WO2023127960A1 - Differentiation suppression agent for muscle tissue-derived cells - Google Patents
Differentiation suppression agent for muscle tissue-derived cells Download PDFInfo
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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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Classifications
-
- 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
Definitions
- 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
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
本発明は、培養肉製造の技術分野に関する。より具体的に培養肉製造のための細胞培養工程において添加される筋組織由来細胞の分化の抑制剤に関する。
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.
食肉生産は、これまで家畜の飼育により行われてきた。一方、家畜の飼育には、大量の穀物及び水を必要とし、広い飼育場を必要とする。近年、気候変動や食料不足の問題が取り上げられており、より環境負荷が低く、生産効率の高い持続可能な食肉生産が望まれるようになってきている。そうした中で、新たな食肉生産方法として、細胞から培養肉を生産する研究開発に注目が集まっている。
Meat production has so far been done by raising livestock. On the other hand, raising livestock requires a large amount of grain and water, and a large breeding area. In recent years, 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. Under such circumstances, research and development to produce cultured meat from cells is attracting attention as a new meat production method.
食肉の代替物として、植物由来の代用肉が知られているが、その食感や味わいは食肉には及んでいない。一方、動物細胞を培養する培養肉では、本来の食肉に近い食感や味わいを達成することができ、また食肉に比較して細菌やウイルス汚染のリスクが低いという利点がある。技術的には培養肉の製造は可能になってきている。しかしながら、これまでの培養肉製造で用いられている細胞培養培地は、基礎研究や医薬品応用で用いられた大規模培養技術を利用するものであり、そのコストや、食肉としての安全性の面から、食品の製造のための使用が難しかった。基礎研究や医薬品応用で用いられた細胞培養培地では、アミノ酸、ビタミン、無機塩及びグルコース等の炭素源を含む基礎培地に、添加成分としてウシ胎児血清（ＦＢＳ）を添加することが一般的である（非特許文献１：Mol Ther. 2004 Mar;9(3):475-82）。一方、ＦＢＳは胎児より採取した血清であるため、大量入手が困難であるとともに、価格、輸送コスト、感染症リスク、及び動物愛護の点で課題がある。そうした課題を解決すべく、ＦＢＳの必須成分を試薬として補った完全合成培地が開発されている（非特許文献２：The Canadian Journal of Chem Engineering Vol.94, (10) October 2016 1855-1862）。しかしながら、こうした完全合成培地には、組み換えタンパク質、ホルモン剤、血清由来成分などが用いられており、食品として使用するには課題があった。
Plant-based meat substitutes are known as meat substitutes, but their texture and taste are not as good as meat. On the other hand, 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. Technically, the production of cultured meat is becoming possible. However, 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. In cell culture media used in basic research and pharmaceutical applications, it is common to add fetal bovine serum (FBS) as an additive component 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 there are also problems in terms of price, transportation costs, infectious disease risk, and animal welfare. In order to solve such problems, a completely synthetic medium supplemented with essential components of FBS as a reagent 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 contain recombinant proteins, hormone agents, serum-derived components, etc., and have problems when used as foods.
培養肉の製造のための細胞培養培地としては、様々なアプローチが試みられてきている。臓器細胞の産生物を利用した培地（特許文献１：特許第６１１１５１０号公報）、藻類産生物を利用する培地（非特許文献３：Scientific Reports. Jan 31;7:41594）、食品残渣の加水分解物を利用した培地（非特許文献４：Food Funct., 2020,11, 2477-2488）、その他の食品原料成分を利用した培地（特許文献２：国際公開第２０２１／１４８９５５号）などが挙げられる。
Various approaches have been attempted as cell culture media for the production of cultivated meat. Medium using organ cell products (Patent Document 1: Patent No. 6111510), medium using algae products (Non-Patent Document 3: Scientific Reports. Jan 31;7:41594), food residue hydrolysis (Non-Patent Document 4: Food Funct., 2020, 11, 2477-2488), media using other food ingredients (Patent Document 2: International Publication No. 2021/148955), etc. .
培養肉の製造に使用する筋組織由来細胞として、増殖性を有する筋芽細胞及び筋衛星細胞を培養する必要があるが、これらの細胞は分化しやすく、通常の培養条件でも増殖性を有さない筋管細胞へと分化する傾向がある。また、ＦＢＳ添加培地で培養することで分化を抑制しつつ増殖させることができるが、培養肉の製造にあたっては、ＦＢＳは価格、感染症リスク、及び動物愛護の観点から利用を避けることが望まれている。そこで、培養肉の製造にあたって、筋組織由来細胞を大量に培養するためには、ＦＢＳ非添加培地で、筋芽細胞及び筋衛星細胞の分化を抑制する必要があった。
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. In addition, 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.
本発明者らが、筋組織由来細胞を培養するにあたり、筋芽細胞及び筋衛星細胞の分化を抑制する成分について鋭意研究を行ったところ、ホエイを添加物として培地へと添加することにより、筋芽細胞及び筋衛星細胞の分化を抑制できることを見出し、本発明に至った。そこで、本発明は以下に関する：
［１］ ホエイを含む、筋組織由来細胞の分化の抑制剤。
［２] 前記筋組織由来細胞が、筋芽細胞、及び衛星細胞からなる群から選択される、項目１に記載の抑制剤。
［３] 前記抑制剤が、筋芽細胞及び衛星細胞から筋管細胞への分化を抑制する、項目１又は２に記載の抑制剤。
［４] 前記筋組織由来細胞が、家畜由来の筋組織由来細胞である、項目１～３のいずれか一項に記載の抑制剤。
［５] 前記抑制剤が、培養肉製造のための培地に添加される、項目１～４のいずれか一項に記載の抑制剤。
［６］ 前記ホエイ濃度が、0.005%～1.0%の濃度である、請求項１～５のいずれか一項に記載の抑制剤。 In culturing muscle tissue-derived cells, the present inventors conducted intensive research on components that inhibit the differentiation of myoblasts and muscle satellite cells. The inventors have found that the differentiation of blast cells and muscle satellite cells can be suppressed, leading to the present invention. Accordingly, the present invention relates to:
[1] A whey-containing inhibitor of differentiation of muscle tissue-derived cells.
[2] The inhibitor according toitem 1, wherein the muscle tissue-derived cells are selected from the group consisting of myoblasts and satellite cells.
[3] The inhibitor according toitem 1 or 2, which inhibits the differentiation of myoblasts and satellite cells into myotube cells.
[4] The inhibitor according to any one ofitems 1 to 3, wherein the muscle tissue-derived cells are livestock-derived muscle tissue-derived cells.
[5] The inhibitor according to any one ofitems 1 to 4, wherein the inhibitor is added to a medium for producing cultured meat.
[6] The inhibitor according to any one of [1] to [5], wherein the whey concentration is 0.005% to 1.0%.
