Medium for cell growth for cultured meat production

Abstract

The purpose of the present invention is to provide a culture medium in which cells to be used for producing a cultured meat can be grown, by adding thereto a food material as an additive. As a result of culturing cells using a food ingredient as an additive, it became possible to obtain high growth potential in fibroblasts and myoblasts by adding egg white or a dried product of egg white as a cell growth promoter. Accordingly, the present invention pertaining to a cell-growing culture medium containing a basic culture medium and egg white as a cell growth promoter was accomplished.

Description

Technical Field

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

Background Art

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

[0003] As a substitute for meat, plant-derived alternative meat is known, but its texture and taste do not match those of real meat. On the other hand, cultured meat produced by culturing animal cells can achieve a texture and taste similar to those of real meat, and has the advantage of a lower risk of bacterial and viral contamination compared to real meat. Technologically, the production of cultured meat has become possible. However, the cell culture media used in previous cultured meat production utilize large-scale culture techniques used in basic research and pharmaceutical applications, and it has been difficult to use them for food production in terms of cost and safety as food. In cell culture media used in basic research and pharmaceutical applications, it is common to add fetal bovine serum (FBS) as an additive to a basal medium containing carbon sources such as amino acids, vitamins, inorganic salts, and glucose. On the other hand, since FBS is serum collected from fetuses, it is difficult to obtain in large quantities and has problems in terms of price, transportation cost, infection risk, and animal welfare. (Non-Patent Document 1: ALTEX. 2018;35(1):99-118.) To solve such problems, a completely synthetic medium supplemented with essential components of FBS as reagents has been developed (Non-Patent Document 2: The Canadian Journal of Chem Engineering 2016, Vol.94, (10) 1855-1862). However, such completely synthetic media use recombinant proteins, steroid hormone agents, serum-derived components, etc., and there are problems in using them as food.

[0004] Various approaches have been attempted for cell culture media for the production of cultured meat. Examples include media using products of organ cells (Patent Document 1: Japanese Patent No. 6111510), media using algal products (Non-Patent Document 3: Scientific Reports. 2017;7:41594), media using hydrolysates of food residues (Non-Patent Document 4: Food Funct., 2020,11, 2477-2488), and media using other food raw material components (Patent Document 2: International Publication No. 2021 / 148955, Non-Patent Document 5: Jpn J Exp Med. 1985 Apr;55(2):45-51, Non-Patent Document 6; Prog Immunobiol Stand. 1971;5:202-8, Non-Patent Document 7: BMC Biotechnology. 2023 Feb 8;23(1):4.).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

[0007] An object of the present invention is to provide a medium capable of culturing a large amount of cells used for the production of cultured meat by adding food raw material components as cell growth promoters. [Means for Solving the Problems]

[0008] As a result of intensive studies by the present inventors on a medium that can be used for the production of cultured meat, it was found that by adding egg white or dried egg white as a cell growth promoter to the medium, high proliferation activity of cells that are raw materials for cultured meat can be achieved, leading to the present invention. Therefore, the present invention relates to the following: [1] A medium for cell proliferation, comprising a basal medium and egg white or dried egg white as a cell growth promoter. [1-2] Use of egg white or dried egg white for the production of a medium for cell proliferation. [1-3] Use of egg white or dried egg white for the production of a cell growth promoter. [2] The medium according to item 1, or the use according to item 1-2, wherein the proliferated cells are cells used for the production of cultured meat. [3] The medium according to item 1 or 2, or the use according to item 1-2, 1-3 or 2, wherein the medium does not contain animal-derived serum. [4] The medium or use 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, or the use according to item 1-2, 1-3, or 2 to 4, wherein the cells include fibroblasts or cells derived from muscle tissue. [6] The medium according to any one of items 1 to 5, or the use according to items 1-2, 1-3, or 2 to 5, wherein the cells are cells derived from cows, pigs, or chickens. [7] A method for preparing cells for the production of cultured meat, comprising: culturing the cells in a medium containing a basal medium and egg white or dried egg white as a cell growth promoter. [8] The method according to item 7, wherein the medium does not contain animal-derived serum. [9] The method according to item 8, wherein the animal-derived serum is fetal bovine serum (FBS).

[10] The method according to any one of items 7 to 9, wherein the cells are cells derived from cows, pigs, or chickens.

[11] The method according to item 10, wherein the cells contain at least one cell selected from the group consisting of fibroblasts and cells derived from muscle tissue.

