WO2023127960A1 - Agent de suppression de la différenciation des cellules issues du tissu musculaire - Google Patents

Agent de suppression de la différenciation des cellules issues du tissu musculaire Download PDF

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WO2023127960A1
WO2023127960A1 PCT/JP2022/048646 JP2022048646W WO2023127960A1 WO 2023127960 A1 WO2023127960 A1 WO 2023127960A1 JP 2022048646 W JP2022048646 W JP 2022048646W WO 2023127960 A1 WO2023127960 A1 WO 2023127960A1
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cells
differentiation
medium
myoblasts
serum
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Japanese (ja)
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亮 瀬川
泰孝 西山
慶一 古澤
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日本ハム株式会社
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • the present invention relates to the technical field of cultured meat production. More specifically, it relates to an inhibitor of differentiation of muscle tissue-derived cells added in a cell culture process for producing cultured meat.
  • raising livestock requires a large amount of grain and water, and a large breeding area.
  • the problems of climate change and food shortages have been taken up, and there is a growing demand for sustainable meat production that has a lower environmental impact and higher production efficiency.
  • research and development to produce cultured meat from cells is attracting attention as a new meat production method.
  • Plant-based meat substitutes are known as meat substitutes, but their texture and taste are not as good as meat.
  • cultured meat which is made by culturing animal cells, can achieve a texture and taste similar to that of the original meat, and has the advantage of being less susceptible to bacterial and viral contamination than meat.
  • the production of cultured meat is becoming possible.
  • the cell culture medium used in cultured meat production so far uses large-scale culture technology used in basic research and pharmaceutical applications, and from the viewpoint of cost and safety as meat, , was difficult to use for the production of food.
  • Non-Patent Document 1 Mol Ther. 2004 Mar;9(3):475-82.
  • FBS fetal bovine serum
  • Non-Patent Document 2 The Canadian Journal of Chem Engineering Vol.94, (10) October 2016 1855-1862).
  • Such completely synthetic media contain recombinant proteins, hormone agents, serum-derived components, etc., and have problems when used as foods.
  • Patent Document 1 Patent No. 6111510
  • Non-Patent Document 3 Scientific Reports. Jan 31;7:41594
  • Food residue hydrolysis Non-Patent Document 4: Food Funct., 2020, 11, 2477-2488
  • Patent Document 2 International Publication No. 2021/148955), etc.
  • Proliferative myoblasts and muscle satellite cells must be cultured as muscle tissue-derived cells to be used in the production of cultured meat. tend to differentiate into non-myotube cells.
  • culturing in FBS-supplemented medium allows proliferation while suppressing differentiation, but in the production of cultured meat, it is desirable to avoid using FBS from the viewpoint of price, risk of infectious disease, and animal welfare. ing. Therefore, in the production of cultured meat, in order to culture a large amount of muscle tissue-derived cells, it was necessary to suppress the differentiation of myoblasts and muscle satellite cells in a medium without FBS.
  • the present invention relates to: [1] A whey-containing inhibitor of differentiation of muscle tissue-derived cells.
  • the inhibitor according to item 1 wherein the muscle tissue-derived cells are selected from the group consisting of myoblasts and satellite cells.
  • the inhibitor according to item 1 or 2 which inhibits the differentiation of myoblasts and satellite cells into myotube cells.
  • the inhibitor according to any one of items 1 to 3 wherein the muscle tissue-derived cells are livestock-derived muscle tissue-derived cells.
  • the inhibitor according to any one of items 1 to 4 wherein the inhibitor is added to a medium for producing cultured meat.
  • FIG. 1 shows photographs taken on day 4 of culturing bovine cheek-derived myoblasts in an FBS-supplemented medium.
  • FIG. 2 shows a photograph taken on day 3 of culturing bovine cheek-derived myoblasts in a serum-free medium.