［１］ ホエイを含む、筋組織由来細胞の分化の抑制剤。
［２] 前記筋組織由来細胞が、筋芽細胞、及び衛星細胞からなる群から選択される、項目１に記載の抑制剤。
［３] 前記抑制剤が、筋芽細胞及び衛星細胞から筋管細胞への分化を抑制する、項目１又は２に記載の抑制剤。
［４] 前記筋組織由来細胞が、家畜由来の筋組織由来細胞である、項目１～３のいずれか一項に記載の抑制剤。
［５] 前記抑制剤が、培養肉製造のための培地に添加される、項目１～４のいずれか一項に記載の抑制剤。
［６］ 前記ホエイ濃度が、0.005%～1.0%の濃度である、請求項１～５のいずれか一項に記載の抑制剤。 In culturing muscle tissue-derived cells, the present inventors conducted intensive research on components that inhibit the differentiation of myoblasts and muscle satellite cells. The inventors have found that the differentiation of blast cells and muscle satellite cells can be suppressed, leading to the present invention. Accordingly, the present invention relates to:
[1] A whey-containing inhibitor of differentiation of muscle tissue-derived cells.
[2] The inhibitor according to
[3] The inhibitor according to
[4] The inhibitor according to any one of
[5] The inhibitor according to any one of
[6] The inhibitor according to any one of [1] to [5], wherein the whey concentration is 0.005% to 1.0%.
ホエイを基礎培地へと添加することにより、筋芽細胞及び筋衛星細胞の筋管細胞への分化を抑制することができる。
By adding whey to the basal medium, differentiation of myoblasts and muscle satellite cells into myotube cells can be suppressed.
本発明は、ホエイを含む、筋組織由来細胞の分化の抑制剤（以下、本発明に係る分化抑制剤ともいう）に関する。また、別の態様では、本発明は、基礎培地と、筋組織由来細胞の分化の抑制剤としてホエイとを含む培養肉製造用の培地にも関する。本発明は、基礎培地と、筋組織由来細胞の分化の抑制剤としてホエイとを含む培地で細胞を培養する工程を含む、培養肉製造のための細胞の調製方法にも関し、調製された細胞から培養肉を製造する方法にも関する。
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). In another aspect, 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
［分化抑制剤］
本発明に係る筋組織由来細胞の分化の抑制剤は、ホエイを含む。ホエイを含有することにより、筋組織由来細胞、特に筋芽細胞及び筋衛星細胞の筋管細胞への分化を抑制することができる。分化を抑制することにより、増殖性を有する筋芽細胞及び筋衛星細胞を維持することができる。また、ホエイは、筋芽細胞及び筋衛星細胞に対して細胞増殖促進作用を有する。ホエイの細胞増殖促進効果及び筋管細胞への分化の抑制効果は、それぞれＦＢＳに匹敵した（図４及び５）。これにより、培養肉製造のための大量の細胞の調製が可能になり、ＦＢＳの代替として使用することが可能になる。本発明の培養肉製造のための細胞を調製する方法は、基礎培地と、筋組織由来細胞の分化抑制剤としてホエイとを含む培地で細胞を培養する工程を含み、これにより、増殖性を有さない筋管細胞への分化を抑制しつつ、増殖させることができる。また、ホエイは食品原料であることから、本発明に係る分化抑制剤は、食品としての安全性が高い。また、ホエイは安価な原料であることから、本発明に係る分化抑制剤を添加した培地は、調製コストも低いという利点も有する。かかる培地で培養された細胞は、食品としての安全性が高いことから培養肉製造に用いることができる。 [Differentiation inhibitor]
The inhibitor of muscle tissue-derived cell differentiation according to the present invention includes 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. Moreover, since whey is a food raw material, the differentiation inhibitor according to the present invention is highly safe as a food. In addition, since whey is an inexpensive raw material, 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.
本発明に係る筋組織由来細胞の分化の抑制剤は、ホエイを含む。ホエイを含有することにより、筋組織由来細胞、特に筋芽細胞及び筋衛星細胞の筋管細胞への分化を抑制することができる。分化を抑制することにより、増殖性を有する筋芽細胞及び筋衛星細胞を維持することができる。また、ホエイは、筋芽細胞及び筋衛星細胞に対して細胞増殖促進作用を有する。ホエイの細胞増殖促進効果及び筋管細胞への分化の抑制効果は、それぞれＦＢＳに匹敵した（図４及び５）。これにより、培養肉製造のための大量の細胞の調製が可能になり、ＦＢＳの代替として使用することが可能になる。本発明の培養肉製造のための細胞を調製する方法は、基礎培地と、筋組織由来細胞の分化抑制剤としてホエイとを含む培地で細胞を培養する工程を含み、これにより、増殖性を有さない筋管細胞への分化を抑制しつつ、増殖させることができる。また、ホエイは食品原料であることから、本発明に係る分化抑制剤は、食品としての安全性が高い。また、ホエイは安価な原料であることから、本発明に係る分化抑制剤を添加した培地は、調製コストも低いという利点も有する。かかる培地で培養された細胞は、食品としての安全性が高いことから培養肉製造に用いることができる。 [Differentiation inhibitor]
The inhibitor of muscle tissue-derived cell differentiation according to the present invention includes 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. Moreover, since whey is a food raw material, the differentiation inhibitor according to the present invention is highly safe as a food. In addition, since whey is an inexpensive raw material, 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.
本発明に係る分化抑制剤は、既知の培地に添加することができる。本発明に係る分化抑制剤は、無血清培地に添加されてもよいし、動物由来血清含有培地に添加されてもよい。培養肉を製造する観点からは、無血清培地に添加することが好ましい。培地にホエイを添加後、フィルターろ過を行うことで、不溶物を取り除いた培地を培養に用いることができる。ホエイを基礎培地へと添加することで、無血清培地であっても、筋組織由来細胞、特に筋芽細胞及び筋衛星細胞の筋管細胞への分化を抑制しつつ、高い細胞増殖活性を達成することができる。したがって、本発明において用いられるホエイは、分化抑制剤であるとともに、細胞増殖促進剤ともいうことができる。本発明に係る分化抑制剤は、培養肉製造のための細胞培養において用いることができる。
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. After adding whey to the medium, the medium from which insoluble matter has been removed can be used for culture by performing filter filtration. By adding whey to the basal medium, even in a serum-free medium, differentiation of muscle tissue-derived cells, especially myoblasts and muscle satellite cells, into myotube cells is suppressed, and high cell proliferation activity is achieved. can do. Therefore, 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.
ホエイ（乳清又は乳漿ともいう）とは、乳から固形分を除いた水溶液をいう。チーズやヨーグルトなどの乳製品を製造する過程で副産物として大量に生成するため安価である。より具体的にレンネットなどの凝固剤を乳汁又は発酵乳汁に添加して凝固させた凝乳から固形分を分離することでホエイが得られる。固形分としては乳脂肪やカゼインなどのタンパク質の一部又は全部が除かれる。ホエイの主成分はラクトグロブリン、ラクトアルブミン、ラクトフェリンであるが、遊離アミノ酸、無機塩類、ビタミンなどの多種の微量成分を含む。
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.