[12] A method for producing cultured meat, comprising the step of aggregating cells prepared by the method according to any one of items 7 to 11.

[13] The production method according to item 12, wherein the prepared cells are aggregated together with at least one substance selected from the group consisting of other cells, blood, tissue, and extracellular matrix.

[14] The production method according to item 13, wherein the other cells are cultured cells or cells obtained from an animal.

[15] The production method according to any one of items 12 to 14, further comprising culturing after aggregation.

[16] A cell growth promoter for the production of cultured meat, containing egg white or dried egg white.

[17] The cell growth promoter according to item 16, which is added to a medium not containing animal-derived serum.

[18] The cell growth promoter according to item 16 or 17, which promotes the growth of at least one cell selected from the group consisting of fibroblasts and cells derived from muscle tissue.

[19] The cell growth promoter according to item 18, wherein the cells are cells derived from cows, pigs, or chickens.

Advantages of the Invention

[0009] By adding egg white or dried egg white to a basal medium, a cell culture medium that enhances the cell proliferation activity of cells can be provided.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

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Figure 5

Modes for Carrying Out the Invention

[0011] The present invention relates to a medium for cell proliferation, comprising a basal medium and egg white or dried egg white as a cell growth promoter. In another aspect, the present invention also relates to a method for preparing cells for culturing meat, comprising the step of culturing cells in a medium for cell proliferation, comprising a basal medium and egg white or dried egg white as a cell growth promoter, and also relates to a method for producing cultured meat from the prepared cells. In yet another aspect, the present invention relates to a cell growth promoter for culturing meat production, comprising egg white or dried egg white.

[0012] [Medium for Cell Proliferation] The culture medium for cell growth according to the present invention contains a basal medium and egg white or dried egg white as a cell growth promoter. Since egg white or dried egg white is a food raw material, the cells cultured in the medium of the present invention have high safety as food. In addition, since egg white is an inexpensive raw material, the medium of the present invention also has the advantage of low preparation cost. By containing egg white or dried egg white, the cell growth activity can be enhanced. Egg white or dried egg white can enhance the cell growth activity by being formulated at a relatively low concentration, for example, 0.001 to 0.5% by mass, preferably 0.003 to 0.2% by mass. By being formulated at a low concentration, aggregation is suppressed and medium preparation becomes easy. Since the cells cultured in such a medium have high safety as food, they can be used for culturing meat production. The medium of the present invention relates to a serum-free medium that contains egg white or dried egg white while not containing animal-derived serum.

[0013] Animal-derived serum refers to serum produced from the blood of animals. The supernatant obtained by coagulating the obtained blood is called serum. As animal-derived serum, it may be serum derived from any animal, for example, animals such as cows, horses, goats, donkeys, rabbits, and birds, but particularly refers to bovine serum (BCS) and fetal bovine serum (FBS). Serum contains serum lipids such as neutral fat, cholesterol, phospholipids, and free fatty acids, in addition to proteins such as albumin and globulin, and further contains hormones, cytokines, growth factors, etc. In particular, fetal serum contains abundant components necessary for cell growth and is generally added to the medium in the research and pharmaceutical fields. A medium that does not contain animal-derived serum is called a serum-free medium. On the other hand, a serum-free medium does not contain animal-derived serum, but may contain purified components derived from serum or may contain recombinant proteins of serum-derived components.

[0014] In the present invention, by adding egg white or dried egg white to a basal medium, high cell growth activity can be achieved even in a serum-free medium. Therefore, the egg white of the present invention can be referred to as a cell growth promoter (sometimes also referred to as a cell culture supplement). The cell growth promoter according to the present invention can be used in cell culture for culturing meat production and can be added to a medium free of animal-derived serum.

[0015] Egg white is a substance obtained by separating the shell and yolk in the eggs of birds. The bird can be of any kind, but from the viewpoint of enabling mass acquisition, domestic fowls such as chickens, quails, turkeys, ducks, geese, ostriches, etc. can be mentioned. Egg white may be an isolate from eggs or a dried product of the isolate. The dried product is usually provided as a dry powder. Dried egg white is preferred because reduction of transportation costs can be expected. The dried product may be prepared using any drying method, but more preferably by freeze-drying. Commercially available dried egg white may be used, or it may be prepared by drying egg white. After egg white or dried egg white is dissolved in the medium, insoluble matter can be removed by filtering using a filter with an appropriate pore size. As an example, it can be filtered using a 0.3 - 0.5 μM, particularly 0.45 μM filter. When using dried egg white as a growth promoter, it is added at an appropriately selected concentration so that the growth rate increases according to the type of cells used. As an example, it is added to the basal medium so as to be 0.001 mass% - 0.5 mass%. From the viewpoint of exerting a growth effect, the concentration of egg white is preferably 0.002 mass% or more, more preferably 0.003 mass% or more. From the viewpoint of the growth promotion effect reaching a plateau or suppressing reduction, it is preferably 0.2 mass% or less, more preferably 0.1 mass% or less. When using an isolate, the addition amount can be determined by converting it to the dried product.

