Cell-growing culture medium for producing cultured meat

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

Cell growth media for cultured meat production

[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.

[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 traditional meat. On the other hand, cultured meat, which is produced by culturing animal cells, can achieve a texture and flavor similar to that of traditional meat and has the advantage of being less susceptible to bacterial and viral contamination than traditional 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). However, because FBS is serum collected from fetuses, it is difficult to obtain in large quantities and poses challenges in terms of price, transportation costs, risk of infectious diseases, and animal welfare. (Non-Patent Document 1: ALTEX. 2018;35(1):99-118.) To solve these problems, fully synthetic media supplemented with essential FBS components as reagents have been developed (Non-Patent Document 2: The Canadian Journal of Chem Engineering 2016, Vol.94, (10) 1855-1862.) However, these fully synthetic media contain recombinant proteins, steroid hormones, serum-derived components, etc., which poses 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. 2017; 7: 41594), media using hydrolyzed food waste (Non-Patent Document 4: Food Funct., 2020, 11, 2477-2488), and media using other food ingredients (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.).

[0005] Patent No. 6111510 International Publication No. 2021 / 148955

[0006] ALTEX. 2018;35(1):99-118.The Canadian Journal of Chem Engineering 2016, Vol.94, (10) 1855-1862Scientific Reports.2017;7:41594Food Funct. 2020,11, 2477-2488Jpn J Exp Med. 1985 Apr;55(2):45-51Prog Immunobiol Stand. 1971;5:202-8BMC Biotechnology . 2023 Feb 8;23(1):4.

[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.

[0008] The present inventors conducted extensive research into media that can be used in the production of cultured meat, and discovered that adding egg white or dried egg white to the medium as a cell growth promoter can achieve high proliferation activity of cells that are the raw material for cultured meat, leading to the present invention. Therefore, the present invention relates to the following: [1] A cell growth medium 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 cell growth medium. [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 grown cells are cells used in 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 items 1-2, 1-3, or 2 to 4, wherein the cells comprise fibroblasts or muscle tissue-derived cells. [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 derived from bovine, porcine, or chicken. [7] A method for preparing cells for cultured meat production, comprising a step of culturing cells in a medium comprising 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 derived from bovine, porcine, or chicken.

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

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

[13] The production method according to item 12, characterized in that the prepared cells are enriched 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, comprising further culturing after enrichment.

[16] A cell proliferation promoter for cultured meat production, comprising egg white or dried egg white.

[17] The cell proliferation promoter according to Item 16, which is added to an animal-derived serum-free medium.

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

[19] The cell proliferation promoter according to Item 18, wherein the cells are derived from bovine, porcine, or chicken.

[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.

[0010] Figure 1 shows the results of screening for food ingredient components that enhance cell proliferation when bovine fibroblasts (A) and bovine myoblasts (B) are cultured in serum-free medium. Figure 2 shows the results of screening for food ingredient components that enhance cell proliferation when chicken fibroblasts (A) and chicken myoblasts (B) are cultured in serum-free medium. Figure 3 shows the cell proliferation rate relative to the cell number in FBS-supplemented medium when porcine fibroblasts are cultured in a serum-free medium containing dried egg white. Figure 4 is a graph showing the concentration-dependent proliferation-promoting effect of egg white as a cell proliferation promoter when chicken fibroblasts (A) and porcine fibroblasts (B) are cultured in serum-free medium. Figure 5 shows the results of molecular weight analysis of freeze-dried egg white samples.

[0011] The present invention relates to a cell growth medium 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 cultured meat production, comprising culturing cells in a cell growth medium comprising a basal medium and egg white or dried egg white 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 egg white or dried egg white.

[0012] [Cell Growth Medium] The cell growth medium of the present invention comprises a basal medium and egg white or dried egg white as a cell growth promoter. Because egg white or dried egg white is a food ingredient, cells cultured in the medium of the present invention are highly safe as a food. Furthermore, because egg white is an inexpensive ingredient, the medium of the present invention also has the advantage of low preparation costs. By including egg white or dried egg white, cell growth activity can be enhanced. Cell growth activity can be enhanced by incorporating egg white or dried egg white at a relatively low concentration, for example, 0.001 to 0.5% by mass, preferably 0.003 to 0.2% by mass. Incorporation at a low concentration inhibits aggregation and facilitates medium preparation. 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 egg white or dried egg white but does not contain animal-derived serum.