本発明に用いられるホエイは、任意の哺乳動物由来のホエイであってよい。一例として、ウシ、ウマ、ヤギ、ヒツジ、ヒト、ロバの乳汁から得られたホエイを使用することができる。ホエイは、液体であってもよいし、ホエイを乾燥させた乾燥粉末であってもよい。分化抑制剤として添加する観点からは、輸送コスト削減が期待できるため乾燥粉末形態が好ましい。乾燥粉末形態のホエイは市販のものを用いてもよいし、乳清を凍結乾燥することで調製してもよい。乾燥粉末のホエイを分化抑制剤として用いる場合、０．００２５質量％～１．０質量％となるように基礎培地に添加される。ホエイの濃度は、分化抑制効果を発揮する観点から、０．００５質量％以上が好ましく、０．０５質量％以上がより好ましく、０．１％以上がさらに好ましい。溶解の効率性の観点から、０．５質量％以下が好ましく、０．２５質量％以下がさらに好ましい。液体のホエイを用いる場合には、乾燥粉末に換算して添加量を決定することができる。
The whey used in the present invention may be whey derived from any mammal. By way of example, 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. From the viewpoint of addition as a differentiation inhibitor, 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. When liquid whey is used, the amount to be added can be determined in terms of dry powder.
［培地］
本発明に係る分化抑制剤を添加する培地は、本技術分野に既知の培地に添加することができる。本発明に係る分化抑制剤は、無血清培地に添加されてもよいし、動物由来血清含有培地に添加されてもよい。これらの培地は、基礎培地に添加剤を添加することで製造されうる。 [Culture medium]
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.
本発明に係る分化抑制剤を添加する培地は、本技術分野に既知の培地に添加することができる。本発明に係る分化抑制剤は、無血清培地に添加されてもよいし、動物由来血清含有培地に添加されてもよい。これらの培地は、基礎培地に添加剤を添加することで製造されうる。 [Culture medium]
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.
基礎培地は、細胞培養するための培地であって、細胞の維持と増殖に必要な最低限の成分を含む培地をいう。基礎培地中に細胞を播種することで、細胞を死滅させることなく維持することができ、細胞を増殖可能であってもよい。基礎培地としては、様々な培地が市販されているが、通常、アミノ酸類、ビタミン類、緩衝剤、無機塩類及び炭素源を含む。アミノ酸としては、必須アミノ酸及び非必須アミノ酸が含まれる。ビタミン類としては、ビタミンＢ１、ビタミンＣ、ニコチン酸、葉酸等が含まれる。緩衝剤としては、ＨＥＰＥＳなどが含まれる。炭素源としてはグルコース等の単糖類、スクロース等の二糖類、オリゴ糖類や多糖類が添加されうる。通常、基礎培地に対して、血清などの添加物を添加することによって、細胞培養培地を調製することができる。基礎培地としては、本技術分野に知られている任意の基礎培地を使用することができ、一例としてダルベッコ改変イーグル培地（ＤＭＥＭ）、イーグル基礎培地（ＢＭＥ）、ＲＰＭＩ１６４０培地、ＤＭＥＭ／Ｆ１２培地、Ｆ１０培地、Ｆ１２ハム培地、ＭＥＭ、Ｍ１９９培地、エイムス培地、イスコフ改変培地、グラスゴー改変培地、フィッシャー培地などが挙げられる。
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. As the 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.
細胞培養にあたっては、通常、基礎培地に添加剤が添加される。従来の細胞培養であれば、ウシ胎児血清（ＦＢＳ）などの血清が添加剤として添加され、動物由来血清含有培地として用いられる。ＦＢＳは、筋組織由来細胞の分化を抑制しつつ、増殖を促進する。ＦＢＳに加えて、細胞の種類などに応じてその他の添加剤を加えてもよい。また、無血清培地や低血清培地においては、ＦＢＳに代わって細胞の増殖性を維持するために必要とされる成分を含む。こうした添加剤としては、例えば、脂質、ホルモン剤、成長因子、サイトカイン、血清由来タンパク質、抗生物質などが挙げられ、無血清培地及び/又は動物由来血清含有培地に添加されうる。ホルモン剤としては、デキサメタゾン等が挙げられる。成長因子としては、ＦＧＦ、ＩＧＦ、インスリン等が挙げられ、その任意のファミリーが使用されてもよい。サイトカイン類としては、ＩＬ－１α、ＩＬ－１βなどが挙げられる。血清由来タンパク質としてはフェチュイン、フィブロネクチン、アルブミン、グロブリンなどが挙げられる。抗生物質としてはペニシリン、ストレプトマイシン等を使用しうる。無血清培地や低血清培地で一般に使用される添加剤である、ＩＴＳ（インスリン-トランスフェリン-亜セレン酸ナトリウム）も本発明に係る分化抑制剤と共に培地、特に無血清培地に添加されうる。
Additives are usually added to the basal medium for cell culture. In conventional 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. In addition to FBS, other additives may be added depending on the cell type. In serum-free or low-serum media, it also contains components necessary for maintaining cell proliferation in place of FBS. Such 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-1α, IL-1β 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.
動物由来血清とは、動物の血液から製造された血清をいう。取得された血液を凝固させて得られた上澄み液を血清という。動物由来血清としては、任意の動物、例えば、ウシ、ウマ、ヤギ、ロバ、ウサギ、トリなどの動物由来の血清であってもよいが、特にウシ血清（ＢＣＳ）、ウシ胎児血清（ＦＢＳ）を指す。血清には、アルブミン、グロブリンなどのタンパク質の他、中性脂肪、コレステロール、リン脂質、遊離脂肪酸などの血清脂質が含まれ、さらにホルモン、サイトカイン、増殖因子などを含む。特に胎児血清は、細胞の増殖に必要とされる成分が豊富に含まれており、研究や医薬分野において、培地へ添加することが一般的である。動物由来血清を含まない培地を無血清培地と呼ぶ。一方で、無血清培地は、動物由来血清を含まないが、血清由来の精製された成分を含んでもよいし、血清由来成分の組換えタンパク質を含んでもよい。
"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. On the other hand, serum-free media do not contain animal-derived serum, but may contain purified serum-derived components or recombinant proteins of serum-derived components.
