Inhibitor of differentiation of tendon tissue-derived cells

Abstract

In order to obtain muscle tissue-derived cells for use in the production of cultured meat, it is necessary to culture proliferative myoblasts and muscle satellite cells. However, these cells easily differentiate, and have a tendency, even in normal culture conditions, to differentiate to myotube cells which are not proliferative. Here, the objective of the present invention is to identify a component that inhibits differentiation of myoblasts and muscle satellite cells in order to culture a large amount of muscle tissue-derived cells for production of cultured meat. Food components used as additives were studied for differentiation suppressing effect, and the study revealed that when whey was added, differentiation of myoblasts and muscle satellite cells was successfully suppressed, and thus a differentiation suppressing agent is provided.

Description

Technical Field

[0001] The present invention relates to the technical field of cultured meat production. More specifically, it relates to an inhibitor for suppressing the differentiation of muscle tissue-derived cells added in the cell culture process for cultured meat production.

Background Art

[0002] Meat production has hitherto been carried out by raising livestock. On the other hand, raising livestock requires large amounts of grain and water and large breeding farms. In recent years, issues such as climate change and food shortages have been taken up, and there has been a growing demand for sustainable meat production with a lower environmental impact and higher production efficiency. Against this backdrop, research and development on producing cultured meat from cells has attracted attention as a new meat production method.

[0003] As a substitute for meat, plant-based alternative meat is known, but its texture and taste do not match those of real meat. On the other hand, cultured meat produced by culturing animal cells can achieve a texture and taste similar to those of real meat, and has the advantage of a lower risk of bacterial and viral contamination compared to real meat. Technologically, the production of cultured meat has become possible. However, the cell culture media used in the production of cultured meat to date utilize large-scale culture techniques used in basic research and pharmaceutical applications, and it has been difficult to use them for food production in terms of cost and safety as food. In cell culture media used in basic research and pharmaceutical applications, it is common to add fetal bovine serum (FBS) as an additive to a basal medium containing carbon sources such as amino acids, vitamins, inorganic salts, and glucose (Non-Patent Document 1: Mol Ther. 2004 Mar;9(3):475-82). On the other hand, since FBS is serum collected from fetuses, it is difficult to obtain in large quantities and has issues in terms of price, transportation cost, infection risk, and animal welfare. To solve such problems, a completely synthetic medium supplemented with essential components of FBS as reagents has been developed (Non-Patent Document 2: The Canadian Journal of Chem Engineering Vol.94, (10) October 2016 1855-1862). However, such completely synthetic media use recombinant proteins, hormonal agents, serum-derived components, etc., and have issues for use as food.

[0004] Various approaches have been attempted for cell culture media for the production of cultured meat. Examples include media using products of organ cells (Patent Document 1: Japanese Patent No. 6111510), media using algal products (Non-Patent Document 3: Scientific Reports. Jan 31;7:41594), media using hydrolysates of food residues (Non-Patent Document 4: Food Funct., 2020,11, 2477-2488), and media using other food raw material components (Patent Document 2: WO 2021 / 148955).

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Japanese Patent No. 6111510 [Patent Document 2] International Publication No. 2021 / 148955 [Non-Patent Document]

[0006] [Non-Patent Document 1] Mol Ther. 2004 Mar;9(3):475-82 [Non-Patent Document 2] The Canadian Journal of Chem Engineering Vol.94, (10) October 2016 1855-1862 [Non-Patent Document 3] Scientific Reports. Jan 31;7:41594 [Non-Patent Document 4] Food Funct., 2020,11, 2477-2488 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] As muscle tissue-derived cells used for the production of cultured meat, it is necessary to culture myoblasts and satellite cells having proliferative ability. However, these cells are prone to differentiation and tend to differentiate into myotube cells that do not have proliferative ability even under normal culture conditions. In addition, although they can be proliferated while suppressing differentiation by culturing in a medium supplemented with FBS, in the production of cultured meat, it is desired to avoid the use of FBS from the viewpoints of price, infectious disease risk, and animal welfare. Therefore, in the production of cultured meat, in order to culture a large amount of muscle tissue-derived cells, it is necessary to suppress the differentiation of myoblasts and satellite cells in a medium without FBS. [Means for Solving the Problems]

