Cell growth media for cultured meat production

Abstract

The purpose of the present invention is to provide a medium, in which cells to be used for producing a cultured meat can be proliferated, by adding a food material as an additive. The present inventors cultured cells with the use of food components as additives and, consequently, found that when whey was added as a cell proliferation promoter, fibroblasts, adipocytes and myoblasts exhibited high proliferation ability. Thus, the present invention, which pertains to a cell proliferation medium comprising a basic medium and whey as a cell proliferation promoter, has been completed.

Description

[Technical Field]

[0001] The present invention relates to the technical field of cultured meat production. More specifically, the present invention relates to a culture medium for growing cells used in cultured meat production, a method for producing the culture medium, a method for preparing cells for cultured meat production, and a cell growth promoter for cultured meat production. [Background technology]

[0002] Meat production has traditionally been carried out through the raising of livestock. However, raising livestock requires large amounts of grain and water, and large farms. In recent years, issues of climate change and food shortages have come to the forefront, and there is a growing desire for sustainable meat production with a lower environmental impact and higher production efficiency. In this context, research and development into the production of cultured meat from cells has been attracting attention as a new method of meat production.

[0003] Plant-based meat substitutes are known as meat substitutes, but their texture and flavor fall short of those of meat. On the other hand, cultured meat, which is produced by culturing animal cells, can achieve a texture and flavor similar to that of real meat and has the advantage of being less susceptible to bacterial and viral contamination than meat. The production of cultured meat is becoming technically feasible. However, the cell culture media used in cultured meat production to date utilize large-scale culture techniques previously used in basic research and pharmaceutical applications. Due to their cost and safety as meat, their use in food production has been difficult. Cell culture media used in basic research and pharmaceutical applications typically contain a basal medium containing amino acids, vitamins, inorganic salts, and a carbon source such as glucose, supplemented with fetal bovine serum (FBS) as an additional ingredient (Non-Patent Document 1: Mol Ther. 2004 Mar;9(3):475-82). However, because FBS is serum collected from fetuses, it is difficult to obtain in large quantities, and there are issues with price, transportation costs, the risk of infectious diseases, and animal welfare. To address these issues, synthetic media supplemented with essential FBS components have been developed (Non-Patent Document 2: The Canadian Journal of Chem Engineering Vol. 94, (10) October 2016 1855-1862). However, these synthetic media contain recombinant proteins, hormones, serum-derived components, and other ingredients, posing challenges for their use as food.

[0004] Various approaches have been attempted to develop cell culture media for the production of cultured meat, including media using organ cell products (Patent Document 1: Japanese Patent No. 6111510), media using algae products (Non-Patent Document 3: Scientific Reports. Jan 31;7:41594), media using hydrolyzed food waste (Non-Patent Document 4: Food Funct., 2020,11, 2477-2488), and media using other food raw material components (Patent Document 2: International Publication No. 2021 / 148955). [Prior art documents] [Patent documents]

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

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

[0007] The objective is to provide a medium capable of culturing large quantities of cells used in the production of cultured meat by adding food ingredient components as cell growth promoters. [Means for solving the problem]

[0008] The present inventors conducted extensive research into media that can be used to produce cultured meat, and discovered that adding whey to a culture medium as a cell proliferation promoter can achieve high proliferation activity of cells that are the raw material for cultured meat, leading to the present invention. [1] A culture medium for cell growth, comprising a basal medium and whey as a cell growth promoter. [2] The medium of item 1, wherein the expanded cells are cells used in cultivated meat production. [3] The medium according to Item 1 or 2, wherein the medium does not contain animal-derived serum. [4] The medium according to Item 3, wherein the animal-derived serum is fetal bovine serum (FBS). [5] The medium according to any one of items 1 to 4, wherein the cells comprise at least one cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells. [6] The medium described in Item 5, wherein the adipose tissue-derived cells are at least one cell selected from the group consisting of adipose stem cells, multilocular adipocytes, and unilocular adipocytes. [7] The medium according to Item 5, wherein the muscle tissue-derived cells are at least one cell selected from the group consisting of myoblasts and muscle satellite cells. [8] The medium according to any one of items 1 to 7, wherein the cells are derived from bovine. [9] The medium according to any one of items 1 to 8, further comprising a food ingredient.

[10] The medium according to Item 9, wherein the food ingredient is selected from egg white, soybean, fish meal, and wheat flour.

[11] A method for preparing cells for cultivated meat production, comprising: The method comprises the step of culturing cells in a medium containing a basal medium and whey as a cell growth promoter.

[12] The method according to Item 11, wherein the medium does not contain animal-derived serum.

[13] The method according to Item 12, wherein the animal-derived serum is fetal bovine serum (FBS).

[14] The method according to any one of items 11 to 13, wherein the medium further contains a food ingredient.

[15] The method according to Item 14, wherein the food ingredient is selected from egg white, soybean, fish meal, and wheat.

[16] The method according to any one of items 11 to 15, wherein the cells are derived from bovine cells.

[17] The method according to any one of items 11 to 16, wherein the cells comprise at least one cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells.

[18] The method according to Item 17, wherein the adipose tissue-derived cells are at least one cell selected from the group consisting of adipose stem cells, multilocular adipocytes, and unilocular adipocytes.

[19] The method according to Item 17, wherein the muscle tissue-derived cells are at least one cell selected from the group consisting of myoblasts and muscle satellite cells.

[20] The method according to Item 19, wherein the cells are myoblasts or muscle satellite cells, and further comprising a step of inducing differentiation of the myoblasts or muscle satellite cells into myotubes.