[0013] Animal-derived serum refers to serum produced from animal blood. The supernatant obtained by clotting the collected blood is called serum. Animal-derived serum may be serum from any animal, such as cow, horse, goat, donkey, rabbit, or bird, but particularly refers to bovine serum (BCS) and fetal bovine serum (FBS). Serum contains proteins such as albumin and globulin, 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 proteins of serum-derived components.

[0014] In the present invention, by adding egg white or dried egg white to a basal medium, high cell proliferation activity can be achieved even in a serum-free medium. Therefore, the egg white of the present invention can be said to be 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.

[0015] Egg white is a substance obtained by separating the shell and yolk from avian eggs. Any type of bird may be used, but from the perspective of mass availability, poultry, such as chicken, quail, turkey, duck, goose, and ostrich, are preferred. Egg white may be isolated from eggs or may be a dried product of the isolate. Dried products are typically provided as dry powders. Dried egg white is preferred because it is expected to reduce transportation costs. Dried products may be prepared using any drying method, but are more preferably prepared by freeze-drying. Commercially available dried egg whites may be used, or they may be prepared by drying egg whites. Egg white or dried egg whites can be dissolved in a medium and then filtered through a filter with an appropriate pore size to remove insoluble matter. For example, filtration can be performed using a 0.3-0.5 μM filter, particularly a 0.45 μM filter. When dried egg white is used as a growth promoter, it is added at a concentration appropriately selected to increase the growth rate depending on the type of cells used. For example, it is added to the basal medium so as to be 0.001% to 0.5% by mass. From the viewpoint of exerting a proliferation effect, the concentration of egg white is preferably 0.002% by mass or more, and more preferably 0.003% by mass or more. From the viewpoint of the proliferation-promoting effect reaching a plateau or suppressing a decrease, the concentration is preferably 0.2% by mass or less, and more preferably 0.1% by mass or less. When an isolated product is used, the amount to be added can be determined in terms of dry matter.

[0016] Egg white is primarily composed of various proteins, including ovalbumin, ovotransferrin, ovomucoid, ovomucin, globulin, lysozyme, and avidin. 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 5,000 to 800,000 Da. While the average molecular weight varies depending on the treatment of the egg white, from the viewpoint of ensuring that it has not been subjected to hydrolysis, particularly enzymatic hydrolysis and chemical hydrolysis, an average molecular weight of 10,000 Da or more, 15,000 Da or more, or 18,000 Da or more is particularly preferred.

[0017] A basal medium is a medium for cell culture that contains the minimum components necessary for cell maintenance and proliferation. Seeding cells in the basal medium 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 that may be added include monosaccharides such as glucose, disaccharides such as sucrose, oligosaccharides, and polysaccharides. Typically, a cell culture medium can be prepared by adding additives such as serum to the basal medium. The basal medium may be any basal medium known in the art, and examples thereof include 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. On the other hand, the cell growth medium of the present invention contains egg white or dried egg white as a cell growth promoter. Therefore, the cell growth medium of the present invention does not contain animal-derived serum but contains egg white or dried egg white as a substitute, and egg white or dried egg white can also be referred to as a serum substitute. In the present invention, additives other than egg white or dried egg white may be added to the medium. Such additives include components known in the art to be added to serum-free media. Additives added in the art to serum-free media include, for example, 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 and IL-1β, which may be added at a concentration of 0.1 to 1000 ng / mL, for example. Serum-derived proteins include fetuin, fibronectin, albumin, and globulin, which may be added at a concentration of 0.0001 to 1%. 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 serum-free medium containing egg white or dried egg white 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 egg white or dried egg white is added to a basal medium as a cell proliferation promoter, additional food ingredient components may be added. Any ingredient can be added as long as it exerts an effect suitable for cell culture. An effect suitable for cell culture refers to, for example, a differentiation-promoting effect, an inhibitory effect, or a proliferation-promoting effect. For example, an ingredient that exhibits higher cell proliferation activity than when egg white or dried egg white is added alone is preferred, and ingredients derived from soybeans, wheat, fish meal, or microorganisms, such as chlorella, may be added. These food-derived ingredients may be added as extracts, or as dry powders, after which insoluble components are removed by filtration. These food ingredients are added to a basal medium at a concentration of 0.0025% to 1.0% by mass. From the viewpoint of exerting a proliferation effect, 0.005% by mass or more is preferred, and 0.01% by mass or more is even more preferred. From the viewpoint of avoiding aggregation of ingredients, 0.5% by mass or less is preferred, and 0.1% by mass or less is even more preferred. The mass ratio of the 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 5:1 to 1:5, and more preferably 3:1 to 1:3.