[0008] When the present inventors conducted intensive research on components that suppress the differentiation of myoblasts and satellite cells in culturing muscle tissue-derived cells, they found that by adding whey as an additive to the culture medium, the differentiation of myoblasts and satellite cells can be suppressed, leading to the present invention. Accordingly, the present invention relates to the following: [1] An inhibitor of differentiation of muscle tissue-derived cells, comprising whey. [2] The inhibitor according to item 1, wherein the muscle tissue-derived cells are selected from the group consisting of myoblasts and satellite cells. [3] The inhibitor according to item 1 or 2, wherein the inhibitor suppresses the differentiation of myoblasts and satellite cells into myotube cells. [4] The inhibitor according to any one of items 1 to 3, wherein the muscle tissue-derived cells are muscle tissue-derived cells from livestock. [5] The inhibitor according to any one of items 1 to 4, wherein the inhibitor is added to a culture medium for culturing meat. [6] The inhibitor according to any one of claims 1 to 5, wherein the whey concentration is in the range of 0.005% to 1.0%. [Effect of the Invention]

[0009] By adding whey to the basal medium, the differentiation of myoblasts and satellite cells into myotube cells can be suppressed. [Brief Description of the Drawings]

[0010]

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[0011] The present invention relates to an inhibitor for suppressing the differentiation of muscle tissue-derived cells containing whey (hereinafter, also referred to as the differentiation inhibitor according to the present invention). In another aspect, the present invention also relates to a medium for culturing meat, which comprises a basal medium and whey as an inhibitor for suppressing the differentiation of muscle tissue-derived cells. The present invention also relates to a method for preparing cells for culturing meat, which includes a step of culturing cells in a medium comprising a basal medium and whey as an inhibitor for suppressing the differentiation of muscle tissue-derived cells, and a method for producing culturing meat from the prepared cells.

[0012] [Differentiation Inhibitor] The inhibitor of the differentiation of muscle tissue-derived cells according to the present invention contains whey. By containing whey, it is possible to inhibit the differentiation of muscle tissue-derived cells, particularly myoblasts and satellite cells, into myotubes. By inhibiting differentiation, proliferative myoblasts and satellite cells can be maintained. In addition, whey has a cell proliferation promoting effect on myoblasts and satellite cells. The cell proliferation promoting effect of whey and the inhibitory effect on differentiation into myotubes are comparable to those of FBS, respectively (Figs. 4 and 5). As a result, it becomes possible to prepare a large number of cells for the production of cultured meat and to use it as an alternative to FBS. The method for preparing cells for the production of cultured meat according to the present invention includes the step of culturing cells in a medium containing a basal medium and whey as an inhibitor of the differentiation of muscle tissue-derived cells, whereby it is possible to proliferate while inhibiting the differentiation into non-proliferative myotubes. In addition, since whey is a food raw material, the differentiation inhibitor according to the present invention has high safety as a food. In addition, since whey is an inexpensive raw material, the medium added with the differentiation inhibitor according to the present invention also has the advantage of low preparation cost. Cells cultured in such a medium can be used for the production of cultured meat because of their high safety as a food.

[0013] The differentiation inhibitor according to the present invention can be added to a known medium. The differentiation inhibitor according to the present invention may be added to a serum-free medium or a medium containing animal-derived serum. From the viewpoint of producing cultured meat, it is preferable to add it to a serum-free medium. After adding whey to the medium, by performing filter filtration, a medium from which insoluble matter has been removed can be used for culturing. By adding whey to the basal medium, even in a serum-free medium, it is possible to achieve high cell proliferation activity while inhibiting the differentiation of muscle tissue-derived cells, particularly myoblasts and satellite cells, into myotubes. Therefore, whey used in the present invention can be said to be both a differentiation inhibitor and a cell proliferation promoter. The differentiation inhibitor according to the present invention can be used in cell culture for the production of cultured meat.