[0016] Egg white is mainly composed of various proteins, such as ovalbumin, ovotransferrin, ovomucoid, ovomucin, globulin, lysozyme, avidin, etc. It may also contain trace components such as free amino acids, inorganic salts, and vitamins. Egg white or dried egg white can be characterized by having an average molecular weight of 5000 - 800000 Da. The average molecular weight varies depending on the treatment of egg white, but from the perspective of ensuring that hydrolysis treatment, especially enzymatic hydrolysis treatment and chemical hydrolysis treatment, are not carried out, it is particularly preferred to have an average molecular weight of 10000 Da or more, 15000 Da or more, or 18000 Da or more.

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

[0018] When culturing cells, 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. On the other hand, the medium for cell growth of the present invention contains egg white or dried egg white as a cell growth promoter. Therefore, the medium for cell growth of the present invention does not contain animal-derived serum, but contains egg white or dried egg white as a substitute therefor, and egg white or dried egg white can also be referred to as a serum substitute. In the present invention, other additives different from egg white or dried egg white may be added to the medium. Examples of such additives include known components added to serum-free media in the art. Examples of additives added to serum-free media in the art include lipids, hormonal agents, growth factors, cytokines, serum-derived proteins, antibiotics, etc. Examples of hormonal agents include dexamethasone. Examples of growth factors include FGF, IGF, and insulin, and any family thereof may be used. Examples of cytokines include IL-1α, IL-1β, etc., and as an example, they can be added at a concentration of 0.1 to 1000 ng / mL. Examples of serum-derived proteins include fetuin, fibronectin, albumin, globulin, etc., and as an example, they can be added at a concentration of 0.0001 to 1%. Examples of antibiotics include penicillin, streptomycin, etc., and as an example, they can be added at a concentration of penicillin 10 to 500 U / mL and streptomycin 10 to 500 μg / mL. ITS (insulin-transferrin-sodium selenite), an additive commonly used in serum-free and low-serum media, can also be added to the serum-free medium containing egg white or dried egg white of the present invention. As an example of the addition amount, a 100-fold concentrated premix solution can be added at a concentration of 0.1 to 5%.

[0019] When egg white or dried egg white is added to the basal medium as a cell growth promoter, additional food raw material components may be further added. Any components can be added as long as they exhibit effects suitable for cell culture. The effects suitable for cell culture refer to, for example, a differentiation promoting effect, a suppressing effect, or a growth promoting effect. As an example, components that have a higher cell growth activity than when egg white or dried egg white is added alone are preferred, and components derived from soybeans, wheat, fish meal, microorganisms, such as those derived from Chlorella, can be added. These food-derived components may be added as extracts, or dried powders may be added and insoluble components removed by filtration. These food raw materials are added to the basal medium so as to be 0.0025% by mass to 1.0% by mass, and from the viewpoint of exerting a growth effect, 0.005% by mass or more is preferred, and 0.01% by mass or more is more preferred. From the viewpoint of avoiding aggregation of components, 0.5% by mass or less is preferred, and 0.1% by mass or less is more preferred. The mass ratio of egg white or dried egg white to other food raw material components can be appropriately selected within the range of 10:1 to 1:10. Preferably it is 5:1 to 1:5, and more preferably 3:1 to 1:3.

[0020] [Cell] The cell culture medium according to the present invention can culture any animal cells. From the viewpoint of manufacturing cultured meat, cells derived from livestock such as cows, pigs, goats, sheep, rabbits, chickens, ostriches, and ducks can be used. In particular, it is preferable to use cells of cows, pigs, and chickens. The cell culture medium according to the present invention can also culture tissues in which cells are aggregated. Animal cells may be primary cells obtained from an animal and subcultured cells subcultured from the primary cells, or may be immortalized cells. Primary cells can be obtained by mincing animal tissues in a medium. They may also be cells differentiated from stem cells such as somatic stem cells, embryonic stem cells, and induced pluripotent stem cells. From the viewpoint of manufacturing cultured meat, it is preferable to culture at least one cell selected from the group consisting of fibroblasts, adipocytes, and cells derived from muscle tissue.