  • Fig. 3 shows myoblasts derived from bovine cheeks cultured in a serum-free medium supplemented with food ingredients (non-additive (-), wheat flour, soybean, egg white, whey) and an FBS-supplemented medium, photographed on day 4. Represents a photograph taken FIG.
  • FIG. 4 shows that bovine cheek-derived myoblasts were cultured in a serum-free medium supplemented with food ingredients (non-additive (-), wheat flour, soybean, egg white, whey) and an FBS-supplemented medium. The results of measuring the ratio of the number of nuclei in myotubes to the total number of nuclei in cultures are shown.
  • FIG. 5 shows that bovine cheek-derived myoblasts were cultured in a serum-free medium supplemented with food ingredients (non-additive (-), wheat flour, soybean, egg white, whey) and an FBS-supplemented medium. The results of determining the cell proliferation promoting effect of the culture based on the number of cells are shown.
  • bovine cheek-derived myoblasts were cultured in a serum-free medium supplemented with food ingredients (non-additive (-), wheat flour, soybean, egg white, whey) and an FBS-supplemented medium.
  • food ingredients non-additive (-), wheat flour, soybean, egg white, whey
  • FBS-supplemented medium The results of comparing gene expression levels of desmin (A) and myogenin (B) in cells are shown.
  • Figure 7 shows that myoblasts derived from bovine cheeks were treated with various concentrations of whey (0.005% by mass, 0.025% by mass, 0.050% by mass, 0.1% by mass, 0.25% by mass). , 0.5% by mass, and 1.0% by mass) were examined for differentiation inhibitory effects.
  • the present invention relates to a whey-containing inhibitor of differentiation of muscle tissue-derived cells (hereinafter also referred to as a differentiation inhibitor according to the present invention).
  • the present invention also relates to a cultured meat production medium comprising a basal medium and whey as an inhibitor of differentiation of muscle tissue-derived cells.
  • the present invention also relates to a method for preparing cells for production of cultured meat, comprising the step of culturing cells in a medium containing a basal medium and whey as an inhibitor of differentiation of muscle tissue-derived cells. It also relates to a method of producing cultivated meat from
  • the inhibitor of muscle tissue-derived cell differentiation according to the present invention includes whey.
  • whey By containing whey, differentiation of muscle tissue-derived cells, particularly myoblasts and muscle satellite cells, into myotube cells can be suppressed. By suppressing differentiation, proliferative myoblasts and muscle satellite cells can be maintained. Whey also has a cell proliferation-promoting effect on myoblasts and muscle satellite cells. The effect of whey on promoting cell growth and suppressing the differentiation into myotubes was comparable to FBS, respectively (Figs. 4 and 5). This allows the preparation of large quantities of cells for cultivated meat production and allows their use as an alternative to FBS.
  • the method of preparing cells for producing cultured meat of the present invention includes a step of culturing cells in a medium containing a basal medium and whey as a differentiation inhibitor for muscle tissue-derived cells, thereby proliferating the cells. It is possible to proliferate while suppressing differentiation into myotube cells.
  • the differentiation inhibitor according to the present invention is highly safe as a food.
  • the medium supplemented with the differentiation inhibitor according to the present invention also has the advantage of low preparation cost. Cells cultured in such a medium are highly safe as food and can be used for cultured meat production.
  • the differentiation inhibitor according to the present invention can be added to known media.
  • the differentiation inhibitor according to the present invention may be added to a serum-free medium or to an animal-derived serum-containing medium. From the viewpoint of producing cultured meat, it is preferably added to a serum-free medium.
  • the medium from which insoluble matter has been removed can be used for culture by performing filter filtration.
  • the whey used in the present invention can be said to be a differentiation inhibitor as well as a cell growth promoter.
  • the differentiation inhibitor according to the present invention can be used in cell culture for producing cultivated meat.