[0014] Whey (also referred to as milk serum or whey) refers to an aqueous solution obtained by removing solids from milk. It is inexpensive because it is produced in large quantities as a byproduct in the process of manufacturing dairy products such as cheese and yogurt. More specifically, whey can be obtained by separating solids from curd that has been coagulated by adding a coagulant such as rennet to milk or fermented milk. As solids, part or all of proteins such as milk fat and casein are removed. The main components of whey are lactoglobulin, lactalbumin, and lactoferrin, but it also contains various trace components such as free amino acids, inorganic salts, and vitamins.

[0015] The whey used in the present invention may be whey derived from any mammal. As an example, whey obtained from the milk of cows, horses, goats, sheep, humans, or donkeys can be used. The whey may be in liquid form or may be a dry powder obtained by drying whey. From the perspective of adding it as a differentiation inhibitor, a dry powder form is preferred because it can be expected to reduce transportation costs. For commercially available dry powder forms of whey, they may be used, or whey may be prepared by freeze-drying. When using dry powder whey as a differentiation inhibitor, it is added to the basal medium so as to be 0.0025% to 1.0% by mass. From the perspective of exerting a differentiation inhibitory effect, the concentration of whey is preferably 0.005% by mass or more, more preferably 0.05% by mass or more, and even more preferably 0.1% or more. From the perspective of dissolution efficiency, it is preferably 0.5% by mass or less, and even more preferably 0.25% by mass or less. When using liquid whey, the addition amount can be determined by converting it to dry powder.

[0016] [Medium] The medium to which the differentiation inhibitor according to the present invention is added can be added to a medium known in the art. The differentiation inhibitor according to the present invention may be added to a serum-free medium or to a medium containing animal-derived serum. These media can be produced by adding additives to a basal medium.

[0017] A basal medium refers to a medium for cell culture that contains the minimum components necessary for cell maintenance and growth. By seeding cells in the basal medium, the cells can be maintained without being killed and may be capable of growing. Various basal media are commercially available. Usually, they contain amino acids, vitamins, buffers, inorganic salts, and a carbon source. The amino acids include essential amino acids and non-essential amino acids. The vitamins include vitamin B1, vitamin C, nicotinic acid, folic acid, etc. The buffers include HEPES, etc. As the carbon source, monosaccharides such as glucose, disaccharides such as sucrose, oligosaccharides, and polysaccharides can be added. Usually, a cell culture medium can be prepared by adding additives such as serum to the basal medium. As the basal medium, any basal medium known in the art can be used. Examples include Dulbecco's Modified Eagle Medium (DMEM), Eagle's Basal Medium (BME), RPMI1640 medium, DMEM / F12 medium, F10 medium, Ham's F12 medium, MEM, M199 medium, Ames medium, Iscove's Modified Medium, Glasgow Modified Medium, Fisher's medium, etc.

[0018] In cell culture, additives are usually added to a basal medium. In conventional cell culture, serum such as fetal bovine serum (FBS) is added as an additive and used as a medium containing animal-derived serum. FBS promotes cell growth while suppressing the differentiation of cells derived from muscle tissue. In addition to FBS, other additives may be added according to the cell type and the like. Also, in serum-free media and low-serum media, components necessary to maintain cell growth are included instead of FBS. Such additives include, for example, lipids, hormonal agents, growth factors, cytokines, serum-derived proteins, antibiotics, etc., and can be added to serum-free media and / or media containing animal-derived serum. Examples of hormonal agents include dexamethasone. Examples of growth factors include FGF, IGF, insulin, etc., and any family thereof may be used. Examples of cytokines include IL-1α, IL-1β, etc. Examples of serum-derived proteins include fetuin, fibronectin, albumin, globulin, etc. As antibiotics, penicillin, streptomycin, etc. can be used. ITS (insulin-transferrin-sodium selenite), which is an additive commonly used in serum-free media and low-serum media, can also be added to a medium, particularly a serum-free medium, together with the differentiation inhibitor according to the present invention.