[21] A method for producing cultured meat, comprising a step of enriching cells prepared by the method according to any one of items 11 to 20.

[22] The production method according to Item 21, characterized in that the prepared cells are accumulated together with at least one substance selected from the group consisting of other cells, blood, tissue, and extracellular matrix.

[23] The method according to Item 22, wherein the other cells are cultured cells or cells obtained from an animal.

[24] The production method according to any one of items 21 to 23, further comprising culturing after enrichment.

[25] Cell growth promoters for cultured meat production, including whey.

[26] The cell growth promoting agent according to Item 25, which is added to an animal-derived serum-free medium.

[27] The cell proliferation promoter according to Item 25 or 26, which promotes proliferation of at least one cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells.

[28] The cell proliferation promoter described in Item 27, wherein the adipose tissue-derived cells are at least one cell selected from the group consisting of adipose stem cells, multilocular adipocytes, and unilocular adipocytes.

[29] The cell proliferation promoter described in Item 27, wherein the muscle tissue-derived cells are at least one cell selected from the group consisting of myoblasts and muscle satellite cells.

[30] The cell proliferation promoter according to any one of items 25 to 29, wherein the cells are bovine-derived cells.

[31] Mixing the basal medium with whey as a cell growth promoter to obtain a medium for cell growth; a step of heat sterilizing the medium A method for producing a medium, comprising:

[32] The method according to Item 31, wherein the heat sterilization step is carried out by boiling.

[33] The method according to Item 31, wherein the heat sterilization step is carried out by a hot plate exchanger or a steam cleaning device. [Effects of the Invention]

[0009] By adding whey to a basal medium, a cell culture medium can be provided that enhances the cell proliferation activity of cells. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the proliferation rates of primary myoblasts and fibroblasts derived from seven cows when cultured in serum-free medium and medium supplemented with 10% FBS. [Figure 2] Figure 2 shows the results of screening food ingredients that promote cell proliferation when bovine myoblasts are cultured in serum-free medium. [Figure 3] Figure 3 shows the results of screening food ingredients that enhance cell proliferation when bovine adipocytes are cultured in serum-free medium. [Figure 4] Figure 4 shows the results of screening food ingredients that promote cell proliferation when bovine fibroblasts are cultured in serum-free medium. (A) shows the results using Holstein-derived fibroblasts, (B) shows the results using fibroblasts derived from a first-generation crossbred cattle (F1), and (C) shows the results using fibroblasts derived from Japanese Black cattle. [Figure 5] FIG. 5 is a graph showing the concentration-dependent proliferation-promoting effect when whey is used as a cell proliferation promoter when bovine myoblasts are cultured in a serum-free medium. [Figure 6] FIG. 6 shows the results of screening for food ingredients that enhance cell proliferation when combined with whey when bovine myoblasts are cultured in serum-free medium. [Figure 7]Figure 7 shows the results of screening food ingredients that enhance cell proliferation when bovine kidney cells are cultured in serum-free medium. [Figure 8] Figure 8 shows photographs of myoblasts grown in 10% FBS-supplemented medium (10% FBS) and whey-supplemented medium (whey), which were subjected to differentiation induction treatment and fluorescently stained for myosin heavy chain (MyHC) and nuclei (DAPI). [Figure 9] Figure 9 shows the cell counts after myoblasts were cultured and grown in a 10% FBS-supplemented medium and a 0.1% whey-supplemented medium, which were prepared by heat-treating an FBS stock solution and a 1% whey solution, respectively, and adding them to a basal medium after heat treatment, as well as in a 10% FBS-supplemented medium and a 0.1% whey-supplemented medium that had not been heat-treated. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to a cell growth medium comprising a basal medium and whey as a cell growth promoter. In another aspect, the present invention also relates to a method for preparing cells for cultured meat production, comprising culturing cells in a cell growth medium comprising a basal medium and whey as a cell growth promoter, and to a method for producing cultured meat from the prepared cells. In yet another aspect, the present invention also relates to a cell growth promoter for cultured meat production, comprising whey. In yet another aspect, the present invention also relates to a method for producing a medium for cultured meat production, comprising mixing a basal medium with whey as a cell growth promoter to obtain a cell growth medium, and heat-sterilizing the medium.

[0012] [Cell growth medium] The cell growth medium of the present invention comprises a basal medium and whey as a cell growth promoter. Because whey is a food ingredient, cells cultured in the medium of the present invention are highly safe as a food. Furthermore, because whey is an inexpensive ingredient, the medium of the present invention also has the advantage of low preparation costs. By including whey, cell growth activity can be enhanced. Because cells cultured in such a medium are highly safe as a food, they can be used in cultured meat production. The medium of the present invention relates to a serum-free medium that contains whey but does not contain animal-derived serum.

[0013] Animal-derived serum refers to serum produced from animal blood. The supernatant obtained by clotting collected blood is called serum. Animal-derived serum can be serum from any animal, such as cow, horse, goat, donkey, rabbit, or chicken, but specifically refers to bovine serum (BCS) and fetal bovine serum (FBS). Serum contains proteins such as albumin and globulins, as well as serum lipids such as neutral fats, cholesterol, phospholipids, and free fatty acids, as well as hormones, cytokines, and growth factors. Fetal serum, in particular, is rich in components required for cell growth and is commonly added to culture media in research and pharmaceutical fields. Culture media that do not contain animal-derived serum are called serum-free media. On the other hand, serum-free media do not contain animal-derived serum, but may contain purified serum-derived components or recombinant serum-derived components. Animal-derived serum is susceptible to heat denaturation, and its activity decreases when sterilized by heat (Figure 9). Therefore, culture media containing animal-derived serum are typically sterilized using filter sterilization or UV sterilization rather than heat sterilization.