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

[0022] Muscle tissue-derived cells are cells that make up muscle tissue and are cells separated from muscle tissue and cultured. Examples of muscle tissue-derived cells include myoblasts, satellite cells, and myotube cells. However, since myotube cells do not have proliferative ability, from the perspective of proliferation, myoblasts and / or satellite cells are preferred. Satellite cells are somatic stem cells contained in muscle, can proliferate, and can differentiate into myoblasts. Myoblasts are cells from which muscle fibers are derived and are mononuclear cells with proliferative ability. When myoblasts differentiate, they fuse with each other to form multinuclear myotube cells, which further mature into muscle fibers. Muscle fibers are composed of myofibrils, which are the structural units composed of actin fibers and myosin fibers, which are proteins that make up muscle. Depending on the isoform of myosin, they are classified into red muscle fibers (type I, IIA) and white muscle fibers (IIB), which contribute to the difference in taste of meat.

[0023] Adipocytes refer to cells that have fat droplets in their cytoplasm, are roughly classified into white adipocytes and brown adipocytes, and also include adipose stem cells, which are their progenitor cells. Adipocytes biosynthesize from glucose to fatty acids and mature and hypertrophy by taking up fatty acids through the fatty acid transport pathway. In the production of cultured meat, the taste can be improved by accumulating adipocytes together with fibroblasts and muscle tissue-derived cells.

[0024] When using bovine cells, especially for the purpose of culturing fibroblasts and myoblasts, egg white or dried egg white can be used. When culturing fibroblasts, it has a relative growth rate exceeding 50% compared to the case of using 10% FBS, is increased compared to the non-added negative control, and has a higher relative growth rate than when using soybeans, kamaboko, and wheat (Figure 1A). Also, when culturing myoblasts, it has a relative growth rate of 34% compared to the case of using 10% FBS, is increased compared to the non-added negative control, and has a higher relative growth rate than when using soybeans, kamaboko, and wheat (Figure 1B). The use of an enzymatically treated egg white preparation instead of FBS for culturing bovine myoblasts has been disclosed (Non-Patent Document 4). In the present invention, it can be said to be a simple method in that bovine fibroblasts and myoblasts can be proliferated even when egg white is used as it is or only dried. When culturing bovine fibroblasts and myoblasts, ITS can be further added in addition to egg white or dried egg white.

[0025] When using chicken cells, especially for the purpose of culturing fibroblasts and myoblasts, egg white or dried egg white can be used. When culturing fibroblasts, it has a relative growth rate of at least 80% compared to the case of using 10% FBS, and depending on the usage concentration, it has a relative growth rate equal to or higher than that with the addition of 10% FBS. By using egg white or dried egg white, it is significantly increased compared to the non-added negative control, and it has been shown that the relative growth rate is higher than that when using wheat, rice, corn, and wheat protein (Figure 2A). In addition, when culturing chicken fibroblasts using soybeans or chlorella, since it has a relative growth rate equivalent to that when adding 10% FBS, these components can also be used as cell growth promoters. Also, when culturing myoblasts using egg white or dried egg white, it has a relative growth rate exceeding 70% compared to the case of using 10% FBS. This relative growth rate is increased compared to the relative growth rate of the non-added negative control, and it has been shown that it is higher than the relative growth rates using wheat, rice bran, chickpeas, skipjack, fish gelatin, shrimp, whey, porcine gelatin, and the non-added negative control (Figure 2B). When culturing chicken skin cells, it is disclosed to use egg white-yolk fraction instead of FBS (Non-Patent Document 5: Jpn J Exp Med. (1985)). In the present invention, for the purpose of manufacturing cultured meat, it is characterized by growing fibroblasts and / or myoblasts. In the culture of chicken myoblasts, usually, FBS has poor substitutability, while egg white or dried egg white has a sufficient growth-promoting effect as a substitute for FBS.

[0026] When using porcine cells, especially for the purpose of culturing fibroblasts, egg white or dried egg white can be used. When culturing fibroblasts, it has a relative growth rate of at least 75% compared to the case of using 10% FBS. By using egg white or dried egg white, it significantly increases compared to the non-added negative control (Figure 3).