[0019] Animal-derived serum refers to serum produced from the blood of an animal. The supernatant obtained by coagulating the obtained blood is called serum. Animal-derived serum may be serum derived from any animal, for example, animals such as cows, horses, goats, donkeys, rabbits, birds, etc., but particularly refers to bovine serum (BCS) and fetal bovine serum (FBS). Serum contains, in addition to proteins such as albumin and globulin, serum lipids such as neutral fat, cholesterol, phospholipid, and free fatty acid, and further contains hormones, cytokines, growth factors, etc. In particular, fetal serum contains abundant components necessary for cell growth and is generally added to the medium in the research and pharmaceutical fields. A medium that does not contain animal-derived serum is called a serum-free medium. On the other hand, a serum-free medium does not contain animal-derived serum, but may contain purified components derived from serum or may contain recombinant proteins of serum-derived components.

[0020] [Cell] The differentiation-inhibiting medium according to the present invention can inhibit differentiation in cells derived from muscle tissue obtained from any animal. From the perspective of culturing meat, cells derived from livestock such as cows, pigs, goats, sheep, rabbits, chickens, ostriches, and ducks can be used. Particularly when using bovine cells, cells of any species among Holstein, Jersey, Japanese Black, Brown Japanese, Shorthorn, Polled Japanese, and their crossbreds may be used. Particularly from the perspective of meat production, cells of meat breeds such as Japanese Black, Brown Japanese, Shorthorn, and Polled Japanese are preferred. Cells derived from the muscle tissue of these animals can be cultured. The cells derived from the muscle tissue of an animal may be primary cells obtained from the animal, subcultured cells subcultured from the primary cells, or established cells. The primary cells can be obtained by mincing animal tissue in a medium. They may also be cells differentiated from stem cells such as somatic stem cells, embryonic stem cells, and induced pluripotent stem cells.

[0021] Skeletal muscle tissue-derived cells are cells that constitute skeletal muscle tissue and are cells isolated from skeletal muscle tissue and cultured. Examples of skeletal muscle tissue-derived cells include myoblasts, satellite cells, and myotube cells. However, since myotube cells do not have proliferative ability, from the perspective of proliferation, myoblasts and / or satellite cells are preferred. Satellite cells are somatic stem cells contained in muscle and are characterized by the expression of the transcription factor Pax7. Satellite cells usually maintain a quiescent state (resting phase) in vivo and differentiate into proliferative myoblasts upon activation by injury or the like. When performing cell culture, depending on the culture conditions, satellite cells can also be proliferated in an undifferentiated state. Myoblasts are mononuclear cells with proliferative ability that can form muscle fibers and are characterized by the expression of MyoD. When myoblasts further differentiate, they fuse with each other to form multinucleated myotube cells, which further mature into muscle fibers. Differentiation into myotube cells can be determined by observing multinucleation and shape (Figs. 1 to 3). Furthermore, differentiation from myoblasts to myotube cells can also be determined by measuring the expression of genes specifically expressed in myotube cells, such as desmin and myogenin (Fig. 6). The differentiation inhibitor of the present invention can inhibit the differentiation from satellite cells to myoblasts and the differentiation from myoblasts to myotube cells, respectively. On the other hand, myoblasts and satellite cells differentiate into myotube cells when they reach confluence and a certain period of time has elapsed.