［細胞］
本発明に係る分化抑制培地は、任意の動物から得られた筋組織由来細胞において分化を抑制することができる。培養肉を製造する観点から、ウシ、ブタ、ヤギ、ヒツジ、ウサギ、ニワトリ、ダチョウ、カモ等の家畜由来の細胞を使用しうる。特にウシの細胞を用いる場合、ホルスタイン種、ジャージー種、黒毛和種、褐毛和種、短角種、無角和種、並びにそれらの交配種のうちの任意の種の細胞であってもよい。特に食肉製造の観点から肉用種である黒毛和種、褐毛和種、短角種、無角和種の細胞が好ましい。これらの動物の筋組織由来細胞を培養することができる。動物由来の筋組織由来細胞は、動物から取得された初代細胞、及び初代細胞から継代された継代細胞であってもよいし、株化された細胞であってもよい。初代細胞は、動物組織を培地中で切り刻むことにより取得することができる。体性幹細胞、胚性幹細胞、誘導多能性幹細胞などの幹細胞から分化された細胞であってもよい。 [cell]
The differentiation-suppressing medium according to the present invention can suppress differentiation in muscle tissue-derived cells obtained from any animal. From the viewpoint of producing cultured meat, cells derived from livestock such as cows, pigs, goats, sheep, rabbits, chickens, ostriches and ducks can be used. In particular, when bovine cells are used, cells of any of Holstein, Jersey, Japanese Black, Japanese Brown, Shorthorn, Japanese Polled, and hybrids thereof may be used. In particular, from the viewpoint of meat production, 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.
本発明に係る分化抑制培地は、任意の動物から得られた筋組織由来細胞において分化を抑制することができる。培養肉を製造する観点から、ウシ、ブタ、ヤギ、ヒツジ、ウサギ、ニワトリ、ダチョウ、カモ等の家畜由来の細胞を使用しうる。特にウシの細胞を用いる場合、ホルスタイン種、ジャージー種、黒毛和種、褐毛和種、短角種、無角和種、並びにそれらの交配種のうちの任意の種の細胞であってもよい。特に食肉製造の観点から肉用種である黒毛和種、褐毛和種、短角種、無角和種の細胞が好ましい。これらの動物の筋組織由来細胞を培養することができる。動物由来の筋組織由来細胞は、動物から取得された初代細胞、及び初代細胞から継代された継代細胞であってもよいし、株化された細胞であってもよい。初代細胞は、動物組織を培地中で切り刻むことにより取得することができる。体性幹細胞、胚性幹細胞、誘導多能性幹細胞などの幹細胞から分化された細胞であってもよい。 [cell]
The differentiation-suppressing medium according to the present invention can suppress differentiation in muscle tissue-derived cells obtained from any animal. From the viewpoint of producing cultured meat, cells derived from livestock such as cows, pigs, goats, sheep, rabbits, chickens, ostriches and ducks can be used. In particular, when bovine cells are used, cells of any of Holstein, Jersey, Japanese Black, Japanese Brown, Shorthorn, Japanese Polled, and hybrids thereof may be used. In particular, from the viewpoint of meat production, 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.
筋組織由来細胞とは、筋組織を構成する細胞であり、筋組織から分離されて培養される細胞である。筋組織由来細胞としては、筋芽細胞、筋衛星細胞、筋管細胞が挙げられるが、筋管細胞は増殖性を有さないため、増殖させる観点からは、筋芽細胞及び/又は筋衛星細胞が好ましい。筋衛星細胞は、筋肉中に含まれる体性幹細胞であり、転写因子Ｐａｘ７の発現により特徴づけられる。筋衛星細胞は、通常、生体内では静止状態（休止期）を維持しており、傷害などで活性化することで増殖性を有する筋芽細胞に分化する。細胞培養を行う場合、培養条件によっては、筋衛星細胞は未分化状態で増殖させることもできる。筋芽細胞とは、筋線維を形成可能な増殖性を有する単核の細胞であり、ＭｙｏＤの発現により特徴づけられる。筋芽細胞がさらに分化すると、筋芽細胞同士が融合し、多核の筋管細胞を形成し、さらに成熟して筋線維となる。筋管細胞への分化は、多核や形状を観察することで決定することができる（図１～３）。さらに、筋芽細胞から筋管細胞への分化は、筋管細胞特異的に発現する遺伝子、例えばデスミン、ミオジェニン等の発現を測定することで決定することもできる（図６）。本発明の分化抑制剤は、筋衛星細胞から筋芽細胞への分化、筋芽細胞から筋管細胞への分化をそれぞれ抑制することができる。一方、筋芽細胞及び筋衛星細胞は、コンフルエントに達して一定時間が経過すると、筋管細胞へと分化する。
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.
［培養肉］
培養肉とは、細胞培養を介して製造される食肉のことをいう。本発明において「培養肉製造のため」とは、培養肉の製造に用いる方法であり、食品衛生的に許容されることが要求される。食品衛生上、動物由来血清、ホルモン剤、遺伝子組み換えタンパク質の使用は避けることが好ましい。一般に食肉とは、筋線維、結合組織、及び脂肪の集合体をいう。一方、培養肉は食肉の構造を模倣することが好ましいが、必ずしも食肉の構成を全て含まなくてもよく、筋組織由来細胞の培養細胞を含むものであればよい。培養肉は複数種の細胞の培養物を含んでもよい。培養肉は、筋組織由来細胞からなる群から選ばれる少なくとも１の培養細胞にくわえ、細胞外マトリクスを含んでもよい。培養肉の製造方法は、例えば以下の
筋組織由来細胞を培養する工程、
培養された細胞を回収し、集積する工程
を含む。培養肉の製造方法は、さらに、分化誘導工程や集積後の培養工程を含んでもよい。本発明は、本発明に係る分化抑制剤を含有する培地で培養された細胞を含む培養肉にも関する。 [Cultivated meat]
Cultured meat refers to meat produced through cell culture. In the present invention, "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. On the other hand, 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.
培養肉とは、細胞培養を介して製造される食肉のことをいう。本発明において「培養肉製造のため」とは、培養肉の製造に用いる方法であり、食品衛生的に許容されることが要求される。食品衛生上、動物由来血清、ホルモン剤、遺伝子組み換えタンパク質の使用は避けることが好ましい。一般に食肉とは、筋線維、結合組織、及び脂肪の集合体をいう。一方、培養肉は食肉の構造を模倣することが好ましいが、必ずしも食肉の構成を全て含まなくてもよく、筋組織由来細胞の培養細胞を含むものであればよい。培養肉は複数種の細胞の培養物を含んでもよい。培養肉は、筋組織由来細胞からなる群から選ばれる少なくとも１の培養細胞にくわえ、細胞外マトリクスを含んでもよい。培養肉の製造方法は、例えば以下の
筋組織由来細胞を培養する工程、
培養された細胞を回収し、集積する工程
を含む。培養肉の製造方法は、さらに、分化誘導工程や集積後の培養工程を含んでもよい。本発明は、本発明に係る分化抑制剤を含有する培地で培養された細胞を含む培養肉にも関する。 [Cultivated meat]
Cultured meat refers to meat produced through cell culture. In the present invention, "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. On the other hand, 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.