[0020] [Cells] 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 cows, pigs, goats, sheep, rabbits, chickens, ostriches, and ducks can be used. It is particularly preferable to use cells from cows, pigs, and chickens. 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 animals, subcultured cells passaged from primary cells, or established cell lines. Primary cells can be obtained by mincing animal tissue in the 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 perspective of producing cultured meat, it is preferable to culture at least one cell selected from the group consisting of fibroblasts, adipocytes, and muscle tissue-derived cells.

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

[0022] 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 source 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.

[0023] Adipocytes are cells that contain lipid droplets in their cytoplasm and are broadly divided into white adipocytes and brown adipocytes, as well as their precursor cells, adipose stem cells. Adipocytes mature and enlarge by biosynthesizing fatty acids from glucose and absorbing fatty acids via the fatty acid transport pathway. In the production of cultured meat, accumulating adipocytes along with fibroblasts and muscle tissue-derived cells can improve the taste.

[0024] When using bovine cells, egg white or dried egg white can be used, particularly for the purpose of culturing fibroblasts and myoblasts. When fibroblasts were cultured, the relative proliferation rate exceeded 50% compared to when 10% FBS was used, which was an increase compared to the unsalted negative control, and was shown to be higher than when soybeans, bonito flakes, and wheat were used ( Figure 1A ). When myoblasts were cultured, the relative proliferation rate increased by 34% compared to when 10% FBS was used, which was an increase compared to the unsalted negative control, and was shown to be higher than when soybeans, bonito flakes, and wheat were used ( Figure 1B ). The use of an enzyme-treated egg white preparation instead of FBS for culturing bovine myoblasts has been disclosed ( Non-Patent Document 4 ). The present invention can be considered a simple method in that bovine fibroblasts and myoblasts can be grown using egg white as is or after only drying treatment. When culturing bovine fibroblasts and myoblasts, ITS may be added in addition to egg white or dried egg white.

[0025] When using chicken cells, egg white or dried egg white can be used, particularly for the cultivation of fibroblasts and myoblasts. When fibroblasts were cultured, the relative proliferation rate was at least 80% higher than when 10% FBS was used, and depending on the concentration used, the relative proliferation rate was equivalent to or higher than when 10% FBS was added. The use of egg white or dried egg white significantly increased proliferation compared to the untreated negative control, and the relative proliferation rate was also higher than when wheat, rice, corn, or wheat protein was used (Figure 2A). Furthermore, when chicken fibroblasts were cultured using soybeans or chlorella, the relative proliferation rate was equivalent to when 10% FBS was added, suggesting that these ingredients can also be used as cell proliferation promoters. Furthermore, when myoblasts were cultured using egg white or dried egg white, the relative proliferation rate exceeded 70% compared to when 10% FBS was used. This relative proliferation rate was increased compared to that of the unsupplemented negative control, and was shown to be higher than the relative proliferation rates using wheat, rice bran, chickpea, bonito, fish gelatin, shrimp, whey, porcine gelatin, and the unsupplemented negative control (Figure 2B). The use of egg white and egg yolk fractions instead of FBS in the cultivation of chicken skin cells has been disclosed (Non-Patent Document 5: Jpn J Exp Med. (1985)). The present invention, which aims to produce cultured meat, is characterized by the proliferation of fibroblasts and / or myoblasts. While FBS is generally a poor substitute for chicken myoblasts in the cultivation of chicken myoblasts, egg white or dried egg white has a sufficient proliferation-promoting effect as a substitute for FBS.

[0026] When using porcine cells, egg white or dried egg white can be used, particularly for the purpose of culturing fibroblasts. When fibroblasts are cultured, the relative proliferation rate is at least 75% compared to when 10% FBS is used. The use of egg white or dried egg white significantly increases proliferation compared to the 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 the method used to produce cultured meat, and it is required to be food hygienically acceptable. From a food hygiene perspective, 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. While cultured meat preferably mimics the structure of meat, it does not necessarily have to contain all of the components of meat; it is sufficient as long as it contains a culture of fibroblasts, muscle tissue-derived cells, or adipocytes. It is more preferable for cultured meat to contain cultures of multiple types of cells. In addition to cultured fibroblasts or muscle tissue-derived cells, cultured meat may also contain an extracellular matrix. A method for producing cultured meat includes, for example, the following steps: Culturing at least one cultured cell selected from the group consisting of fibroblasts or muscle tissue-derived cells; and Recovering and concentrating the cultured cells. A method for producing cultured meat may further include a differentiation induction step or a culturing step after concentration. The present invention also relates to cultured meat comprising cells cultured in the cell growth medium of the present invention.