[0022] [Cultured meat] Cultured meat refers to meat produced through cell culture. In the present invention, "for the production of cultured meat" refers to a method used for the production of cultured meat, which is required to be acceptable from the perspective of food hygiene. From the perspective of food hygiene, it is preferably avoided to use animal-derived serum, hormonal agents, and recombinant proteins. Generally, meat refers to an aggregate of muscle fibers, connective tissue, and fat. On the other hand, cultured meat preferably mimics the structure of meat, but does not necessarily contain all the components of meat, and may contain cultured cells of skeletal muscle tissue-derived cells. Cultured meat may contain a culture of multiple types of cells. Cultured meat may contain an extracellular matrix in addition to at least one cultured cell selected from the group consisting of skeletal muscle tissue-derived cells. Methods for producing cultured meat are, for example, as follows A step of culturing cells derived from muscle tissue, A step of collecting and accumulating the cultured cells is included. The method for producing cultured meat may further include a differentiation induction step and a culturing step after accumulation. The present invention also relates to cultured meat containing cells cultured in a medium containing a differentiation inhibitor according to the present invention.

[0023] The culturing of cells is performed by seeding the cells in a medium containing whey as a differentiation inhibitor in a basal medium. The culturing is carried out under conditions well known in the art, for example, in a 37°C 5% CO2 incubator. The culturing may be monolayer culture or suspension culture. The proliferated cells can be recovered as a culture by trypsin treatment or the like, and subculture may be further performed after recovery. The culturing of cells can also be carried out by seeding the cells on a detachable construct and culturing. The construct to which the proliferated cells are attached can be recovered as a culture. Such a construct can be constructed with an extracellular matrix such as collagen, elastin, fibronectin, laminin, entactin, etc., and the constructs to which cells are attached can be accumulated to form cultured meat.

[0024] The accumulation step includes molding a culture of one or more types of recovered cells. The culture molded in the accumulation step may be a single piece of meat such as a steak, or may be a branched meat, or may be minced. The accumulation step includes accumulating a culture of cells together with at least one substance selected from the group consisting of other cells, blood, and tissue. The other cells may be cultured cells or cells collected from an animal. Co-culture can also be performed after accumulation. As an example, a culture of one or more types of recovered cells can be mixed and seeded on an extracellular matrix for co-culture. As the extracellular matrix, collagen, elastin, fibronectin, laminin, entactin, etc. can be used.

[0025] The integration process may integrate the culture of the recovered one or more types of cells with blood and / or tissue. The tissue may be obtained from an animal or cultured. As an example, blood, adipose tissue, muscle tissue, etc. separated during the meat processing process may be integrated with the culture to produce cultured meat.

[0026] The differentiation induction process may be performed after cell culture, or before, during, or after the integration process. By the differentiation induction process, mononuclear satellite cells and myoblasts can be differentiated into multinuclear myotube cells and further matured into muscle fibers. Differentiation induction includes culturing in a medium not containing the differentiation inhibitor of the present invention. It may be performed by a method known in the art, but a method of culturing under a high carbon dioxide concentration is known. For example, by culturing in a whey-free medium under a CO2 atmosphere of 5-10% (v / v), differentiation into myotube cells can be promoted.

[0027] All documents mentioned in this specification are hereby incorporated by reference in their entirety. The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description in the claims. Changes to the present invention, such as addition, deletion, and substitution of the constituent elements of the present invention, can be made on the condition that the gist of the present invention is not deviated from.

Examples

[0028] Test 1: Collection of satellite cells Bovine myoblasts were collected from cheek muscle using the following procedure. After washing the collected tissue with ethanol and PBS, it was minced finely using scissors in a clean bench. The muscle tissue was digested by shaking culture at 37 °C for 1.5 hours in Dulbecco's modified Eagle medium supplemented with 0.2% collagenase II. The reaction was stopped by adding 20% FBS to the reaction solution after digestion. The digestion solution was centrifuged at 80×g for 3 minutes to remove floating tissue with forceps, and then the supernatant was collected. The supernatant obtained by centrifuging again at 80×g for 3 minutes was passed through a nylon mesh (100 μm) for cell separation. The precipitate obtained by centrifuging the filtrate at 1500×g for 5 minutes was suspended in Dulbecco's modified Eagle medium containing 20% FBS. The cell suspension was passed through a 100-μm nylon mesh and then again through a 40-μm nylon mesh, and the filtrate was centrifuged at 1500×g for 5 minutes. The precipitate was left standing on ice in erythrocyte lysing solution for 5 minutes to remove blood cells. After washing twice with phosphate buffer, it was pooled in Dulbecco's modified Eagle medium containing 10% FBS and seeded in a culture dish. The proliferated cells were used for the test.