  • Whey also called whey or whey refers to an aqueous solution obtained by removing solids from milk. It is cheap because it is produced in large quantities as a by-product in the process of manufacturing dairy products such as cheese and yogurt. More specifically, whey is obtained by adding a coagulant such as rennet to milk or fermented milk and separating the solids from the curdled milk. Part or all of proteins such as milk fat and casein are excluded from the solid content.
  • the main components of whey are lactoglobulin, lactalbumin and lactoferrin, but it also contains various minor components such as free amino acids, inorganic salts and vitamins.
  • the whey used in the present invention may be whey derived from any mammal.
  • Whey obtained from cow, horse, goat, sheep, human and donkey milk can be used.
  • Whey may be liquid or may be dry powder obtained by drying whey.
  • a dry powder form is preferable because it can be expected to reduce transportation costs.
  • Whey in dry powder form may be commercially available or may be prepared by freeze-drying whey. When dry powder whey is used as a differentiation inhibitor, it is added to the basal medium at 0.0025% to 1.0% by mass.
  • 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 viewpoint of exhibiting an effect of inhibiting differentiation. From the viewpoint of dissolution efficiency, it is preferably 0.5% by mass or less, more preferably 0.25% by mass or less.
  • the amount to be added can be determined in terms of dry powder.
  • the medium to which the differentiation inhibitor according to the present invention is added can be added to any known medium in this technical field.
  • the differentiation inhibitor according to the present invention may be added to a serum-free medium or to an animal-derived serum-containing medium. These media can be produced by adding additives to a basal medium.
  • a basal medium is a medium for culturing cells, and refers to a medium that contains the minimum components necessary for the maintenance and growth of cells. Seeding the cells in a basal medium may keep the cells from dying and allow the cells to grow.
  • Various media are commercially available as basal media, but they usually contain amino acids, vitamins, buffers, inorganic salts and a carbon source.
  • Amino acids include essential amino acids and non-essential amino acids.
  • Vitamins include vitamin B1, vitamin C, nicotinic acid, folic acid and the like. Buffers include HEPES and the like.
  • As a carbon source monosaccharides such as glucose, disaccharides such as sucrose, oligosaccharides and polysaccharides can be added.
  • a cell culture medium can usually be prepared by adding an additive such as serum to a basal medium.
  • a basal medium any basal medium known in the art can be used, examples being Dulbecco's Modified Eagle's Medium (DMEM), Eagle's Basal Medium (BME), RPMI 1640 medium, DMEM/F12 medium, F10 Medium, F12 Ham's medium, MEM, M199 medium, Ames medium, Iscove's modified medium, Glasgow's modified medium, Fisher's medium and the like.
  • DMEM Dulbecco's Modified Eagle's Medium
  • BME Eagle's Basal Medium
  • RPMI 1640 medium DMEM/F12 medium
  • F10 Medium F12 Ham's medium
  • MEM M199 medium
  • Ames medium Iscove's modified medium
  • Glasgow's modified medium Fisher's medium and the like.
  • Additives are usually added to the basal medium for cell culture.
  • serum such as fetal bovine serum (FBS) is added as an additive and used as an animal-derived serum-containing medium.
  • FBS promotes proliferation while suppressing differentiation of muscle tissue-derived cells.
  • other additives may be added depending on the cell type.
  • FBS serum-free or low-serum media, it also contains components necessary for maintaining cell proliferation in place of FBS.
  • additives include, for example, lipids, hormones, growth factors, cytokines, serum-derived proteins, antibiotics, etc., and can be added to serum-free media and/or animal-derived serum-containing media.
  • Hormone agents include dexamethasone and the like.
  • Growth factors include FGF, IGF, insulin, etc., and any family thereof may be used.
  • Cytokines include IL-1 ⁇ , IL-1 ⁇ and the like.
  • Serum-derived proteins include fetuin, fibronectin, albumin, globulin, and the like. Penicillin, streptomycin and the like can be used as antibiotics.