[0014] In the present invention, adding whey to a basal medium can achieve high cell proliferation activity even in a serum-free medium. Therefore, the whey of the present invention can be referred to as a cell proliferation promoter (sometimes referred to as a cell culture supplement). The cell proliferation promoter of the present invention can be used in cell culture for cultured meat production and can be added to an animal-derived serum-free medium. The cell proliferation promoting effect of whey is not reduced by boiling (Figure 9). Therefore, heat sterilization can be used when using a medium containing whey. Equipment such as a plate heat exchanger or a steam cleaner can be used for heat sterilization of the medium. Industrial culture, which requires large-scale culture, requires a simple sterilization method for the medium. If heat sterilization is possible, after medium preparation, the prepared medium can be directly heat sterilized using a steam cleaner or sterilized in the flow path using a plate heat exchanger, and then introduced directly into a culture vessel, simplifying the process from medium preparation to culture. Heat-denaturable components can be sterilized separately using filter sterilization or UV sterilization, and then added to the heat-sterilized medium.

[0015] Whey (also called whey or milk serum) is the aqueous solution remaining after removing solids from milk. It is produced in large quantities as a by-product in the production of dairy products such as cheese and yogurt, making it inexpensive. More specifically, whey is obtained by adding a coagulant such as rennet to milk or fermented milk, coagulating it, and then separating the solids from the curds. The solids removed include some or all of the milk fat and proteins such as casein. The main components of whey are lactoglobulin, lactalbumin, and lactoferrin, but it also contains a variety of trace components such as free amino acids, inorganic salts, and vitamins.

[0016] The whey used in the present invention may be whey derived from any mammal. For example, whey obtained from the milk of cows, horses, goats, sheep, humans, or donkeys can be used. Bovine whey is particularly useful due to its availability. Whey may be in liquid form or in the form of a dried powder. From the perspective of adding it as a cell proliferation promoter, a dried powder form is preferred because it is expected to reduce transportation costs. Commercially available dried powder whey may be used, or it may be prepared by freeze-drying whey. When using dried powder whey as a proliferation promoter, it is added to the basal medium at a concentration of 0.0025% to 1.0% by mass. From the perspective of exerting a proliferation effect, the whey concentration is preferably 0.025% by mass or more, more preferably 0.05% by mass or more. From the perspective of reaching a plateau in the proliferation-promoting effect, it is preferably 0.8% by mass or less, more preferably 0.5% by mass or less. When using liquid whey, the amount to be added can be determined by converting it to a dry powder (FIG. 5).

[0017] Basal media are culture media for cell culture that contain the minimum components necessary for cell maintenance and proliferation. Seeding cells in basal media allows cells to be maintained without dying and may also allow cells to proliferate. Various basal media are commercially available, and typically contain amino acids, vitamins, buffers, inorganic salts, and a carbon source. Amino acids include essential and non-essential amino acids. Vitamins include vitamin B1, vitamin C, nicotinic acid, folic acid, and the like. Buffers include HEPES and the like. Carbon sources may include monosaccharides such as glucose, disaccharides such as sucrose, oligosaccharides, and polysaccharides. Cell culture media can typically be prepared by adding additives such as serum to basal media. The basal medium may be any basal medium known in the art, including, for example, Dulbecco's modified Eagle's medium (DMEM), Basal Eagle's medium (BME), RPMI1640 medium, DMEM / F12 medium, F10 medium, F12 Ham's medium, MEM, M199 medium, Ames' medium, Iscove's modified medium, Glasgow's modified medium, and Fisher's medium.

[0018] During cell culture, a cell growth promoter is added to the basal medium. In conventional cell culture, serum such as fetal bovine serum (FBS) is added as a cell growth promoter (Figure 1). On the other hand, the cell growth medium of the present invention contains whey as a cell growth promoter. For example, the cell growth medium of the present invention does not contain animal-derived serum, but contains whey as a substitute. In the present invention, additives other than whey may be added to the medium. Such additives include components known in the art to be added to serum-free media. Examples of additives added to serum-free media in the art include lipids, hormones, growth factors, cytokines, serum-derived proteins, antibiotics, etc. Hormones include dexamethasone, etc. Growth factors include FGF, IGF, and insulin, and any family thereof may be used. Cytokines include IL-1α, IL-1β, etc., which may be added to a concentration of, for example, 0.1 to 1000 ng / ml. Serum-derived proteins include fetuin, fibronectin, albumin, and globulin, which may be added at a concentration of 0.0001 to 1%, for example. Antibiotics include penicillin and streptomycin, which may be added at concentrations of 10 to 500 U / ml for penicillin and 10 to 500 μg / ml for streptomycin, for example. ITS (insulin-transferrin-sodium selenite), an additive commonly used in serum-free and low-serum media, may also be added to the whey-containing serum-free medium of the present invention. For example, a 100-fold concentrated premix solution may be added at a concentration of 0.1 to 5%.