細胞の培養は、基礎培地に分化抑制剤としてホエイとを含む培地に、細胞を播種することで行われる。培養は、本技術分野において周知の条件、例えば３７℃５％ＣＯ2インキュベータ内で培養される。培養は平板培養であってもよいし、浮遊培養であってもよい。増殖した細胞は、トリプシン処理などにより細胞を培養物として回収することができ、回収後さらに継代培養を行ってもよい。細胞の培養は、剥離可能な構築物に細胞を播種して培養することもできる。増殖した細胞が付着された構築物を培養物として回収することができる。かかる構築物は、コラーゲン、エラスチン、フィブロネクチン、ラミニン、エンタクチンなどの細胞外マトリクスで構築することができ、細胞が付着した構築物を集積して培養肉を成型してもよい。
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.
集積工程は、回収された１又は複数種の細胞の培養物を成型することを含む。集積工程で成型された培養物は、ステーキなどのように１枚肉であってもよいし、枝肉であってもよいし、ミンチ状であってもよい。集積工程は、細胞の培養物を、他の細胞、血液及び組織からなる群から選ばれる少なくとも１の物質と合わせて集積することを含む。他の細胞としては、培養細胞であってもよいし、動物から採取された細胞であってもよい。集積後にさらに共培養を行うこともできる。一例として、回収された１又は複数種の細胞の培養物を混合し、細胞外マトリクスに播種して共培養することができる。細胞外マトリクスとしては、コラーゲン、エラスチン、フィブロネクチン、ラミニン、エンタクチンなどを使用しうる。
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.
集積工程は、回収された１又は複数種の細胞の培養物を、血液及び/又は組織と集積してもよい。組織としては、動物から取得されたものであっても、培養されたものであってもよい。一例として、食肉の処理過程で分離された血液、脂肪組織や筋組織などを培養物と集積して培養肉を製造してもよい。
In the accumulating step, 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. For example, cultured meat may be produced by accumulating blood, adipose tissue, muscle tissue, or the like separated during meat processing with a culture.
分化誘導工程は、細胞培養後に行われてもよいし、集積工程の前、集積工程中、集積工程後に行われてもよい。分化誘導工程により、単核の筋衛星細胞及び筋芽細胞を多核の筋管細胞へと分化させ、さらに筋線維として成熟させることができる。分化誘導は、本発明の分化抑制剤を含まない培地中で培養することを含む。本技術分野に既知の方法により行われてよいが、高い二酸化炭素濃度下で培養する手法が知られており、一例として５～１０％（ｖ／ｖ）のＣＯ2雰囲気下でホエイ非含有培地中で培養することにより、筋管細胞への分化を促進することができる。
The differentiation-inducing step may be performed after cell culturing, or may be performed before, during, or after the enrichment step. By the differentiation-inducing 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.
本明細書において言及される全ての文献はその全体が引用により本明細書に取り込まれる。以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。
All documents referred to in this specification are hereby incorporated by reference in their entirety. The embodiments of the invention described below are for illustrative purposes only and are not intended to limit the scope of the invention. The technical scope of the present invention is limited only by the description of the claims. Modifications of the present invention, such as additions, deletions and replacements of constituent elements of the present invention, can be made without departing from the gist of the present invention.
試験１：筋芽細胞の採取
ウシ筋芽細胞は頬肉を用いて、以下の工程により細胞を採取した。採取した組織をエタノールとＰＢＳで洗浄後に、クリーンベンチ内ではさみを用いて細かく刻んだ。０．２％コラゲナーゼＩＩを添加したダルベッコ改変イーグル培地中で、３７℃で１．５時間振とう培養を行い筋組織を消化させた。消化後の反応液に２０％ＦＢＳを加える事で反応を停止させた。消化液を８０×ｇで３分間遠心分離し浮遊組織をピンセットで除いた上で、上澄み液を分取した。再度８０×ｇで３分間遠心分離して得た上澄み液を細胞分別用のナイロンメッシュ（１００μｍ）に通した。ろ過液を１５００×ｇで５分間遠心分離して得た沈殿を、２０％ＦＢＳを含むダルベッコ改変イーグル培地中で懸濁した。細胞懸濁液を１００μｍのナイロンメッシュに通した後、再度４０μｍのナイロンメッシュに通し、ろ液を１５００×ｇで５分間遠心分離した。沈殿を赤血球溶解液で氷上で５分間静置し、血球細胞を除去した。リン酸緩衝液で２回洗浄した上で、１０％ＦＢＳを含むダルベッコ改変イーグル培地中にプールさせ、培養皿に播種した。増殖した細胞を試験に用いた。 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 μm) 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 μm nylon mesh, it was again passed through a 40 μm 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.
ウシ筋芽細胞は頬肉を用いて、以下の工程により細胞を採取した。採取した組織をエタノールとＰＢＳで洗浄後に、クリーンベンチ内ではさみを用いて細かく刻んだ。０．２％コラゲナーゼＩＩを添加したダルベッコ改変イーグル培地中で、３７℃で１．５時間振とう培養を行い筋組織を消化させた。消化後の反応液に２０％ＦＢＳを加える事で反応を停止させた。消化液を８０×ｇで３分間遠心分離し浮遊組織をピンセットで除いた上で、上澄み液を分取した。再度８０×ｇで３分間遠心分離して得た上澄み液を細胞分別用のナイロンメッシュ（１００μｍ）に通した。ろ過液を１５００×ｇで５分間遠心分離して得た沈殿を、２０％ＦＢＳを含むダルベッコ改変イーグル培地中で懸濁した。細胞懸濁液を１００μｍのナイロンメッシュに通した後、再度４０μｍのナイロンメッシュに通し、ろ液を１５００×ｇで５分間遠心分離した。沈殿を赤血球溶解液で氷上で５分間静置し、血球細胞を除去した。リン酸緩衝液で２回洗浄した上で、１０％ＦＢＳを含むダルベッコ改変イーグル培地中にプールさせ、培養皿に播種した。増殖した細胞を試験に用いた。 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 μm) 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 μm nylon mesh, it was again passed through a 40 μm 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.
試験２：筋芽細胞の血清含有培地での培養
試験１において採取した筋芽細胞を、ダルベッコ改変イーグル培地に、1％ペニシリン－ストレプトマイシン溶液、２０％ＦＢＳ、２ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子を添加した血清含有培地で３７℃・５％ＣＯ2雰囲気下で培養を行った。培養４日目に顕微鏡観察を行った（図１）。血清含有培地で筋芽細胞を培養すると、増殖してコンフルエントに達し、培養４日目にはコンフルエントに達した筋芽細胞が融合し多核の筋管細胞へと分化した。 Test 2: Cultivation of Myoblasts in Serum-Containing Medium Myoblasts harvested inTest 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.