[0028] The cells are cultured by seeding them in the cell growth medium of the present invention, i.e., a medium containing a basal medium and 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, at 37°C and CO ２ The cells are cultured in an incubator^. Culture may be plate culture or suspension culture. The proliferated cells can be harvested as a culture by trypsinization or other methods, and may be further subcultured after harvest. Cells can also be cultured by seeding them onto a detachable construct. The construct to which the proliferated cells have attached can be harvested as a culture. Such constructs can be constructed with extracellular matrices such as collagen, elastin, fibronectin, laminin, and entactin, and the constructs to which the cells have attached may be accumulated to form cultivated meat.

[0029] 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 purpose.

[0030] 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.

[0031] The differentiation induction step may be performed after cell culture, or may be performed before, during, or after the accumulation step. The differentiation induction step allows mononuclear muscle satellite cells and myoblasts to differentiate into multinuclear myotubes, which can then be matured into muscle fibers or allow lipid droplets to accumulate in the cytoplasm. Differentiation induction may be performed by methods known in the art, and one example is a technique of culturing cells under a high carbon dioxide concentration, such as 5 to 10% (v / v) CO ２ By culturing the cells under this atmosphere, differentiation into myotubes can be promoted.

[0032] [Method for producing medium] The medium for cell growth according to the present invention is prepared by a production method comprising the following steps: a step of mixing a basal medium with egg white or dried egg white as a cell growth promoter to obtain a medium for cell growth; and a step of sterilizing the medium. The production method according to the present invention may also comprise a step of heat sterilization or filter sterilization of components susceptible to thermal denaturation, followed by addition to the medium.

[0033] 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.

[0034] Example 1: Cell Preparation (1) Harvesting of Bovine Myoblasts Bovine myoblasts were harvested using the following process. The harvested muscle tissue was washed with ethanol and phosphate buffered saline (PBS) and then minced using 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 (DMEM) supplemented with 0.2% collagenase II (Worthington). The digested reaction solution was stopped by adding 20% ​​FBS. The digested solution was centrifuged at 80 × g for 3 minutes, the floating tissue was removed with tweezers, and the supernatant was collected. The supernatant obtained by centrifuging again at 80 × g for 3 minutes was passed through a nylon mesh (100 μm) for cell separation. The filtrate was centrifuged at 1500 g for 5 minutes, and the resulting precipitate was suspended in Dulbecco's Modified Eagle's Medium containing 20% ​​FBS. The cell suspension was passed through a 100 μm nylon mesh and then again through a 40 μm nylon mesh, and the filtrate was centrifuged at 1500 × g for 5 minutes. The precipitate was left 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 DMEM medium containing 20% ​​FBS and 4 ng / mL human basic fibroblast growth factor (bFGF), and seeded onto culture dishes. The expanded cells were used for the study.

[0035] (2) Collection of bovine and porcine fibroblasts Fibroblasts were collected from the skin tissue of domestic cattle and domestic pigs using the following process. After washing the tissue with ethanol and PBS, the dermis layer was peeled off in a clean bench and isolated. The isolated tissue was finely minced with scissors, placed in a culture dish containing DMEM 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 experiment.

[0036] (3) Collection of Chicken Myoblasts Fibroblasts were collected from 18-day-old chicken embryos using the following procedure. Breast tissue was minced with scissors and placed in DMEM medium containing 2 mg / mL collagenase II (Sigma) for 1 hour at 37°C. The tissue was suspended using a 10 mL syringe equipped with an 18G needle, and the suspension was passed through a 100 μm filter. The filtered suspension was centrifuged at 1500 rpm for 5 minutes, and the resulting precipitate was suspended in DMEM medium containing 20% ​​FBS and 4 ng / mL bFGF. The cell suspension was seeded onto tissue culture dishes. After overnight culture, non-adherent cells were seeded onto collagen-coated culture dishes and passaged twice for use as myoblasts.