[0027] [Cultured meat] Cultured meat refers to meat produced through cell culture. In the present invention, "for the production of cultured meat" refers to a method used for the production of cultured meat, which is required to be acceptable from the perspective of food hygiene. From the perspective of food hygiene, it is preferably to avoid the use of animal-derived serum, hormonal agents, and recombinant proteins. Generally, meat refers to an aggregate of muscle fibers, connective tissue, and fat. On the other hand, although it is preferable for cultured meat to mimic the structure of meat, it does not necessarily have to contain all the components of meat, and it may include a culture of fibroblasts or muscle tissue-derived cells or adipocytes. It is more preferable to include a culture of multiple types of cells. Cultured meat may contain an extracellular matrix in addition to cultured cells of fibroblasts or muscle tissue-derived cells. The method for producing cultured meat includes, for example, the following: A step of culturing at least one cultured cell selected from the group consisting of fibroblasts or muscle tissue-derived cells A step of recovering and accumulating the cultured cells is included. The method for producing cultured meat may further include a differentiation induction step or a culture step after accumulation. The present invention also relates to cultured meat containing cells cultured in the cell growth medium according to the present invention.

[0028] The culture of cells is carried out by seeding the cells in the cell growth medium according to the present invention, that is, a basal medium and a medium containing egg white or dried egg white as a cell growth promoter. The culture is carried out under conditions well known in the art, for example, in a 37 °C CO 2 incubator. The culture may be a monolayer culture or a suspension culture. The proliferated cells can be recovered as a culture by trypsin treatment or the like, and subculture may be further carried out after recovery. The culture of cells can also be carried out by seeding the cells on a detachable construct and culturing. The construct to which the proliferated cells are attached can be recovered as a culture. Such a construct can be constructed with an extracellular matrix such as collagen, elastin, fibronectin, laminin, and entactin, and the constructs to which the cells are attached can be accumulated to form cultured meat.

[0029] The integration process includes molding a culture of one or more types of recovered cells. The culture molded in the integration process may be a single piece of meat such as a steak, or it may be minced meat, or it may be ground meat. The integration process includes integrating a culture of cells with at least one substance selected from the group consisting of other cells, blood, and tissue. The other cells may be cultured cells or cells collected from an animal. More specifically, it can be molded in combination with other cells cultured in the cell growth medium according to the present invention. As an example, muscle tissue-derived cells cultured in the cell growth medium according to the present invention can be integrated with adipose tissue-derived cells and / or fibroblasts cultured in the cell growth medium according to the present invention. Co-culture can also be performed after integration. As an example, a culture of one or more types of recovered cells can be mixed, seeded on the extracellular matrix, and co-cultured. As the extracellular matrix, collagen, elastin, fibronectin, laminin, entactin, etc. can be used. Also for the medium at this time, the cell growth medium of the present invention can be used.

[0030] The integration process may integrate a culture of one or more types of recovered cells with blood and / or tissue. The tissue may be obtained from an animal or may be cultured. As an example, blood, adipose tissue, muscle tissue, etc. separated during the processing of meat can be integrated with the culture to produce cultured meat.

[0031] The differentiation induction process may be performed after cell culture, or may be performed before, during, or after the integration process. By the differentiation induction process, mononuclear satellite cells and myoblasts can be differentiated into multinucleated myotube cells, further matured as muscle fibers, or lipid droplets can be accumulated in the cytoplasm. Differentiation induction may be performed by methods known in the art. As an example, a method of culturing under a high carbon dioxide concentration is known. As an example, culturing in an atmosphere of 5-10% (v / v) CO 2 By culturing in the atmosphere, differentiation into myotube cells can be promoted.

[0032] [Method for manufacturing medium] The culture medium for cell growth according to the present invention is prepared by a production method including the following steps: A step of mixing a basal medium and egg white or dried egg white as a cell growth promoter to obtain a culture medium for cell growth; A step of sterilizing the culture medium. The production method according to the present invention may include a step of performing heat sterilization or filter sterilization on components liable to heat denaturation and adding them to the culture medium.

[0033] All documents referred to in this specification are hereby incorporated by reference in their entirety. The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the claims. Modifications of the present invention, for example, addition, deletion, and substitution of the constituent elements of the present invention, can be made on the condition that the gist of the present invention is not deviated from.