[0029] Test 2: Culture of satellite cells in serum-containing medium The myoblasts collected in Test 1 were cultured in Dulbecco's modified Eagle medium supplemented with 1% penicillin-streptomycin solution, 20% FBS, and 2 ng / ml human basic fibroblast growth factor in a 37 °C and 5% CO2 atmosphere. Microscopic observation was performed on the 4th day of culture (Figure 1). When myoblasts were cultured in serum-containing medium, they proliferated and reached confluence, and on the 4th day of culture, the confluent myoblasts differentiated into multinucleated myotube cells by fusion.

[0030] Test 3: Culture of satellite cells in serum-free medium The myoblasts collected in Test 1 were cultured in Dulbecco's Modified Eagle Medium supplemented with 1% penicillin-streptomycin solution, 1% ITS liquid medium supplement, 2 ng / ml human basic fibroblast growth factor, a lipid additive for cell culture (Sigma, L0288), and 0.2% BSA in a 37°C and 5% CO2 atmosphere. Microscopic observation was performed on the third day of culture (Figure 2). When myoblasts were cultured in serum-free medium, many cells maintained mononuclear myoblasts on the third day of culture, but some myoblasts fused and differentiated into multinucleated myotube cells.

[0031] Test 4: Exploration of food materials that inhibit myotube differentiation in serum-free medium As the serum-free medium, Dulbecco's Modified Eagle Medium supplemented with penicillin-streptomycin solution, ITS liquid medium supplement, 2 ng / ml human basic fibroblast growth factor, a lipid additive for cell culture, and 0.2% BSA was used (the same as in Test 3). As food raw material components, egg white, soy, whey, and wheat flour (all dry powders) were used. Each type of food component was dissolved at 0.1% in the serum-free medium, and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components for use in the test. The cells collected in Test 1 were seeded at about 5×10 3 cells / cm 3 and cultured in a CO2 incubator set at 37°C and a CO2 concentration of 5%. Differentiation into myotubes was confirmed by microscopic observation on the fourth day of culture (Figure 3). As a control, the same experiment was conducted using an unsupplemented medium (-) and a medium supplemented with 10% FBS. The cell count and cell morphology when cultured with the addition of egg white, soy, and wheat flour were not significantly different from those cultured in the unsupplemented medium. On the other hand, when cultured with the addition of whey, the cell count was as high as that in the 10% FBS supplemented group, but the number of differentiated and fused cells was small. Also, the shape maintained the shape at the time of isolation. Therefore, whey exerted a differentiation inhibitory effect and a cell proliferation effect.

[0032] Test 5: Quantification of the myotube differentiation inhibitory effect during serum-free medium culture The myotube formation rate of the cultures in Test 4 was quantified. Samples stained for nuclei and myosin heavy chain were observed with an all-in-one microscope manufactured by Keyence Corporation, and the ratio of the number of nuclei in myotubes to the total number of nuclei in the field of view was measured as an index of differentiation (fusion index). Immunostaining of myosin heavy chain was performed according to the following procedure. After washing the cells once with phosphate-buffered saline (PBS), they were fixed overnight at 4°C with 4% paraformaldehyde. After washing three times with PBS, permeabilization was carried out with 1% Triton X-100 / PBS for 5 minutes at room temperature. After washing three times with PBS, blocking was performed for 30 minutes at room temperature using a commercially available blocking solution for immunostaining (KAC, CTKN001). The primary antibody reaction was carried out for 1 hour at room temperature with a solution obtained by diluting an anti-myosin heavy chain monoclonal antibody (Clone MF20) to 1 μg / mL. After washing three times with PBS, the secondary antibody reaction was carried out for 30 minutes at room temperature using a 500-fold diluted solution of Alexa 488 goat anti-mouse IgG (abcam, ab150117). After washing with PBS, nuclear staining was performed with DAPI, followed by observation with a fluorescence microscope to determine the differentiation index (Figure 4). Also, the cell growth effect was examined based on the cell count (Figure 5). In the whey-added group, growth was promoted and the differentiation rate was low, to the same extent as in the 10% FBS-added group.