[0019] When whey is added to a basal medium as a cell proliferation promoter, additional food ingredients may be added. Any ingredient can be added as long as it exhibits an effect suitable for cell culture. An example of an effect suitable for cell culture is a differentiation-inhibiting effect or a proliferation-promoting effect. For example, ingredients that exhibit higher cell proliferation activity than whey alone are preferred, and ingredients derived from egg white, soybeans, wheat flour, fish meal, or, for example, bonito flakes can be added. These food-derived ingredients may be added as extracts, or as dry powders, with insoluble components removed by filtration. To achieve a high cell proliferation-promoting effect, combinations of whey and soybeans, whey and bonito flakes, and whey and egg white are preferred, with whey and egg white and whey and soybeans being particularly preferred (Figure 6). Adding these combinations to a basal medium as a cell proliferation promoter can exhibit a higher cell proliferation-promoting effect than 10% fetal bovine serum (FBS). The dry powders of egg white, soybeans, wheat flour, and bonito flakes are added to the basal medium at a concentration of 0.0025% to 1.0% by mass. From the viewpoint of exerting a proliferation effect, these food raw material components are preferably 0.005% by mass or more, more preferably 0.01% by mass or more. From the viewpoint of avoiding aggregation of components, the content is preferably 0.5% by mass or less, more preferably 0.1% by mass or less. The mass ratio of whey to other food raw material components can be appropriately selected within the range of 10:1 to 1:10. It is preferably 5:1 to 1:5, and more preferably 3:1 to 1:3.

[0020] [cell] The cell culture medium of the present invention can be used to culture any animal cells. From the perspective of producing cultured meat, cells derived from livestock such as cattle, pigs, goats, sheep, rabbits, chickens, ostriches, and ducks can be used. In particular, when using bovine cells, cells from any of the following breeds may be used: Holstein, Jersey, Japanese Black, Japanese Brown, Shorthorn, Japanese Polled, and their crossbreeds. However, from the perspective of meat production, cells from meat breeds such as Japanese Black, Japanese Brown, Shorthorn, and Japanese Polled are preferred. Any cell from these animals can be cultured (Figure 4). The cell culture medium of the present invention can also be used to culture tissues containing cell aggregates. Animal cells may be primary cells obtained from an animal, subcultured cells passaged from primary cells, or established cell lines. Primary cells can be obtained by mincing animal tissue in the medium. Cells may also be cells differentiated from stem cells such as somatic stem cells, embryonic stem cells, and induced pluripotent stem cells. From the perspective of producing cultured meat, it is preferable to culture at least one cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells (Figures 2 to 4).

[0021] Fibroblasts are cells that make up connective tissue and produce extracellular matrix components such as collagen and elastin. Fibroblasts present in muscle are specifically called myofibroblasts. Myofibroblasts form the connective tissue that surrounds bundles of muscle fibers in skeletal muscles. Myofibroblasts express α-SMA, produce extracellular matrix, and can accumulate fat, contributing to the texture and taste of food.

[0022] Adipose tissue-derived cells are cells that constitute adipose tissue and are isolated from adipose tissue and cultured. Adipose tissue-derived cells are at least one cell selected from the group consisting of adipose stem cells, multilocular adipocytes, and unilocular adipocytes. Adipose stem cells are mesenchymal stem cells that have the ability to differentiate into various cells and can differentiate into muscle cells, adipocytes, and connective tissue cells. Multilocular adipocytes, also known as brown adipocytes, contribute to fat burning in the body. Unilocular adipocytes, also known as white adipocytes, can store lipid droplets within the cells. Adipose tissue-derived cells contain fat, which contributes to the taste of meat.

[0023] Muscle tissue-derived cells are cells that make up muscle tissue and are isolated from muscle tissue and cultured. Examples of muscle tissue-derived cells include myoblasts, satellite cells, and myotubes. However, myotubes do not proliferate, so myoblasts and / or satellite cells are preferred from the perspective of proliferation. Satellite cells are somatic stem cells contained in muscle and can proliferate and differentiate into myoblasts. Myoblasts are mononuclear cells that are the origin of muscle fibers and have the ability to proliferate. When myoblasts differentiate, they fuse with each other to form multinucleated myotubes, which then mature into muscle fibers. Myofibers are composed of myofibrils, which are made up of actin and myosin filaments, which are proteins that make up muscle. They are classified into red muscle fibers (type I and type IIA) and white muscle fibers (type IIB) based on the myosin isoform, which contributes to the difference in the taste of meat.

[0024] [Cultured meat] Cultured meat refers to meat produced through cell culture. In the present invention, "for the production of cultured meat" refers to the method used to produce cultured meat, which is required to be food hygienically acceptable. From the standpoint of food hygiene, it is preferable to avoid the use of animal-derived serum, hormones, and genetically modified proteins. Generally, meat refers to an aggregate of muscle fibers, connective tissue, and fat. On the other hand, it is preferable for cultured meat to mimic the structure of meat, but it does not necessarily have to contain all of the components of meat, as long as it contains at least one cultured cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells. It is more preferable for it to contain a culture of multiple types of cells. Cultured meat may contain an extracellular matrix in addition to at least one cultured cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells. Methods for producing cultured meat include, for example, the following methods. A step of culturing at least one cultured cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells. A process of collecting and enriching the cultured cells The method for producing cultured meat may further include a differentiation induction step and a culture step after enrichment. The present invention also relates to cultured meat containing cells cultured in the cell proliferation medium of the present invention.