Example

[0034] Example 1: Preparation of cells (1) Collection of bovine myoblasts Bovine myoblasts were collected through the following steps. The collected muscle tissue was washed with ethanol and phosphate-buffered saline (PBS), and then minced finely using scissors in a clean bench. The minced tissue was digested by shaking culture at 37 °C for 1.5 h in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.2% collagenase II (Worthington). The reaction was stopped by adding 20% fetal bovine serum (FBS) to the digested reaction solution. The digestion solution was centrifuged at 80×g for 3 min to remove floating tissue with forceps, and then the supernatant was collected. The supernatant obtained by centrifuging again at 80×g for 3 min was passed through a nylon mesh (100 μm) for cell separation. The filtrate was centrifuged at 1500 g for 5 min, and the obtained precipitate was suspended in DMEM 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 min. The precipitate was left standing on ice in erythrocyte lysate for 5 min to remove blood cells. After washing twice with phosphate-buffered saline, the cells were pooled in DMEM medium containing 20% FBS and 4 ng / mL human basic fibroblast growth factor (bFGF), seeded in a culture dish, and the proliferated cells were used for the test.

[0035] (2) Collection of bovine and porcine fibroblasts Fibroblasts were collected from the skin tissues of domestic cattle and domestic pigs through the following steps. After washing the tissues with ethanol and PBS, the dermal layer was peeled off and isolated in a clean bench. The isolated tissue was minced finely with scissors and placed in a culture dish containing DMEM medium with 10% FBS, and cultured in a CO 2 incubator at 37 °C for several days. The migrated cells were collected and used for the experiment.

[0036] (3) Collection of chicken myoblasts Fibroblasts were collected from 18-day-old chicken fertilized eggs by the following procedure. The breast tissue was minced with scissors and placed in DMEM medium containing 2 mg / mL collagenase II (Sigma), and subjected to an enzymatic reaction at 37°C for 1 hour. After suspending the tissue with a 10 mL syringe equipped with an 18G needle, the suspension was passed through a 100 μm filter. The precipitate after centrifuging the filtered suspension at 1500 rpm for 5 minutes was suspended in DMEM medium containing 20% FBS and 4 ng / mL bFGF. The cell suspension was seeded in a tissue culture dish. The non-adherent cells after overnight culture were seeded in a collagen-coated culture dish, and those subcultured twice were used as myoblasts.

[0037] Example 2: Search for food materials that enhance cell proliferation in serum-free medium (1) Bovine cells Using bovine myoblasts and fibroblasts derived from livestock, components that promote proliferation in serum-free medium were explored. As the serum-free medium, DMEM medium supplemented with 1% penicillin-streptomycin (PS) solution, 1% ITS liquid medium supplement (insulin-transferrin-sodium selenite), 2 ng / mL bFGF, and a lipid additive for cell culture (Sigma) was used. As food ingredient components, egg white, soybean, wheat flour, and dried bonito flakes (all in dry powder form) were used. Each type of food component was dissolved in the serum-free medium at 0.1% (only 0.02% for dried bonito flakes), and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components and used in the test. Myoblasts were seeded at about 5×10 3 cells / cm 3 onto a collagen-coated culture dish and cultured for 3 days. The one with 10% FBS added to DMEM medium was used as a positive control. Fibroblasts were seeded at 1×10 4 cells / cm 3 onto a tissue culture dish and cultured for 3 to 4 days. The one with 10% FBS added to DMEM medium was used as a positive control. CO 2 was set at a concentration of 5% in a CO 2After culturing in an incubator, the number of viable cells obtained by trypsin treatment was counted, and the ratio (relative growth rate (%)) of the number of cells at the time of using each culture solution to that at the time of using the serum-containing medium was calculated. The results are shown in Fig. 1. When culturing bovine fibroblasts (A), the negative control without addition was 30%, while when each food component was added, it increased to 59% (egg white), 48% (soybean), 40% (dried bonito flakes), and 37% (wheat flour). In particular, when using egg white, a high cell growth rate could be achieved. When culturing bovine myoblasts (B), the negative control without addition was 20%, while when each food component was added, it was 34% (egg white), 24% (soybean), and 19% (wheat). In particular, when using egg white, a high cell growth rate could be achieved.