試験１において採取した筋芽細胞を、ダルベッコ改変イーグル培地に、1％ペニシリン－ストレプトマイシン溶液、２０％ＦＢＳ、２ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子を添加した血清含有培地で３７℃・５％ＣＯ2雰囲気下で培養を行った。培養４日目に顕微鏡観察を行った（図１）。血清含有培地で筋芽細胞を培養すると、増殖してコンフルエントに達し、培養４日目にはコンフルエントに達した筋芽細胞が融合し多核の筋管細胞へと分化した。 Test 2: Cultivation of Myoblasts in Serum-Containing Medium Myoblasts harvested in
試験３：筋芽細胞の無血清培地での培養
試験１において採取した筋芽細胞を、ダルベッコ改変イーグル培地に、１％ペニシリン－ストレプトマイシン溶液、１％ＩＴＳ液体培地サプリメント）、２ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子、細胞培養用脂質添加剤（sigma, L0288）、０．２％ＢＳＡを添加した無血清培地で３７℃・５％ＣＯ2雰囲気下で培養を行った。培養３日目に顕微鏡観察を行った（図２）。無血清培地で筋芽細胞を培養すると、培養３日目には多くの細胞は単核の筋芽細胞を維持しているが、一部の筋芽細胞は融合し多核の筋管細胞へと分化した。 Test 3: Cultivation of myoblasts in serum-free medium Myoblasts harvested inTest 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.
試験１において採取した筋芽細胞を、ダルベッコ改変イーグル培地に、１％ペニシリン－ストレプトマイシン溶液、１％ＩＴＳ液体培地サプリメント）、２ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子、細胞培養用脂質添加剤（sigma, L0288）、０．２％ＢＳＡを添加した無血清培地で３７℃・５％ＣＯ2雰囲気下で培養を行った。培養３日目に顕微鏡観察を行った（図２）。無血清培地で筋芽細胞を培養すると、培養３日目には多くの細胞は単核の筋芽細胞を維持しているが、一部の筋芽細胞は融合し多核の筋管細胞へと分化した。 Test 3: Cultivation of myoblasts in serum-free medium Myoblasts harvested in
試験４：無血清培地での筋管分化を抑制させる食品素材の探索
無血清培地としては、ダルベッコ改変イーグル培地に、ペニシリン－ストレプトマイシン溶液、ＩＴＳ液体培地サプリメント、２ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子、細胞培養用脂質添加剤、０．２％ＢＳＡを加えたものを使用した(試験３と同様)。
食品原料成分としては、卵白・大豆・ホエイ・小麦粉（いずれも乾燥粉末)を用いた。各種食品成分は無血清培地に対して０．１％で溶かし、遠心分離後の上清を０．４５μｍのフィルターろ過して不溶成分を除去したものを試験に用いた。試験１で採取した細胞を、約５×１０3ｃｅｌｌｓ／ｃｍ3で播種し、３７℃・ＣＯ2濃度５％に設定したＣＯ2インキュベータ内で培養した。筋管への分化は、培養４日目の顕微鏡観察を行う事で確認した（図３）。対照として、未添加培地（－）、１０％ＦＢＳ添加培地を用いて同じ実験を行った。卵白、大豆、小麦粉を添加して培養した場合の細胞数及び細胞形態は、未添加培地で培養した場合と大差がなかった。一方で、ホエイを添加して培養した場合、細胞数は１０％ＦＢＳ添加群と同等に多い一方で、分化して融合した細胞の数が少なかった。また形状も単離した際の形状を維持した。したがって、ホエイは、分化抑制効果と細胞増殖効果を発揮した。 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 μm filter to remove insoluble components. The cells collected inTest 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.
無血清培地としては、ダルベッコ改変イーグル培地に、ペニシリン－ストレプトマイシン溶液、ＩＴＳ液体培地サプリメント、２ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子、細胞培養用脂質添加剤、０．２％ＢＳＡを加えたものを使用した(試験３と同様)。
食品原料成分としては、卵白・大豆・ホエイ・小麦粉（いずれも乾燥粉末)を用いた。各種食品成分は無血清培地に対して０．１％で溶かし、遠心分離後の上清を０．４５μｍのフィルターろ過して不溶成分を除去したものを試験に用いた。試験１で採取した細胞を、約５×１０3ｃｅｌｌｓ／ｃｍ3で播種し、３７℃・ＣＯ2濃度５％に設定したＣＯ2インキュベータ内で培養した。筋管への分化は、培養４日目の顕微鏡観察を行う事で確認した（図３）。対照として、未添加培地（－）、１０％ＦＢＳ添加培地を用いて同じ実験を行った。卵白、大豆、小麦粉を添加して培養した場合の細胞数及び細胞形態は、未添加培地で培養した場合と大差がなかった。一方で、ホエイを添加して培養した場合、細胞数は１０％ＦＢＳ添加群と同等に多い一方で、分化して融合した細胞の数が少なかった。また形状も単離した際の形状を維持した。したがって、ホエイは、分化抑制効果と細胞増殖効果を発揮した。 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 μm filter to remove insoluble components. The cells collected in
試験５：無血清培地培養時の筋管分化抑制効果の定量
試験４の培養物の筋管形成率を、定量化した。核及びミオシン重鎖を染色したサンプルをキーエンス社製のオールインワン顕微鏡で観察し、視野における全核数に対する筋管中の核数の割合を測定して分化の指標（fusion index）とした。ミオシン重鎖は、下記の工程により免疫染色を行った。細胞をリン酸緩衝液（ＰＢＳ）で１回洗浄した後、４％パラホルムアルデヒドで４°Ｃで１晩固定した。ＰＢＳで３回洗浄後、１％ Ｔｒｉｔｏｎ Ｘ－１００／ＰＢＳで室温５分間透過処理を行った。ＰＢＳで３回洗浄後、免疫染色用の市販のブロッキング溶液（ケー・エー・シー，ＣＴＫＮ００１）を用いて室温で３０分間ブロッキングを行った。抗ミオシン重鎖モノクローナル抗体（Ｃｌｏｎｅ ＭＦ２０）を１μｇ／ｍＬに希釈した液で室温１時間にわたり１次抗体反応を行った。ＰＢＳで３回洗浄後、Ａｌｅｘａ ４８８ ｇｏａｔ ａｎｔｉ ｍｏｕｓｅ ＩｇＧ （ａｂｃａｍ， ａｂ１５０１１７ ） の５００倍希釈液を用いて室温で３０分間にわたり２次抗体反応を行った。ＰＢＳ洗浄後にＤＡＰＩで核染色を行い、蛍光顕微鏡による観察を行い、分化の指標を決定した（図４）。また、細胞数から細胞増殖効果についても検討した（図５）。ホエイ添加群では、１０％ＦＢＳ添加群と同程度に、増殖が促進され、かつ分化率が低かった。 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. After washing three times with PBS, permeabilization was performed with 1% Triton X-100/PBS for 5 minutes at room temperature. After washing with PBS three times, blocking was performed at room temperature for 30 minutes using a commercially available blocking solution for immunostaining (KAC, CTKN001). A primary antibody reaction was carried out at room temperature for 1 hour with an anti-myosin heavy chain monoclonal antibody (Clone MF20) diluted to 1 μg/mL. After washing with PBS three times, secondary antibody reaction was carried out for 30 minutes at room temperature using a 500-fold dilution of Alexa 488 goat anti-mouse IgG (abcam, ab150117). After washing with PBS, nuclear staining was performed with DAPI, observation was performed with a fluorescence microscope, and the index of differentiation was determined (Fig. 4). Moreover, the cell proliferation effect was also examined from the number of cells (Fig. 5). In the whey-added group, proliferation was promoted and the differentiation rate was low to the same extent as the 10% FBS-added group.