[0025] Cells are cultured by seeding them in the cell growth medium of the present invention, i.e., a medium containing a basal medium and whey as a cell growth promoter. Culture is performed under conditions well known in the art, for example, in a CO2 incubator at 37°C. Culture may be plate culture or suspension culture. Proliferated cells can be recovered as a culture by trypsin treatment or other methods, and may be further subcultured after recovery. Cell culture may also be performed by seeding cells onto a detachable construct. The construct to which the proliferated cells have attached can be recovered as a culture. Such constructs can be constructed with extracellular matrices such as collagen, elastin, fibronectin, laminin, and entactin, and cell-attached constructs may be accumulated to form cultivated meat.

[0026] The enrichment step involves shaping the culture of one or more types of cells recovered. The culture formed in the enrichment step may be a single piece of meat, such as a steak, a carcass, or minced meat. The enrichment step involves enriching the cell culture with at least one substance selected from the group consisting of other cells, blood, and tissues. The other cells may be cultured cells or cells collected from animals. More specifically, the cell culture may be shaped together with other cells cultured in the cell growth medium of the present invention. For example, muscle tissue-derived cells cultured in the cell growth medium of the present invention may be enriched with adipose tissue-derived cells and / or fibroblasts cultured in the cell growth medium of the present invention. Co-culture may also be performed after enrichment. For example, the culture of one or more types of cells recovered may be mixed and seeded on an extracellular matrix for co-culture. Examples of the extracellular matrix include collagen, elastin, fibronectin, laminin, and entactin. The cell growth medium of the present invention may also be used as the medium for this process.

[0027] In the accumulation step, the culture of one or more types of cells that have been collected may be accumulated with blood and / or tissue. The tissue may be obtained from an animal or may be cultured. For example, blood, adipose tissue, muscle tissue, etc. separated during meat processing may be accumulated with the culture to produce cultured meat.

[0028] The differentiation induction step may be performed after cell culture, or may be performed before, during, or after the enrichment step. The differentiation induction step allows mononuclear muscle satellite cells and myoblasts to differentiate into multinuclear myotubes, which can then mature into muscle fibers. Differentiation induction may be performed by methods known in the art, including, for example, culturing under a high carbon dioxide concentration. For example, culturing under a 5-10% (v / v) CO2 atmosphere can promote differentiation into myotubes.

[0029] [Culture medium manufacturing method] The cell growth medium of the present invention is prepared by a manufacturing method comprising the following steps: Mixing the basal medium with whey as a cell growth promoter to obtain a medium for cell growth; A step of heat sterilizing the medium. The production method of the present invention may further include a step of filter-sterilizing components susceptible to thermal denaturation and adding the resulting mixture to the medium. The production of cultured meat requires the mass cultivation of cells, necessitating the preparation of a large amount of medium. Large-scale medium must be sterilized before cell seeding, and heat sterilization, which is simple and allows for large-scale processing, is preferred. Cell growth medium prepared by mixing a basal medium with whey is resistant to denaturation by heat treatment and can therefore be subjected to heat treatment. Heat treatment can be selected as desired as long as the activity of the whey added to the medium is not lost; for example, boiling can be used. The heating temperature is selected appropriately to kill the target bacteria, and can be between 60°C and 180°C, for example. To achieve sufficient sterilization, 75°C or higher is preferred, and 100°C or higher is more preferred. To prevent denaturation of the medium, 150°C or lower is preferred, and 130°C or lower is more preferred. The heat sterilization time can be selected appropriately to achieve sufficient sterilization. For example, heat treatment is performed for 0.5 seconds to 60 minutes. Heat sterilization can be performed during the process of introducing the medium prepared in the medium preparation tank into the culture tank through the flow path. For example, a plate heat exchanger can be used to perform heat sterilization in the flow path.

[0030] All documents mentioned in this specification are incorporated herein by reference in their entirety. The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, can be made without departing from the spirit of the present invention. [Example]

[0031] Test 1: Cell collection (1) Collection of myoblasts Bovine myoblasts were harvested from the longissimus muscle using the following procedure. After washing the tissue with ethanol and phosphate buffered saline (PBS), it was minced with scissors in a clean bench. The muscle tissue was digested by shaking at 37°C for 1.5 hours in Dulbecco's modified Eagle's medium supplemented with 0.2% collagenase II (Worthington). The digestion reaction was stopped by adding 20% ​​FBS to the reaction solution. The digestion solution was centrifuged at 80 × g for 3 minutes, the floating tissue was removed with tweezers, and the supernatant was collected. The supernatant was then passed through a nylon mesh (100 μm) for cell separation. The filtrate was centrifuged at 1500 × g for 5 minutes, and the resulting pellet was suspended in Dulbecco's modified Eagle's medium containing 20% ​​FBS. The cell suspension was passed through a 100 μm nylon mesh, then again through a 40 μm nylon mesh, and the filtrate was centrifuged at 1500 × g for 5 minutes. The pellet was then incubated on ice for 5 minutes with red blood cell lysis solution (pluriSelect Life Science) to remove blood cells. After washing twice with phosphate buffer, the cells were pooled in Dulbecco's modified Eagle's medium containing 10% FBS and seeded onto culture dishes. The expanded cells were used for testing.

[0032] (2) Collection of adipocytes Bovine adipocytes were harvested from adipose tissue near the intestine using the following procedure. After washing with ethanol and PBS, the tissue was minced with scissors in a clean bench. The adipose tissue was digested by shaking in Dulbecco's modified Eagle's medium supplemented with 0.2% collagenase I (GIBCO) for 1 hour. 20% FBS was added to the digested reaction solution, and the mixture was centrifuged at 180 × g for 10 minutes. The floating tissue was removed with tweezers, and the supernatant was collected. The supernatant was passed through a nylon mesh (100 μm) for cell separation and centrifuged at 420 × g for 5 minutes. The precipitate was incubated on ice with red blood cell lysis solution for 5 minutes to remove blood cells. After washing twice with phosphate buffer, the cells were pooled in Dulbecco's modified Eagle's medium containing 10% FBS and seeded onto culture dishes. The expanded cells were used for the experiments.