[0037] Example 2: Search for food materials that promote cell proliferation in serum-free medium (1) Bovine cells Myoblasts and fibroblasts derived from domestic cattle were used to search for components that promote proliferation in serum-free medium. The serum-free medium used was 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). 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% (except for dried bonito flakes, which were 0.02%). After centrifugation, the supernatant was filtered through a 0.45 μm filter to remove insoluble components, and the resulting solution was used for the test. Approximately 5 × 10 myoblasts were used. ３ cells / cm ３ The cells were seeded on a collagen-coated culture dish and cultured for 3 days. A positive control was a DMEM medium supplemented with 10% FBS. Fibroblasts were cultured at 1 × 10 ４ cells / cm３ The cells were seeded on tissue culture dishes and cultured for 3 to 4 days. A positive control was DMEM medium supplemented with 10% FBS. The medium was incubated at 37°C and CO ２ CO concentration set to 5% ２ After culturing in an incubator, the number of viable cells obtained by trypsin treatment was counted, and the ratio of the cell count when each culture medium was used to the number when serum-containing medium was used (relative proliferation rate (%)) was calculated. The results are shown in Figure 1. When bovine fibroblasts were cultured (A), the proliferation rate for the unsupplemented negative control was 30%, while when each food component was added, it increased to 59% (egg white), 48% (soybean), 40% (bonito flakes), and 37% (wheat flour). A particularly high cell proliferation rate was achieved when egg white was used. When bovine myoblasts were cultured (B), the proliferation rate for the unsupplemented negative control was 20%, while when each food component was added, it increased to 34% (egg white), 24% (soybean), and 19% (wheat). A particularly high cell proliferation rate was achieved when egg white was used.

[0038] (2) Chicken fibroblasts. Chicken fibroblast cell line (DF-1) obtained from ATCC was used to search for components that promote proliferation in serum-free medium. 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. Serum-containing medium supplemented with 10% FBS was used as a positive control. Wheat flour, rice flour, corn flour, soybean, egg white, and chlorella (all dried powders) were used as food ingredients. Each food ingredient was dissolved in serum-free medium at 0.5%, and the resulting supernatant was filtered through a 0.45 μm filter to remove insoluble components. The resulting solution was diluted to 0.1, 0.02, or 0.01% in serum-free medium and used in the test. Approximately 6 × 10 ４ cells / cm ３ The cells were seeded and incubated at 37°C and CO ２ CO concentration set to 5% ２Cells were cultured in an incubator. After 3 days of culture, they 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 in each culture medium was calculated relative to that in serum-containing medium. Various food ingredients were added to chicken fibroblast cultures at 0.1% to 0.01% levels, and their effects on cell proliferation were evaluated. The results are shown in Figure 2(A). At 0.1%, soybeans, chlorella, and egg white exhibited cell proliferation comparable to that observed in serum-containing medium, but at 0.01%, cell proliferation activity was only observed in egg white. BMC Biotechnol (2023) Feb 8;23(1):4 (Non-Patent Document 7) and Prog Immunobiol Stand (1971) 5:202-8 (Non-Patent Document 6) suggest that egg white may have a greater effect on cell proliferation than soybeans and chlorella, which have been reported as serum replacements. Furthermore, Food Funct., (2020) 11, 2477-2488 (Non-Patent Document 4) reported that egg white hydrolysate had a slight growth-promoting effect, but compared to that report, it was found that the amount of egg white added could be reduced. Furthermore, it was found that growth was equivalent to that in serum-containing medium even without the use of growth factors.

[0039] (3) Chicken myoblasts. Chicken myoblasts were used to explore components that promote proliferation in serum-free media. The serum-free medium used consisted of DMEM supplemented with 4 μM insulin, 1% penicillin-streptomycin (PS) solution, 0.2% BSA, 1% lipid additive for cell culture, and 5 ng / mL bFGF. Serum-containing medium supplemented with 10% FBS and 5 ng / mL bFGF was used as a positive control. The food ingredients used were wheat flour, rice bran, chickpeas, dried bonito flakes, fish gelatin, shrimp powder, egg white, and porcine gelatin (all dried powders). Each food ingredient was dissolved in 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 for testing. The serum-containing medium was a positive control, in which 10% FBS and 5 ng / mL bFGF were added to DMEM medium. ４ cells / cm３ The cells were seeded onto collagen-coated plates and cultured for 3 days. The viable cells obtained by trypsinization were counted, and the ratio of the cell counts in each culture medium to those in serum-containing medium was calculated. The results are shown in Figure 2(B). Chicken myoblasts also showed the greatest growth-promoting effect when egg white was added, with 73% growth compared to serum-containing medium.