試験４の培養物の筋管形成率を、定量化した。核及びミオシン重鎖を染色したサンプルをキーエンス社製のオールインワン顕微鏡で観察し、視野における全核数に対する筋管中の核数の割合を測定して分化の指標（fusion index）とした。ミオシン重鎖は、下記の工程により免疫染色を行った。細胞をリン酸緩衝液（ＰＢＳ）で１回洗浄した後、４％パラホルムアルデヒドで４°Ｃで１晩固定した。ＰＢＳで３回洗浄後、１％ Ｔｒｉｔｏｎ Ｘ－１００／ＰＢＳで室温５分間透過処理を行った。ＰＢＳで３回洗浄後、免疫染色用の市販のブロッキング溶液（ケー・エー・シー，ＣＴＫＮ００１）を用いて室温で３０分間ブロッキングを行った。抗ミオシン重鎖モノクローナル抗体（Ｃｌｏｎｅ ＭＦ２０）を１μｇ／ｍＬに希釈した液で室温１時間にわたり１次抗体反応を行った。ＰＢＳで３回洗浄後、Ａｌｅｘａ ４８８ ｇｏａｔ ａｎｔｉ ｍｏｕｓｅ ＩｇＧ （ａｂｃａｍ， ａｂ１５０１１７ ） の５００倍希釈液を用いて室温で３０分間にわたり２次抗体反応を行った。ＰＢＳ洗浄後にＤＡＰＩで核染色を行い、蛍光顕微鏡による観察を行い、分化の指標を決定した（図４）。また、細胞数から細胞増殖効果についても検討した（図５）。ホエイ添加群では、１０％ＦＢＳ添加群と同程度に、増殖が促進され、かつ分化率が低かった。 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. After washing three times with PBS, permeabilization was performed with 1% Triton X-100/PBS for 5 minutes at room temperature. After washing with PBS three times, blocking was performed at room temperature for 30 minutes using a commercially available blocking solution for immunostaining (KAC, CTKN001). A primary antibody reaction was carried out at room temperature for 1 hour with an anti-myosin heavy chain monoclonal antibody (Clone MF20) diluted to 1 μg/mL. After washing with PBS three times, secondary antibody reaction was carried out for 30 minutes at room temperature using a 500-fold dilution of Alexa 488 goat anti-mouse IgG (abcam, ab150117). After washing with PBS, nuclear staining was performed with DAPI, observation was performed with a fluorescence microscope, and the index of differentiation was determined (Fig. 4). Moreover, the cell proliferation effect was also examined from the number of cells (Fig. 5). In the whey-added group, proliferation was promoted and the differentiation rate was low to the same extent as the 10% FBS-added group.
試験６：無血清培地培養時の筋管分化抑制効果の定量
各種培地で増殖培養させた筋芽細胞の分化状態の確認を行った。無血清培地は、ダルベッコ改変イーグル培地に、ペニシリン－ストレプトマイシン溶液、ＩＴＳ液体培地サプリメント、５ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子、細胞培養用脂質添加剤、０．０５％ＢＳＡを加えたものを使用した。食品成分添加培地は無血清培地をベースとして各種食品成分を０．１％添加し、遠心分離後の上清を０．４５μｍのフィルターろ過して不溶成分を除去したものを試験に用いた。血清含有培地には、ダルベッコ改変イーグル培地に、ペニシリン－ストレプトマイシン溶液、５ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子、１０％ＦＢＳを加えたものを使用した。
約５×１０３ｃｅｌｌｓ／ ｃｍ３の細胞を各種培地を含む培養皿に播種し、３７℃・ＣＯ２濃度５％に設定したＣＯ２インキュベータ内で培養した。培養２日後の筋分化マーカーの発現をｑＰＣＲにより評価した。 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 μm 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.
各種培地で増殖培養させた筋芽細胞の分化状態の確認を行った。無血清培地は、ダルベッコ改変イーグル培地に、ペニシリン－ストレプトマイシン溶液、ＩＴＳ液体培地サプリメント、５ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子、細胞培養用脂質添加剤、０．０５％ＢＳＡを加えたものを使用した。食品成分添加培地は無血清培地をベースとして各種食品成分を０．１％添加し、遠心分離後の上清を０．４５μｍのフィルターろ過して不溶成分を除去したものを試験に用いた。血清含有培地には、ダルベッコ改変イーグル培地に、ペニシリン－ストレプトマイシン溶液、５ｎｇ／ｍｌヒト塩基性線維芽細胞増殖因子、１０％ＦＢＳを加えたものを使用した。
約５×１０３ｃｅｌｌｓ／ ｃｍ３の細胞を各種培地を含む培養皿に播種し、３７℃・ＣＯ２濃度５％に設定したＣＯ２インキュベータ内で培養した。培養２日後の筋分化マーカーの発現をｑＰＣＲにより評価した。 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 μm 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.
ｑＰＣＲ方法
ＴＲＩｚｏｌ Ｒｅａｇｅｎｔ（インビトロジェン）により細胞からＲＮＡ抽出を行い、ＰｒｉｍｅＳｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ Ｋｉｔ（タカラバイオ)を用いた逆転写反応によりｃＤＮＡを合成した。ｃＤＮＡを鋳型にしてＢｉｏＲａｄ社製のリアルタイムＰＣＲ機を用いてターゲット遺伝子の増幅を行った。遺伝子増幅は、ＴＢ ＧｒｅｅｎII Ｋｉｔ（タカラバイオ）と表１に対応したプライマーを使用した。ターゲット遺伝子の発現量は、内在性コントロール遺伝子との発現比較により評価した（ΔCt法）。血清含有培地でのマーカー発現を１とした時の、培養液間の各種遺伝子発現比較を行った。
ホエイを添加した培地において、筋管細胞に特異的に発現するデスミン及びミオジェニンの発現量が、対照の１０％ＦＢＳと同等であった一方、食品成分未添加の無血清培地（無血清）や、他の食品成分（大豆粉末、小麦粉、卵白）を添加した培地においては、デスミンの発現が、対照の１０％ＦＢＳと比較し約３倍増加し、ミオジェニンの発現が対照の１０％ＦＢＳと比較し約２倍増加した。ホエイは、１０％ＦＢＳと同等に、筋芽細胞から筋管細胞への分化を抑制していることが示された。
RNA was extracted from the cells by qPCR method TRIzol Reagent (Invitrogen), and cDNA was synthesized by reverse transcription reaction using PrimeScript RT reagent Kit (Takara Bio). Using the cDNA as a template, the target gene was amplified using a BioRad real-time PCR machine. 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 the endogenous control gene (ΔCt method). When marker expression in serum-containing medium was set to 1, various gene expression comparisons between cultures were performed.