[0033] (3) Collection of fibroblasts Fibroblasts were harvested from bovine skin tissue using the following process. After washing the tissue with ethanol and PBS, the dermis layer was removed and isolated in a clean bench. The isolated tissue was finely minced with scissors and placed in a culture dish containing Dulbecco's modified Eagle's medium containing 10% FBS, and cultured in a CO2 incubator at 37°C for several days. The migrated cells were collected and used in the experiments.

[0034] Test 2: Comparison of proliferation potential between serum-free and serum-containing media (i) Culture medium The serum-free medium used was Dulbecco's modified Eagle's medium supplemented with 1% penicillin-streptomycin solution, 1% ITS liquid medium supplement, 2 ng / ml human basic fibroblast growth factor, and lipid additive for cell culture (Sigma, L0288). The serum-containing medium used was Dulbecco's modified Eagle's medium supplemented with a penicillin-streptomycin solution and 10% FBS.

[0035] (ii) Growth test In this study, proliferation tests were performed on primary bovine myoblasts and fibroblasts derived from seven different individuals in serum-free and serum-containing media. 3 cells / cm 3 Cells were seeded and cultured in a CO2 incubator set at 37°C and 5% CO2. After 3 days of culture, the number of viable cells obtained by trypsinization was counted, and the ratio of the number of cells after proliferation to the number of initially seeded cells was calculated. All data for primary bovine myoblasts and fibroblasts derived from seven different individuals were averaged (Figure 1). Cells cultured in serum-free medium had low cell proliferation activity, while cells cultured in FBS-containing medium had high cell proliferation activity. Cultures cultured in FBS-containing medium had approximately four times the cell number of cultures in serum-free medium.

[0036] Test 3: Search for food ingredients that enhance cell growth in serum-free medium (1) Exploratory tests using bovine myoblasts (i) Culture medium Test medium was prepared by adding food ingredients as additives to the serum-free medium prepared in Test 2. The food ingredients used were egg white, soybeans, whey, wheat flour, and dried bonito flakes (all dried powders). Each food ingredient was dissolved in the serum-free medium at 0.1% (only dried bonito flakes were dissolved at 0.02%), and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components, which was then used in the test. Medium without any additives and medium supplemented with 10% FBS were used as controls.

[0037] (ii) Screening Test Using myoblasts derived from Holstein cows, we searched for components that promote proliferation in serum-free medium. 3 cells / cm 3 Cells were seeded and cultured in a CO2 incubator set at 37°C and 5% CO2. After 3 days of culture, the number of viable cells obtained by trypsinization was counted, and the ratio of the number of cells in the food ingredient-supplemented medium to the number of cells in serum-free medium was calculated (Figure 2). When cultured in a medium supplemented with whey, the cell number was higher than in a culture in a serum-free medium, although it was lower than in a culture in a 10% FBS-supplemented medium. Furthermore, when cultured in a medium supplemented with egg white, the cell number was higher than in a culture in a serum-free medium, although it was lower than in a culture in a 10% FBS-supplemented medium.

[0038] (2) Exploratory tests using bovine adipocytes (i) Culture medium Test medium was prepared by adding food ingredients to the serum-free medium prepared in Test 2. The food ingredients used were egg white, soybean, whey, and wheat flour (all in dry powder form). Each food ingredient was dissolved in the serum-free medium at 0.1% concentration, and after centrifugation, the supernatant was filtered through a 0.45 μm filter to remove insoluble components, and this was used in the test. Additive-free (serum-free) medium and medium supplemented with 10% FBS were used as controls.

[0039] (ii) Screening Test Using Japanese Black cattle-derived adipocytes, we searched for components that promote proliferation in serum-free medium. 4 cells / cm3 Cells were seeded and cultured in a CO2 incubator set at 37°C and 5% CO2. After 4 days of culture, the number of viable cells obtained by trypsinization was counted, and the ratio of the number of cells in the food component-supplemented medium to the number of cells in the serum-free medium was calculated (Figure 3). When cultured in the whey-supplemented medium, the cell number was higher than in the serum-free medium, although it was lower than in the culture in the 10% FBS-supplemented medium. On the other hand, other food components did not affect cell proliferation activity.

[0040] (3) Exploratory tests using bovine fibroblasts (i) Culture medium Test medium was prepared by adding food ingredients as additives to the serum-free medium prepared in Test 2. The food ingredients used were egg white, soybeans, whey, wheat flour, and dried bonito flakes (all dried powders). Each food ingredient was dissolved in serum-free medium at 0.1% (only dried bonito flakes were dissolved at 0.02%), and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components, which was then used in the test. As controls, medium without additives (serum-free) and medium supplemented with 10% FBS were used.

[0041] (ii) Screening Test We searched for components that promote proliferation in serum-free medium using fibroblasts derived from Holstein, Japanese Black, and F1 (first generation crossbred cattle). 3 cells / cm 3Cells were seeded and cultured in a CO2 incubator set at 37°C and 5% CO2. After 3 days of culture, viable cells were counted by trypsinization, and the ratio of cell numbers in the food ingredient-supplemented medium to those in serum-free medium was calculated (Figure 4A: Holstein, B: F1, C: Japanese Black). The cell proliferation activity of food ingredients varied depending on the type of cattle, but the trends were consistent. In Holstein and Japanese Black cattle, cell numbers were higher in whey-supplemented medium than in serum-free medium, although lower than in cultures supplemented with 10% FBS. In contrast, in F1 cattle, cell numbers were comparable to those in 10% FBS-supplemented medium. Furthermore, cell numbers were higher in egg white-supplemented medium than in serum-free medium, although lower than in cultures supplemented with 10% FBS.