[0033] Test 6: Quantification of the myotube differentiation inhibitory effect during serum-free medium culture The differentiation state of myoblasts grown in various media was confirmed. As the serum-free medium, Dulbecco's Modified Eagle Medium supplemented with penicillin-streptomycin solution, ITS liquid medium supplement, 5 ng / ml human basic fibroblast growth factor, lipid additive for cell culture, and 0.05% BSA was used. The food ingredient-added medium was prepared by adding 0.1% of various food ingredients to the serum-free medium, filtering the supernatant after centrifugation through a 0.45-μm filter to remove insoluble components, and using this for the test. As the serum-containing medium, Dulbecco's Modified Eagle Medium supplemented with penicillin-streptomycin solution, 5 ng / ml human basic fibroblast growth factor, and 10% FBS was used. Approximately 5×10 3 cells / cm 3The cells were seeded in a culture dish containing various media and cultured in a CO2 incubator set at 37°C and 5% CO2 concentration. The expression of muscle differentiation markers after 2 days of culture was evaluated by qPCR.

[0034] qPCR method RNA was extracted from the cells using TRIzol Reagent (Invitrogen), and cDNA was synthesized by reverse transcription reaction using PrimeScript RT reagent Kit (Takara Bio). Using the cDNA as a template, amplification of the target gene was performed using a real-time PCR machine manufactured by Bio-Rad. For gene amplification, TB Green II Kit (Takara Bio) and primers corresponding to Table 1 were used. The expression level of the target gene was evaluated by comparing the expression with that of the endogenous control gene (ΔCt method). Comparative analysis of the expression of various genes among the culture media was performed with the marker expression in the serum-containing medium set as 1.

Table 1

[0035] Test 7: Examination of the concentration of whey that exhibits a differentiation inhibitory effect The effects on growth and differentiation were evaluated when the whey addition concentration in the food ingredient-added medium was changed. As the food ingredient-added medium, Dulbecco's Modified Eagle Medium supplemented with penicillin-streptomycin solution, ITS liquid medium supplement, 2 ng / ml human basic fibroblast growth factor, lipid additive for cell culture, and 0.2% BSA was used. Whey powders at various concentrations (0.005 mass%, 0.025 mass%, 0.05 mass%, 0.1 mass%, 0.25 mass%, 0.5 mass%, 1.0 mass%) were dissolved, and the supernatant after centrifugation was filtered through a 0.45-μm filter to remove insoluble components, which was then used in the test. Approximately 7.5×10 3 cells / cm 3 of cells were seeded and cultured in a CO2 incubator set at 37°C and a CO2 concentration of 5%. On the 4th day of culture, cell counting and calculation of the myotube formation rate (fusion index) were performed. The cell count was the number of viable cells obtained by trypsin treatment. The ratio of myotube cells was determined by immunostaining against myosin heavy chain, and the ratio of myosin-positive cells to the total number of nuclei in the visual field was measured as an index of differentiation. Whey exhibited a differentiation-inhibiting effect at a concentration of 0.005 mass%, and the differentiation-inhibiting effect was dose-dependently exhibited up to 1.0 mass%.

Claims

1. An inhibitor for suppressing the differentiation of muscle tissue-derived cells, comprising whey, added to a medium for culturing meat such that the medium contains 0.005% to 1.0% by mass of whey in terms of dry powder, and suppressing the differentiation from myoblasts and satellite cells into myotube cells, said inhibitor.

2. The inhibitor according to claim 1, wherein the muscle tissue-derived cells are muscle tissue-derived cells from livestock.

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