[0038] (2) Chicken fibroblasts Using the chicken fibroblast cell line (DF-1) obtained from ATCC, components that promote the growth of serum-free medium were explored. The serum-free medium used was DMEM medium supplemented with 4 μM insulin, 1% penicillin-streptomycin (PS) solution, 0.2% BSA, and 1% lipid additive for cell culture. As the serum-containing medium, the one obtained by adding 10% FBS to DMEM medium was used as a positive control. As food raw material components, wheat flour, rice flour, corn flour, soybeans, egg white, and chlorella (all were dry powders) were used. Each food component was dissolved at 0.5% in the serum-free medium, and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components, and then diluted to 0.1%, 0.02%, and 0.01% in the serum-free medium for use in the test. Approximately 6×10 4 cells / cm 3 of cells were seeded, and at 37 °C and CO 2 concentration set at 5% CO 2They were cultured in an incubator. Three days after culturing, the cells were stained with 0.5% crystal violet / 20% methanol. The stained cells were eluted with 100% methanol, and the absorbance at 590 nm was measured using a plate reader. The absorbance of each culture solution was calculated relative to the absorbance when using the serum-containing medium. Various food components were added at 0.1% to 0.01% to the culture of chicken fibroblasts, and the effects on cell growth were evaluated. The results are shown in Fig. 2(A). When added at 0.1%, cell growth comparable to that in the presence of serum was confirmed in soybeans, chlorella, and egg white. However, when added at 0.01%, cell growth activity was confirmed only in egg white. Even when compared with soybeans and chlorella, for which serum replacement has been reported in BMC Biotechnol (2023) Feb 8;23(1):4 (Non-Patent Document 7) and Prog Immunobiol Stand (1971) 5:202-8 (Non-Patent Document 6), it was suggested that the growth-promoting effect of egg white might be high. Also, in Food Funct., (2020) 11, 2477-2488 (Non-Patent Document 4), a slight growth-promoting effect was reported for egg white hydrolysate, but it was found that the amount of egg white added could be reduced compared with that report. Moreover, it was found that growth could be achieved equivalently to that in the serum-containing medium without using growth factors.

[0039] (3) Chicken myoblasts Using chicken myoblasts, a search was conducted for components that promote the growth of serum-free medium. The serum-free medium used was DMEM medium supplemented with 4 μM insulin, 1% penicillin-streptomycin (PS) solution, 0.2% BSA, 1% lipid additive for cell culture, and 5 ng / mL bFGF. As the serum-containing medium, a positive control was used, which was DMEM medium supplemented with 10% FBS and 5 ng / mL bFGF. As food raw material components, wheat flour, rice bran, chickpeas, dried bonito, fish gelatin, shrimp powder, egg white, and porcine gelatin (all in dry powder form) were used. Various food components were dissolved in the serum-free medium at 0.1% (0.025% for egg white only), and the supernatant after centrifugation was filtered through a 0.45 μm filter and used in the test. As the serum-containing medium, a positive control was used, which was DMEM medium supplemented with 10% FBS and 5 ng / mL bFGF. The myoblasts were approximately 2×104 cells / cm 3 Cells were seeded onto collagen-coated plates, and after culturing for 3 days, the number of viable cells obtained by trypsin treatment was counted, and the ratio of the number of cells at the time of using each culture medium to the number of cells at the time of using the serum-containing medium was calculated. The results are shown in Fig. 2(B). Even in chicken myoblasts, the growth-promoting effect was highest when egg white was added, and a 73% growth compared to that in the presence of serum was confirmed.

[0040] Example 3: Comparison with serum-containing medium (porcine fibroblasts) As the serum-free medium, DMEM medium supplemented with 4 μM insulin, 1% penicillin-streptomycin (PS) solution, 0.2% BSA, and 1% lipid additive for cell culture was used. As the serum-containing medium, DMEM medium supplemented with 10% FBS was used as a positive control. Egg white as a food raw material component was dissolved in the serum-free medium at 0.025%, and the supernatant after centrifugation was filtered through a 0.45-μm filter to remove insoluble components, and about 6×10 4 cells / cm 3 cells were seeded, and cultured at 37 °C in a CO 2 incubator with a set CO 2 concentration of 5% for 3 days, and then Cell counting kit solution (Dojindo) was added. Color development reaction was carried out in the CO 2 incubator for 2 hours, and the absorbance at 450 nm was measured. The ratio of the absorbance at the time of using each culture medium to the absorbance at the time of using the serum-containing medium was calculated. The results are shown in Fig. 3. When culturing porcine fibroblasts, no cell growth was observed in the non-added negative control (DMEM), while a high cell growth rate was achieved when egg white was used as a food component.