[0040] Example 3: Comparison with serum-containing medium (porcine fibroblasts) The serum-free medium used was DMEM medium supplemented with 4 uM insulin, 1% penicillin-streptomycin (PS) solution, 0.2% BSA, and 1% lipid additive for cell culture. The serum-containing medium used was DMEM medium supplemented with 10% FBS, which was used as a positive control. Egg white, as a food ingredient, was dissolved in serum-free medium at 0.025%, and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components, and the resulting solution was collected at approximately 6 × 10 ４ cells / cm ３ The cells were seeded and incubated at 37°C and CO ２ CO concentration set to 5% ２ The cells were cultured in an incubator for 3 days, and then a cell counting kit solution (Doujin) was added. ２ The color reaction was carried out in an incubator for 2 hours, and the absorbance at 450 nm was measured. The ratio of the absorbance when each culture medium was used to the absorbance when serum-containing medium was used was calculated. The results are shown in Figure 3. When porcine fibroblasts were cultured, no cell growth was observed in the unsupplemented negative control (DMEM), whereas a high cell growth rate was achieved when egg white was used as a food ingredient.

[0041] Example 4: Verification of Effective Concentration A search for components that promote proliferation in serum-free medium was conducted using chicken fibroblast cell line (DF-1) and porcine fibroblast cells. 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 a serum-containing medium, DMEM medium supplemented with 10% FBS was used as a positive control. As a food ingredient, egg white was dissolved in serum-free medium at 0.2%, and the resulting supernatant was centrifuged and filtered through a 0.45 μm filter to remove insoluble components. The resulting solution was diluted with serum-free medium to 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, and 0.0016%. Approximately 6 × 10 ４ cells / cm ３ The cells were seeded and incubated at 37°C and CO ２ CO concentration set to 5% ２ After culturing in an incubator for 3 days, a Cell Counting Kit solution (Doujin) was added. ２ The color reaction was carried out in an incubator for 2 hours, and the absorbance at 450 nm was measured. The ratio of the absorbance when using each culture medium to the absorbance when using serum-containing medium was calculated. For pig and chicken fibroblasts, proliferation activity was confirmed to be more than 70% of that when serum was added at 0.003% or higher. It was found that the amount of egg white added could be reduced for pig and chicken cells, even compared to the report in Food Funct., (2020) 11, 2477-2488 (Non-Patent Document 4).

[0042] Example 5: Analysis of Molecular Weight of Egg White Powder Dissolved Solution 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). The flow path was equilibrated by flowing 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 of the sample solution and standard solution were injected, and the absorption wavelength at 214 nm was detected at each elution time. 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 relative to the molecular weight of the standard using GPC analysis software (Shimadzu Corporation). The sample solution 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. The standard solutions were ovalbumin, ovomucoid, cytochrome c, aptinin, (Pro-Pro-Gly) ５ A solution containing 0.5 mg / mL of each was used. The peak of the egg white solution was detected at a retention time shorter than the retention time of the cytochrome c peak in the standard solution (27.7 minutes), indicating that the main component of the egg white solution was larger than cytochrome c (105 aa). When calculated using analytical software, the average molecular weight was approximately 37,000 Da (equivalent to 336 aa). This was a larger molecular weight than the reported molecular weight (15 aa or less) in Food Funct., (2020) 11, 2477-2488 (Non-Patent Document 4).

Claims

1. A medium for cell growth comprising a basal medium and egg white or dried egg white as a cell growth promoter.

2. The medium of claim 1, wherein the expanded cells are cells used in cultivated meat production.

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

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

5. The medium of claim 1, wherein the cells comprise fibroblasts or muscle tissue-derived cells.

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

7. A method for preparing cells for cultured meat production, comprising the step of culturing the cells in a medium comprising a basal medium and egg white or dried egg white as a cell growth promoter.

8. The method of claim 7, wherein the medium is free of animal-derived serum.

9. The method of claim 8, wherein the animal-derived serum is fetal bovine serum (FBS).

10. The method of claim 7, wherein the cells are derived from a cow, a pig, or a chicken.

11. The method of claim 10, wherein the cells comprise at least one cell selected from the group consisting of fibroblasts and muscle tissue-derived cells.

12. A method for producing cultured meat, comprising a step of enriching cells prepared by the method according to any one of claims 7 to 11.

13. The method according to claim 12, 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.

14. The method according to claim 13, wherein the other cells are cultured cells or cells obtained from an animal.

15. The method of claim 12, further comprising culturing after enrichment.

16. Cell growth promoters for cultured meat production, containing egg white or dried egg white.

17. The cell proliferation promoting agent according to claim 16, which is added to an animal-derived serum-free medium.

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

19. The cell proliferation promoter according to claim 18, wherein the cells are derived from cows, pigs, or chickens.

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