[0042] Test 4: Examination of whey concentration added to serum-free medium (i) Culture medium The serum-free medium used was Dulbecco's modified Eagle's medium supplemented with penicillin-streptomycin solution, ITS liquid medium supplement, 2 ng / ml human basic fibroblast growth factor, 0.1% lipid additive for cell culture, and BSA. The food ingredient used was whey (dry powder). Various food ingredients were dissolved in serum-free medium at various concentrations (1.0%, 0.5%, 0.25%, 0.1%, 0.05%, 0.025%, 0.01%, 0.005%, and 0%). After centrifugation, the supernatant was filtered through a 0.45 μm filter to remove insoluble components and used for the test.

[0043] (ii) Testing Using Holstein-derived myoblasts, we investigated the whey concentration that promotes proliferation in serum-free medium. 3 cells / cm 3The cells were seeded and cultured in a CO2 incubator set at 37°C and a CO2 concentration of 5%. After three days of culture, the number of viable cells obtained by trypsinization was counted, and the ratio of the number of cells in the food ingredient-supplemented medium to the number of cells in the serum-free medium was calculated (Figure 5). As a cell proliferation promoter, whey began to exert a cell proliferation-promoting effect from 0.005% by mass, and the cell proliferation-promoting effect plateaued at 0.1% by mass.

[0044] Test 5: Examination of the combined effects of whey and food ingredients added to serum-free medium (bovine myoblasts) (i) Culture medium Test medium was prepared by adding food ingredients as additives to the serum-free medium used in Test 4. The food ingredients used were egg white, soybeans, wheat flour, and dried bonito flakes (all dried powders). Each food ingredient was dissolved in serum-free medium at 0.1% (only dried bonito flakes at 0.02%), and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components. This was then used for the test. As negative controls, medium without added food ingredients (serum-free) and medium supplemented with 10% FBS were used. The following tests were performed on the above medium with and without the addition of 0.1% whey. Note that whey was added before filtering. (ii) Testing Using Holstein-derived myoblasts, we searched for components that promote proliferation in serum-free medium. 3 cells / cm 3 The cells were seeded and cultured in a CO2 incubator set at 37°C and 5% CO2. After 3 days of culture, the number of viable cells obtained by trypsinization was counted, and the ratio of the number of cells in the food ingredient-supplemented medium to the number of cells in the serum-free medium was calculated (Figure 6). Whey also exhibited a proliferation-promoting effect when combined with other food ingredients. The proliferation-promoting effect was additive, and when combined with soybeans and egg white, which also have proliferation-promoting effects, it exhibited a proliferation-promoting effect comparable to that of 10% FBS.

[0045] Comparative Example 1: Search for food materials that enhance cell proliferation in serum-free medium (bovine kidney cell line) (i) Culture medium Test medium was prepared by adding food ingredients as additives to the serum-free medium prepared in Test 2. The food ingredients used were egg white, soybean, whey, and wheat flour (all in dry powder form). Each food ingredient was dissolved in serum-free medium at 0.1% concentration, and after centrifugation, the supernatant was filtered through a 0.45 μm filter to remove insoluble components, and this was used in the test. As controls, medium without additives (serum-free) and medium supplemented with 10% FBS were used. (ii) Screening Test Using a bovine kidney cell line (MDBK) obtained from ATCC, we searched for components that promote proliferation in serum-free medium. 4 cells / cm 3 The cells were seeded and cultured in a CO2 incubator set at 37°C and a CO2 concentration of 5%. After 4 days of culture, the number of viable cells obtained by trypsinization was counted, and the ratio of the number of cells in the food ingredient-supplemented medium to the number of cells in the serum-free medium was calculated (Figure 7). The food ingredient did not have a proliferation-promoting effect on bovine kidney cells.

[0046] Test 6: Differentiation of myoblasts cultured in whey medium Using Holstein-derived myoblasts, we confirmed that cells grown in a food-supplemented medium could be induced to differentiate into myotubes. The food-supplemented medium consisted of 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, with 0.1% whey powder dissolved in the medium. After centrifugation, the supernatant was filtered through a 0.45 μm filter to remove insoluble components and used for the test. The serum-containing control medium consisted of Dulbecco's modified Eagle's medium supplemented with penicillin-streptomycin solution and 10% FBS. The differentiation-inducing medium consisted of Dulbecco's modified Eagle's medium supplemented with penicillin-streptomycin solution and 2% horse serum. Approximately 0.75 × 10 4 cells / cm 3The cells were seeded and cultured for 4 days in a CO2 incubator set at 37°C and 5% CO2, then the medium was changed to differentiation-inducing medium and cultured for an additional 6 days. Differentiation into myotubes was confirmed by immunostaining for myosin heavy chain.