  • ITS insulin-transferrin-sodium selenite
  • an additive commonly used in serum-free or low-serum media can also be added to media, particularly serum-free media, together with the differentiation inhibitor of the present invention.
  • Animal-derived serum refers to serum manufactured from animal blood.
  • the supernatant obtained by coagulating the obtained blood is called serum.
  • the animal-derived serum may be serum derived from any animal such as bovine, horse, goat, donkey, rabbit, chicken, etc., especially bovine serum (BCS) and fetal bovine serum (FBS). Point.
  • Serum contains proteins such as albumin and globulin, serum lipids such as neutral lipids, cholesterol, phospholipids and free fatty acids, and further contains hormones, cytokines, growth factors and the like.
  • Fetal serum in particular, is rich in components required for cell growth and is generally added to culture media in the fields of research and medicine.
  • a medium that does not contain animal-derived serum is called a serum-free medium.
  • serum-free media do not contain animal-derived serum, but may contain purified serum-derived components or recombinant proteins of serum-derived components.
  • the differentiation-suppressing medium according to the present invention can suppress differentiation in muscle tissue-derived cells obtained from any animal.
  • cells derived from livestock such as cows, pigs, goats, sheep, rabbits, chickens, ostriches and ducks can be used.
  • bovine cells when bovine cells are used, cells of any of Holstein, Jersey, Japanese Black, Japanese Brown, Shorthorn, Japanese Polled, and hybrids thereof may be used.
  • cells of Japanese Black, Japanese Brown, Shorthorn, and Japanese Polled which are breeds for meat, are preferable.
  • Muscle tissue-derived cells from these animals can be cultured.
  • the animal-derived muscle tissue-derived cells may be primary cells obtained from an animal, passaged cells subcultured from the primary cells, or established cells.
  • Primary cells can be obtained by mincing animal tissue in culture medium.
  • Cells differentiated from stem cells such as somatic stem cells, embryonic stem cells, and induced pluripotent stem cells may also be used.
  • Muscle tissue-derived cells are cells that constitute muscle tissue, and are cells that are separated from muscle tissue and cultured. Examples of muscle tissue-derived cells include myoblasts, muscle satellite cells, and myotubes. Since myotubes do not have proliferative properties, from the viewpoint of proliferation, myoblasts and/or muscle satellite cells are preferred. is preferred. Muscle satellite cells are somatic stem cells contained in muscle and characterized by the expression of the transcription factor Pax7. Muscle satellite cells normally maintain a quiescent state (resting phase) in vivo, and differentiate into proliferative myoblasts when activated by injury or the like. When performing cell culture, muscle satellite cells can be grown in an undifferentiated state depending on the culture conditions.
  • Myoblasts are proliferative mononuclear cells capable of forming muscle fibers and are characterized by the expression of MyoD. As the myoblasts differentiate further, they fuse to form multinucleated myotube cells, which further mature into myofibers. Differentiation into myotube cells can be determined by observing multinucleation and shape (Figs. 1-3). Furthermore, differentiation from myoblasts to myotubes can also be determined by measuring the expression of genes specifically expressed in myotubes, such as desmin and myogenin (Fig. 6). The differentiation inhibitor of the present invention can inhibit the differentiation of muscle satellite cells into myoblasts and the differentiation of myoblasts into myotube cells. On the other hand, myoblasts and muscle satellite cells differentiate into myotubes after a certain period of time has passed after reaching confluency.
  • Cultured meat refers to meat produced through cell culture.
  • "for production of cultured meat” means a method used for production of cultured meat, and is required to be food hygienically acceptable. From the viewpoint of food hygiene, it is preferable to avoid using animal-derived serum, hormone agents, and genetically modified proteins.
  • Meat generally refers to a collection of muscle fibers, connective tissue, and fat.
  • cultured meat preferably imitates the structure of meat, but does not necessarily contain all the components of meat, as long as it contains cultured muscle tissue-derived cells. Cultured meat may comprise cultures of multiple types of cells.