[0041] Example 4: Verification of effective concentration Using chicken fibroblast cell line (DF-1) and porcine fibroblasts, we searched for components that promote the growth of serum-free medium. As the serum-free medium, DMEM medium supplemented with 4 μM insulin, 1% penicillin-streptomycin (PS) solution, 0.2% BSA, and 1% lipid additive for cell culture was used. As the serum-containing medium, DMEM medium supplemented with 10% FBS was used as a positive control. As a food raw material component, egg white was dissolved in the serum-free medium at 0.2%, and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components, and then diluted to 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.0016% in the serum-free medium. Approximately 6×10 4 cells / cm 3 of cells were seeded, and cultured in an incubator at 37°C with a CO 2 concentration set at 5%. 2 After culturing for 3 days in the incubator, Cell counting kit solution (Dojindo) was added. A color reaction was carried out in the CO 2 incubator for 2 hours, and the absorbance at 450 nm was measured. The ratio of the absorbance of each culture solution to the absorbance when using the serum-containing medium was calculated. For porcine and chicken fibroblasts, a growth activity of 70% or more of that in the presence of serum was confirmed at 0.003% or more. It was found that the amount of egg white added could be reduced even compared with the report of Food Funct., (2020) 11, 2477-2488 (Non-Patent Document 4) in porcine and chicken cells.

[0042] Example 5: Analysis of the molecular weight of egg white powder lysate The molecular weight distribution of the egg white solution was measured by gel filtration chromatography. A Superdex 30 Increase 10 / 300 GL column (cytiva) was installed in a column oven set at 37 °C in a high-performance liquid chromatography (HPLC: Shimadzu Corporation), and the flow path was equilibrated by flowing a phosphate buffer (pH = 7.4) containing 280 mM NaCl (hereinafter referred to as the eluent) at a flow rate of 0.5 mL / min. Subsequently, 50 μL each of the sample solution and the standard solution were injected, the absorption wavelength at 214 nm was detected for each elution time, and the molecular weight distribution was confirmed from the obtained peaks. The average molecular weight was calculated from a calibration curve created from the retention time with respect to the molecular weight of the standard product using GPC analysis software (Shimadzu Corporation). The sample solution used was prepared by dissolving 0.2% egg white powder in water, passing it through a 0.45 μm filter, and then diluting it 10-fold with the eluent. As the standard solution, a solution containing 0.5 mg / mL each of ovalbumin, ovomucoid, cytochrome c, apurinine, and (Pro-Pro-Gly) 5 was used. Since the peak of the egg white solution was detected at a retention time shorter than that of the cytochrome c peak (27.7 minutes) in the standard solution, it was found that the main component of the egg white solution was larger than cytochrome c (105 aa). As calculated by the analysis software, the average molecular weight was approximately 37,000 Da (equivalent to 336 aa). This was a larger molecular weight compared to the report (15 aa or less) in Food Funct., (2020) 11, 2477-2488 (Non-Patent Document 4).

Claims

1. A cell growth medium for growing cells for cultured meat production, comprising a basal medium and egg white or dried egg white as a cell growth promoter, The cells for producing cultured meat are fibroblasts or myoblasts derived from livestock, The medium is free of animal-derived serum.

2. The medium of claim 1 , wherein the animal-derived serum is fetal bovine serum (FBS).

3. The medium according to claim 1 or 2, wherein the cells are derived from a cow, a pig, or a chicken.

4. 1. A method for preparing cells for cultivated meat production, comprising: The method includes culturing cells in a medium containing a basal medium and egg white or a dried egg white as a cell growth promoter, the medium being free of animal-derived serum; The method, wherein the cells are fibroblasts or myoblasts derived from livestock.

5. The method of claim 4, wherein the animal-derived serum is fetal bovine serum (FBS).

6. The method of claim 4, wherein the cells are derived from a cow, a pig, or a chicken.

7. A method for producing cultured meat, comprising a step of enriching cells prepared by the method according to any one of claims 4 to 6.

8. The method for producing a cell according to claim 7, 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.

9. The method according to claim 8 , wherein the other cells are cultured cells or cells obtained from an animal.

10. The method according to claim 7, further comprising culturing after enrichment.

11. A cell proliferation promoter for cultured meat production, comprising egg white or dried egg white, The cells for producing cultured meat are fibroblasts or myoblasts derived from livestock, The growth promoting agent is for addition to an animal-derived serum-free medium.

12. The proliferation-promoting agent according to claim 11 , wherein the cells are derived from a cow, a pig, or a chicken.

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