9 , wherein the proteoglycan comprises aggrecan.

11. The composition of claim 9 or 10, wherein the proteoglycan comprises an animal proteoglycan.

12. 12. The composition of claim 11, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray.

13. The composition according to any one of claims 9 to 12, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000.

14. The composition according to any one of claims 9 to 13, wherein the sugar chain of the proteoglycan is chondroitin sulfate.

15. A medium for use in promoting the proliferation of myoblasts, comprising the composition for use in promoting the proliferation of myoblasts according to any one of claims 9 to 14.

16. A culture medium kit for use in promoting the proliferation of myoblasts, comprising the composition for use in promoting the proliferation of myoblasts according to any one of claims 9 to 14 and a culture medium.

17. A method for inhibiting myoblast differentiation, comprising the step of culturing myoblasts in the presence of proteoglycan.

18. The differentiation-inhibiting method according to claim 17, wherein after the culture, the proportion of myoblasts among all cells is 90% or more.

19. The differentiation-inhibiting method according to claim 17 or 18, wherein after the culture, the proportion of myotube cells among all cells is 10% or less.

20. The differentiation-inhibiting method according to any one of claims 17 to 19, wherein the concentration of the proteoglycan is 200 to 3000 µg / ml.

21. The differentiation inhibiting method according to any one of claims 17 to 20, wherein the culturing is performed until the total number of cells after the culturing is 4 to 6 times the total number of cells at the start of the culturing.

22. The differentiation-inhibiting method according to any one of claims 17 to 21, wherein the culture is carried out in the presence of serum.

23. The differentiation inhibiting method according to claim 22 , wherein the serum concentration is greater than 0%.

24. The differentiation-inhibiting method according to any one of claims 17 to 23, wherein the culture is a suspension culture.

25. The differentiation-inhibiting method according to any one of claims 17 to 24, wherein the proteoglycan includes aggrecan.

26. The differentiation-inhibiting method according to any one of claims 17 to 25, wherein the proteoglycan includes an animal proteoglycan.

27. The differentiation-inhibiting method according to any one of claims 17 to 26, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000.

28. The differentiation-inhibiting method according to any one of claims 17 to 27, wherein the sugar chain of the proteoglycan is chondroitin sulfate.

29. The differentiation-inhibiting method according to any one of claims 17 to 28, wherein the proteoglycan is an animal proteoglycan.

30. 30. The method for inhibiting differentiation according to claim 29, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, and ray.

31. 30. The method of any one of claims 17 to 29, wherein the proteoglycan is heterologous to the species of the myoblast.

32. A method for promoting myoblast proliferation, comprising the step of culturing myoblasts in the presence of proteoglycan.

33. The proliferation promoting method according to claim 32, wherein after the culture, the proportion of myoblasts among all cells is 90% or more.

34. The method for promoting proliferation according to claim 32 or 33, wherein after the culture, the proportion of myotube cells among all cells is 10% or less.

35. The proliferation-promoting method according to any one of claims 32 to 34, wherein the concentration of the proteoglycan is 200 to 3000 μg / ml.

36. The proliferation promotion method according to any one of claims 32 to 35, wherein the culture is performed until the total number of cells after the culture is 4 to 6 times the total number of cells at the start of the culture.

37. The method for promoting proliferation according to any one of claims 32 to 36, wherein the culture is carried out in the presence of serum.

38. The method for promoting proliferation according to claim 37, wherein the serum concentration is greater than 0%.

39. The proliferation promoting method according to any one of claims 32 to 38, wherein the culture is a suspension culture.

40. 40. The method for promoting proliferation according to any one of claims 32 to 39, wherein the proteoglycan comprises aggrecan.

41. The growth-promoting method according to any one of claims 32 to 40, wherein the proteoglycan comprises an animal proteoglycan.

42. The method for promoting proliferation according to any one of claims 32 to 41, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000.

43. The method for promoting proliferation according to any one of claims 32 to 42, wherein the sugar chain of the proteoglycan is chondroitin sulfate.

44. The method for promoting proliferation according to any one of claims 32 to 43, wherein the proteoglycan is an animal proteoglycan.

45. 45. The method for promoting growth according to claim 44, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, and ray.

46. 46. The method of any one of claims 32 to 45, wherein the proteoglycan is heterologous to the species of the myoblasts.

47. comprising two or more myoblasts, the two or more myoblasts are adhered via an extracellular matrix; A cell population, wherein the extracellular matrix comprises proteoglycans heterologous to the species of the myoblasts.

48. The cell population of claim 47, comprising a cell cluster containing the two or more myoblasts.

49. The cell population described in claim 47 or 48, wherein the proportion of myoblasts among all cells in the cell population is 90% or more.

50. Contains myotube cells, 50. The cell population according to any one of claims 47 to 49, wherein the proportion of myotube cells among all cells in the cell population is 10% or less.

51. 51. The cell population of any one of claims 47 to 50, wherein the proteoglycan comprises aggrecan.

52. 52. The cell population of any one of claims 47 to 51, wherein the proteoglycan comprises an animal proteoglycan.

53. 53. The cell population of any one of claims 52, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray.

54. The cell population according to any one of claims 47 to 53, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000.

55. The cell population described in any one of claims 47 to 54, wherein the sugar chain of the proteoglycan is chondroitin sulfate.

56. A cell population containing myoblasts, obtained by the differentiation-inhibiting method according to any one of claims 17 to 31 or the proliferation-promoting method according to any one of claims 32 to 46.

57. Culturing myoblasts; and inducing myotubes from the cultured myoblasts, A method for producing myotubes, wherein the culture is carried out by the differentiation-inhibiting method according to any one of claims 17 to 31 or the proliferation-promoting method according to any one of claims 32 to 46.

58. comprising two or more myotubes; the two or more myotubes are adhered via an extracellular matrix; A cell population, wherein the extracellular matrix comprises proteoglycans heterologous to the species of the myotube cells.

59. The cell population of claim 58, comprising a cell mass containing the two or more myotube cells.

60. The cell population described in claim 58 or 59, wherein the proportion of myotube cells among all cells in the cell population is 50% or more.

61. Contains myoblasts, 61. A cell population according to any one of claims 58 to 60, wherein the proportion of myoblasts among all cells in the cell population is 50% or less.

62. 62. The cell population of any one of claims 58 to 61, wherein the proteoglycan comprises aggrecan.

63. 63. The cell population of any one of claims 58 to 62, wherein the proteoglycan comprises an animal proteoglycan.

64. 64. The cell population of any one of claims 63, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray.

65. The cell population according to any one of claims 58 to 64, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000.

66. The cell population described in any one of claims 58 to 65, wherein the sugar chain of the proteoglycan is chondroitin sulfate.

67. A cell population containing myotubes obtained by the method for producing myotubes described in claim 57.

68. 68. A method for producing cultured meat, a cultured meat product, a processed meat, or a meat product, comprising producing the cultured meat, a cultured meat product, a processed meat, or a meat product from a cell population comprising the myotube cells of any one of claims 58 to 67.

69. Culturing myoblasts; and inducing myotubes from the cultured myoblasts, The method of claim 68, wherein the culture is carried out by a differentiation-inhibiting method according to any one of claims 17 to 31 or a proliferation-promoting method according to any one of claims 32 to 46.

70. 68. A cultured meat, processed cultured meat, processed meat, or meat product comprising a cell population comprising the myotube cells of any one of claims 58 to 67.

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