  • the cultured meat may contain an extracellular matrix in addition to at least one cultured cell selected from the group consisting of muscle tissue-derived cells.
  • the method for producing cultured meat includes, for example, the following steps of culturing muscle tissue-derived cells, collecting and accumulating the cultured cells.
  • the method for producing cultured meat may further include a differentiation-inducing step and a culture step after accumulation.
  • the present invention also relates to cultured meat containing cells cultured in a medium containing the differentiation inhibitor according to the present invention.
  • Cells are cultured by inoculating cells in a medium containing whey as a differentiation inhibitor in a basal medium. Cultures are cultivated under conditions well known in the art, eg, in a 37° C. 5% CO 2 incubator. The culture may be plate culture or suspension culture. The proliferated cells can be recovered as a culture by trypsin treatment or the like, and the cells may be further subcultured after recovery. Cells can also be cultured by seeding cells on a detachable construct. Constructs with attached proliferating cells can be harvested as cultures. Such constructs can be constructed from extracellular matrices such as collagen, elastin, fibronectin, laminin, entactin, etc., and the constructs with attached cells may be accumulated to form cultured meat.
  • extracellular matrices such as collagen, elastin, fibronectin, laminin, entactin, etc.
  • the accumulation step includes forming a culture of one or more types of collected cells.
  • the culture formed in the accumulation step may be a piece of meat such as steak, a carcass, or a minced meat.
  • the accumulating step includes accumulating the cell culture together with at least one substance selected from the group consisting of other cells, blood and tissue. Other cells may be cultured cells or cells collected from animals. Co-cultivation can also be performed after enrichment. As an example, harvested cultures of one or more types of cells can be mixed and seeded onto an extracellular matrix for co-cultivation. Extracellular matrices that can be used include collagen, elastin, fibronectin, laminin, entactin, and the like.
  • the collected culture of one or more types of cells may be accumulated with blood and/or tissue.
  • the tissue may be obtained from an animal or cultured.
  • cultured meat may be produced by accumulating blood, adipose tissue, muscle tissue, or the like separated during meat processing with a culture.
  • the differentiation-inducing step may be performed after cell culturing, or may be performed before, during, or after the enrichment step.
  • mononuclear muscle satellite cells and myoblasts can be differentiated into multinucleated myotube cells and further matured as muscle fibers.
  • Differentiation induction includes culturing in a medium that does not contain the differentiation inhibitor of the present invention. Although it may be carried out by methods known in the art, methods of culturing under high carbon dioxide concentrations are known, such as 5-10% (v/v) CO 2 atmosphere in whey-free medium Differentiation into myotube cells can be promoted by culturing in them.
  • Test 1 Collection of myoblasts Bovine myoblasts were collected from cheek meat by the following steps. After washing the collected tissue with ethanol and PBS, it was minced with scissors in a clean bench. Shaking culture was performed at 37° C. for 1.5 hours in Dulbecco's modified Eagle's medium supplemented with 0.2% collagenase II to digest the muscle tissue. The reaction was stopped by adding 20% FBS to the reaction solution after digestion. The digestive fluid was centrifuged at 80 ⁇ g for 3 minutes, floating tissue was removed with tweezers, and the supernatant was collected. The supernatant obtained by centrifugation at 80 ⁇ g for 3 minutes was passed through a nylon mesh (100 ⁇ m) for cell separation.
  • the precipitate obtained by centrifuging the filtrate at 1500 ⁇ g for 5 minutes was suspended in Dulbecco's modified Eagle's medium containing 20% FBS. After the cell suspension was passed through a 100 ⁇ m nylon mesh, it was again passed through a 40 ⁇ m nylon mesh, and the filtrate was centrifuged at 1500 ⁇ g for 5 minutes. The precipitate was placed on ice with erythrocyte lysate for 5 minutes to remove blood cells. After washing twice with phosphate buffer, they were pooled in Dulbecco's modified Eagle's medium containing 10% FBS and seeded in culture dishes. Proliferated cells were used for testing.