In the medium supplemented with whey, the expression levels of desmin and myogenin, which are specifically expressed in myotube cells, were equivalent to those of the control 10% FBS. In media supplemented with other food ingredients (soybean flour, wheat flour, egg white), desmin expression increased approximately 3-fold compared to the 10% FBS control, and myogenin expression increased compared to the 10% FBS control. increased by about a factor of two. Whey was shown to suppress differentiation from myoblasts to myotube cells to the same extent as 10% FBS.
ＴＲＩｚｏｌ Ｒｅａｇｅｎｔ（インビトロジェン）により細胞からＲＮＡ抽出を行い、ＰｒｉｍｅＳｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ Ｋｉｔ（タカラバイオ)を用いた逆転写反応によりｃＤＮＡを合成した。ｃＤＮＡを鋳型にしてＢｉｏＲａｄ社製のリアルタイムＰＣＲ機を用いてターゲット遺伝子の増幅を行った。遺伝子増幅は、ＴＢ ＧｒｅｅｎII Ｋｉｔ（タカラバイオ）と表１に対応したプライマーを使用した。ターゲット遺伝子の発現量は、内在性コントロール遺伝子との発現比較により評価した（ΔCt法）。血清含有培地でのマーカー発現を１とした時の、培養液間の各種遺伝子発現比較を行った。
試験７：分化抑制効果を発揮するホエイの濃度の検討
食品成分添加培地のホエイ添加濃度を変化させた時の、増殖・分化への影響を評価した。食品成分添加培地は、ダルベッコ改変イーグル培地に、ペニシリン－ストレプトマイシン溶液、ITS液体培地サプリメント、２ｎｇ／ｍｌ ヒト塩基性線維芽細胞増殖因子、細胞培養用脂質添加剤、０．２％ ＢＳＡを添加したものを用いた。各種濃度のホエイ粉末（０．００５質量％、０．０２５質量％、０．０５質量％、０．１質量％、０．２５質量％、０．５質量％、１．０質量％）を溶解させ、遠心分離後の上清を０．４５μｍのフィルターろ過して不溶成分を除去したものを試験に用いた。約７．５×１０3cells/ cm3の細胞を播種し、３７℃・ＣＯ２濃度５％に設定したＣＯ２インキュベータ内で培養した。培養４日目に細胞数計測と筋管形成率（ｆｕｓｉｏｎ ｉｎｄｅｘ）の算出を行った。細胞数は、トリプシン処理により得た生細胞数をカウントした。筋管細胞の割合は、ミオシン重鎖に対する免疫染色を行い、視野における全核数に対するミオシン陽性細胞の割合を測定して分化の指標とした。ホエイは、０．００５質量％の濃度で分化抑制効果を発揮し、１．０質量％まで用量依存的に分化抑制効果を発揮した。 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 μm 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.
食品成分添加培地のホエイ添加濃度を変化させた時の、増殖・分化への影響を評価した。食品成分添加培地は、ダルベッコ改変イーグル培地に、ペニシリン－ストレプトマイシン溶液、ITS液体培地サプリメント、２ｎｇ／ｍｌ ヒト塩基性線維芽細胞増殖因子、細胞培養用脂質添加剤、０．２％ ＢＳＡを添加したものを用いた。各種濃度のホエイ粉末（０．００５質量％、０．０２５質量％、０．０５質量％、０．１質量％、０．２５質量％、０．５質量％、１．０質量％）を溶解させ、遠心分離後の上清を０．４５μｍのフィルターろ過して不溶成分を除去したものを試験に用いた。約７．５×１０3cells/ cm3の細胞を播種し、３７℃・ＣＯ２濃度５％に設定したＣＯ２インキュベータ内で培養した。培養４日目に細胞数計測と筋管形成率（ｆｕｓｉｏｎ ｉｎｄｅｘ）の算出を行った。細胞数は、トリプシン処理により得た生細胞数をカウントした。筋管細胞の割合は、ミオシン重鎖に対する免疫染色を行い、視野における全核数に対するミオシン陽性細胞の割合を測定して分化の指標とした。ホエイは、０．００５質量％の濃度で分化抑制効果を発揮し、１．０質量％まで用量依存的に分化抑制効果を発揮した。 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 μm 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.
Claims (6)
- ホエイを含む、筋組織由来細胞の分化の抑制剤。 An inhibitor of differentiation of muscle tissue-derived cells, including whey.
- 前記筋組織由来細胞が、筋芽細胞、及び衛星細胞からなる群から選択される、請求項１に記載の抑制剤。 The inhibitor according to claim 1, wherein the muscle tissue-derived cells are selected from the group consisting of myoblasts and satellite cells.
- 前記抑制剤が、筋芽細胞及び衛星細胞から筋管細胞への分化を抑制する、請求項１又は２に記載の抑制剤。 The inhibitor according to claim 1 or 2, which inhibits differentiation from myoblasts and satellite cells to myotube cells.
- 前記筋組織由来細胞が、家畜由来の筋組織由来細胞である、請求項１～３のいずれか一項に記載の抑制剤。 The inhibitor according to any one of claims 1 to 3, wherein the muscle tissue-derived cells are livestock-derived muscle tissue-derived cells.
- 前記抑制剤が、培養肉製造のための培地に添加される、請求項１～４のいずれか一項に記載の抑制剤。 The inhibitor according to any one of claims 1 to 4, wherein the inhibitor is added to a medium for producing cultivated meat.
- 前記ホエイ濃度が、０．００５％～１．０％の濃度である、請求項１～５のいずれか一項に記載の抑制剤。 The inhibitor according to any one of claims 1 to 5, wherein the whey concentration is 0.005% to 1.0%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05508542A (en) * | 1990-07-13 | 1993-12-02 | グロペップ リミテッド | growth promoter |
| JPH09512536A (en) * | 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 | Modified milk growth factor |
| JP2009507044A (en) * | 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 | Milk-derived composition and use for enhancing muscle mass or strength |
-
2022
- 2022-12-28 WO PCT/JP2022/048646 patent/WO2023127960A1/en active Application Filing
- 2022-12-28 JP JP2023571227A patent/JPWO2023127960A1/ja active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05508542A (en) * | 1990-07-13 | 1993-12-02 | グロペップ リミテッド | growth promoter |
| JPH09512536A (en) * | 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 | Modified milk growth factor |
| JP2009507044A (en) * | 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 | Milk-derived composition and use for enhancing muscle mass or strength |
Also Published As
| Publication number | Publication date |
|---|---|
| JPWO2023127960A1 (en) | 2023-07-06 |
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