[0047] Immunostaining was performed according to the following steps. 1. Cells were washed once with phosphate buffered saline (PBS) and then fixed by incubating overnight at 4°C with 4% paraformaldehyde. 2. After washing three times with PBS, the cells were treated with 1% Triton X-100 / PBS at room temperature for 5 minutes for equivalent treatment. 3. After washing three times with PBS, blocking was performed for 30 minutes at room temperature using a commercially available blocking solution for immunostaining (KAC). 4. The primary antibody reaction was carried out by treating the sections in a solution containing 1 μg / mL of anti-myosin heavy chain monoclonal antibody (Clone MF20) at room temperature for 1 hour. 5. After washing three times with PBS, the sections were treated with a 500-fold diluted solution of Alexa 488-labeled goat anti-mouse IgG (Abcam, ab150117) at room temperature for 30 minutes to carry out the secondary antibody reaction. After washing with PBS, the nuclei were stained with DAPI and observed using a Keyence all-in-one fluorescence microscope (Figure 8). After proliferation in both whey-supplemented medium and 10% FBS-supplemented medium, differentiation into multinucleated myotubes expressing myosin heavy chains was confirmed.

[0048] Test 7: Whey heat tolerance test A medium was prepared using heat-treated whey and serum, and the effect of heat treatment on cell proliferation was evaluated. A 1% whey solution prepared by dissolving in water and inactivated FBS were used. 10 ml of whey solution and FBS were dispensed into 50 ml tubes, submerged in a boiling pot for 5 minutes, cooled, and the liquid on the tube walls was spun down to prepare the heated component. Unheated components, which were not heat-treated, were used as a control. The food ingredient-supplemented medium used was Dulbecco's modified Eagle's medium supplemented with penicillin-streptomycin solution, ITS liquid medium supplement, 5ng / ml human basic fibroblast growth factor, and lipid additives for cell culture, to which 1 / 10 of the whey solution was added. After centrifugation, the supernatant was filtered through a 0.45μm filter to remove insoluble components, and the resultant was used for the test. The serum-containing medium used was Dulbecco's modified Eagle's medium to which penicillin-streptomycin solution was added, with 1 / 10 the amount of FBS added. 5 x 10 myoblasts 3 cells / cm 3 The cells were seeded onto culture dishes containing various media and cultured in a CO2 incubator set at 37°C and 5% CO2. After 3 days of culture, the number of viable cells obtained by trypsinization was counted (Figure 9). While the cell proliferation promoting effect of whey was not affected even when heated, the cell proliferation promoting effect of FBS was reduced by heating.

Claims

1. A cell growth medium for growing cells used in cultured meat production, comprising a basal medium and 0.025% by mass to 1.0% by mass of whey in dry powder equivalent as a cell growth promoter, The medium, wherein the cells comprise at least one cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells.

2. The medium of claim 1 , wherein the medium is free of animal-derived serum.

3. The medium according to claim 2 , wherein the animal-derived serum is fetal bovine serum (FBS).

4. The medium according to any one of claims 1 to 3, wherein the adipose tissue-derived cells are at least one cell selected from the group consisting of adipose stem cells, multilocular adipocytes, and unilocular adipocytes.

5. The medium according to any one of claims 1 to 3, wherein the muscle tissue-derived cells are at least one cell selected from the group consisting of myoblasts and muscle satellite cells.

6. The medium according to any one of claims 1 to 5, wherein the cells are derived from a bovine.

7. The medium according to any one of claims 1 to 6, further comprising a food ingredient.

8. 8. The medium according to claim 7, wherein the food ingredient is selected from egg white, soybean, fish meal, and wheat flour.

9. 1. A method of preparing cells for cultivated meat production, comprising: The method includes culturing cells in a medium containing a basal medium and 0.025% by mass to 1.0% by mass of whey in terms of dry powder as a cell growth promoter, The method, wherein the cells comprise at least one cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells.

10. The method of claim 9 , wherein the medium is free of animal-derived serum.

11. The method of claim 10, wherein the animal-derived serum is fetal bovine serum (FBS).

12. The method according to any one of claims 9 to 11, wherein the medium further comprises a food ingredient component.

13. 13. The method of claim 12, wherein the food ingredient is selected from egg white, soybean, fish meal, and wheat.

14. The method according to any one of claims 9 to 13, wherein the cells are derived from a bovine.

15. The method according to any one of claims 9 to 14, wherein the adipose tissue-derived cells are at least one cell selected from the group consisting of adipose stem cells, multilocular adipocytes, and unilocular adipocytes.

16. The method according to any one of claims 9 to 14, wherein the muscle tissue-derived cells are at least one cell selected from the group consisting of myoblasts and muscle satellite cells.

17. The method according to claim 16, wherein the cells are myoblasts or muscle satellite cells, and further comprises a step of inducing differentiation of the myoblasts or muscle satellite cells into myotubes.

18. A method for producing cultured meat, comprising a step of enriching cells prepared by the method of any one of claims 9 to 17.

19. The method of claim 18, wherein the prepared cells are accumulated together with at least one substance selected from the group consisting of other cells, blood, tissue, and extracellular matrix.

20. The method of claim 19 , wherein the other cells are cultured cells or cells obtained from an animal.

21. The method according to any one of claims 18 to 20, further comprising culturing after enrichment.

22. A method for producing a medium for growing cells for producing cultured meat, comprising the steps of: mixing a basal medium with 0.025% to 1.0% by mass of whey, calculated as a dry powder, as a cell growth promoter to obtain a medium for cell growth; a step of heat sterilizing the medium wherein the cells comprise at least one cell selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells.

23. 23. The method of claim 22, wherein the heat sterilization step is performed by boiling.

24. 23. The method of claim 22, wherein the heat sterilizing step is performed by a hot plate exchanger or a steam cleaning device.

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