  • Test 2 Cultivation of Myoblasts in Serum-Containing Medium
  • Myoblasts harvested in Test 1 were cultured in Dulbecco's Modified Eagle Medium, 1% penicillin-streptomycin solution, 20% FBS, 2 ng/ml human basic fibroblast proliferation. Cultivation was performed in a serum-containing medium supplemented with factors at 37° C. and 5% CO 2 atmosphere. Microscopic observation was performed on day 4 of culture (Fig. 1). When myoblasts were cultured in a serum-containing medium, they proliferated and reached confluence, and on the fourth day of culture, the confluent myoblasts fused and differentiated into multinucleated myotube cells.
  • Test 3 Cultivation of myoblasts in serum-free medium
  • Myoblasts harvested in Test 1 were cultured in Dulbecco's modified Eagle medium, 1% penicillin-streptomycin solution, 1% ITS liquid medium supplement), 2 ng/ml human basic Cultivation was performed in a serum-free medium supplemented with fibroblast growth factor, a lipid additive for cell culture (sigma, L0288), and 0.2% BSA at 37° C. and 5% CO 2 atmosphere. Microscopic observation was performed on day 3 of culture (Fig. 2). When myoblasts were cultured in serum-free medium, most of the cells maintained mononuclear myoblasts on the third day of culture, but some myoblasts fused to form multinucleated myotubes. Differentiated.
  • Test 4 Search for food materials that suppress myotube differentiation in serum-free medium
  • Serum-free medium consists of Dulbecco's modified Eagle's medium, penicillin-streptomycin solution, ITS liquid medium supplement, and 2 ng/ml human basic fibroblast proliferation.
  • Factors, cell culture lipid additives, plus 0.2% BSA were used (as in Test 3).
  • Egg whites, soybeans, whey, and wheat flour (all dry powders) were used as raw food ingredients.
  • Various food components were dissolved in a serum-free medium at a concentration of 0.1%, and the supernatant after centrifugation was filtered through a 0.45 ⁇ m filter to remove insoluble components.
  • Test 1 The cells collected in Test 1 were seeded at about 5 ⁇ 10 3 cells/cm 3 and cultured in a CO 2 incubator set at 37° C. and a CO 2 concentration of 5%. Differentiation into myotubes was confirmed by microscopic observation on day 4 of culture (Fig. 3). As controls, the same experiment was performed using a non-supplemented medium (-) and a 10% FBS-supplemented medium. The cell number and cell morphology when cultured with the addition of egg white, soybean, and wheat flour were not significantly different from those cultured with the non-supplemented medium. On the other hand, when whey was added and cultured, the number of cells was as large as in the 10% FBS-added group, but the number of differentiated and fused cells was small. Also, the shape was maintained as it was when isolated. Therefore, whey exerted anti-differentiation and cell proliferation effects.
  • Test 5 Quantification of myotube differentiation inhibitory effect during culture in serum-free medium The myotube formation rate of the culture in Test 4 was quantified. A sample stained for nuclei and myosin heavy chain was observed with an all-in-one microscope manufactured by Keyence, and the ratio of the number of nuclei in myotubes to the total number of nuclei in the visual field was measured and used as a differentiation index (fusion index). The myosin heavy chain was immunostained by the following steps. Cells were washed once with phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde overnight at 4°C.
  • PBS phosphate buffered saline
  • Test 6 Determination of myotube differentiation inhibitory effect during culture in serum-free medium The state of differentiation of myoblasts grown and cultured in various media was confirmed.
  • Serum-free medium was Dulbecco's modified Eagle's medium supplemented with penicillin-streptomycin solution, ITS liquid medium supplement, 5 ng/ml human basic fibroblast growth factor, cell culture lipid supplement, 0.05% BSA. used.
  • the food component-added medium was a serum-free medium containing 0.1% of various food components, and the supernatant after centrifugation was filtered through a 0.45 ⁇ m filter to remove insoluble components.
  • the serum-containing medium used was Dulbecco's modified Eagle's medium supplemented with penicillin-streptomycin solution, 5 ng/ml human basic fibroblast growth factor, and 10% FBS. Approximately 5 ⁇ 10 3 cells/cm 3 of cells were seeded in a culture dish containing various media and cultured in a CO 2 incubator set at 37° C. and a CO 2 concentration of 5%. Expression of muscle differentiation markers after 2 days of culture was assessed by qPCR.
  • Test 7 Investigation of Whey Concentration that Demonstrates Differentiation Inhibitory Effect The effect on growth and differentiation was evaluated when the concentration of whey added to the food component-added medium was changed.
  • the food ingredient-supplemented medium is Dulbecco's modified Eagle's 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 powder of various concentrations (0.005% by mass, 0.025% by mass, 0.05% by mass, 0.1% by mass, 0.25% by mass, 0.5% by mass, 1.0% by mass) is dissolved After centrifugation, the supernatant was filtered through a 0.45 ⁇ m filter to remove insoluble components and used for the test.
  • Cells were seeded at about 7.5 ⁇ 10 3 cells/cm 3 and cultured in a CO 2 incubator set at 37° C. and a CO 2 concentration of 5%. On day 4 of culture, cell count and myotube formation rate (fusion index) were calculated. The number of viable cells obtained by trypsinization was counted.
  • the ratio of myotube cells was determined by immunostaining for myosin heavy chain and measuring the ratio of myosin-positive cells to the total number of nuclei in the visual field as an indicator of differentiation.
  • Whey exhibited a differentiation inhibitory effect at a concentration of 0.005% by mass, and dose-dependently exhibited a differentiation inhibitory effect up to 1.0% by mass.

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Abstract

Afin d'obtenir des cellules issues du tissu musculaire pour la production de viande cultivée, il est nécessaire de cultiver des myoblastes prolifératifs et des cellules satellites musculaires. Cependant, ces cellules se différencient facilement et possèdent une tendance, même dans des conditions de culture normales, à se différencier en cellules de type myotube qui ne sont pas prolifératives. L'objectif de la présente invention est d'identifier un composant qui inhibe la différenciation des myoblastes et des cellules satellites musculaires afin de cultiver une grande quantité de cellules issues du tissu musculaire pour la production de viande cultivée. Les composants alimentaires utilisés comme additifs ont été étudiés pour leur effet de suppression de la différenciation, et l'étude a révélé que lorsque du lactosérum était ajouté, la différenciation des myoblastes et des cellules satellites musculaires était supprimée avec succès, et qu'un agent de suppression de la différenciation était donc présent.
PCT/JP2022/048646 2021-12-28 2022-12-28 Agent de suppression de la différenciation des cellules issues du tissu musculaire WO2023127960A1 (fr)

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Citations (3)

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JPH05508542A (ja) * 1990-07-13 1993-12-02 グロペップ　リミテッド 成長促進剤
JPH09512536A (ja) * 1994-04-28 1997-12-16 グロペップ プロプライエタリー リミテッド 修飾乳成長因子
JP2009507044A (ja) * 2005-09-09 2009-02-19 マレー ゴールバーン コーオペラティブ コー リミテッド 乳由来組成物、及び筋肉量または筋力を増強するための使用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05508542A (ja) * 1990-07-13 1993-12-02 グロペップ　リミテッド 成長促進剤
JPH09512536A (ja) * 1994-04-28 1997-12-16 グロペップ プロプライエタリー リミテッド 修飾乳成長因子
JP2009507044A (ja) * 2005-09-09 2009-02-19 マレー ゴールバーン コーオペラティブ コー リミテッド 乳由来組成物、及び筋肉量または筋力を増強するための使用

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