WO2025005124A1 - Cell-growing culture medium for producing cultured meat - Google Patents

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

Media for cell growth in cultured meat production

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.

Until now, meat production has been carried out by raising livestock. However, raising livestock requires large amounts of grain and water, and requires 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 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.

Plant-based meat substitutes are known as meat substitutes, but their texture and taste are not comparable to meat. On the other hand, cultured meat, which is made by culturing animal cells, can achieve a texture and taste close to that of real meat, and has the advantage of being less susceptible to bacterial and viral contamination than meat. It is becoming technically possible to produce cultured meat. However, the cell culture medium used in cultured meat production to date uses large-scale culture techniques used in basic research and pharmaceutical applications, and it has been difficult to use it for food production due to its cost and safety as meat. In cell culture media used in basic research and pharmaceutical applications, fetal bovine serum (FBS) is generally added as an additive to a basal medium containing amino acids, vitamins, inorganic salts, and carbon sources such as glucose. On the other hand, since FBS is serum collected from fetuses, it is difficult to obtain it in large quantities, and there are issues in terms of price, transportation costs, risk of infectious diseases, and animal welfare. (Non-patent literature 1: ALTEX. 2018;35(1):99-118.) To solve these problems, fully synthetic media have been developed that supplement the essential components of FBS as reagents (Non-patent literature 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 problems when used as food.

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

Patent No. 6111510 International Publication No. 2021/148955

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

The aim is to provide a medium capable of mass culturing cells for use in the production of cultured meat by adding food ingredient components as cell growth promoters.

The present inventors conducted extensive research into culture media that can be used to produce cultured meat, and discovered that adding egg white or dried egg white 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. Thus, the present invention relates to the following:
[1] A medium for cell growth 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 producing a medium for cell growth.
[1-3] Use of egg white or dried egg white for the production of a cell proliferation promoter.
[2] The medium according to item 1, or the use according to items 1-2, wherein the expanded cells are cells used in cultivated meat production.
[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 2-4, 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 a cow, a pig, or a chicken.
[7] A method for preparing cells for cultivated meat production, comprising:
The method comprises a step of culturing 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 derived from a cow, a pig, or a 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 accumulated together with at least one substance selected from the group consisting of other cells, blood, tissues, and extracellular matrices.
[14] The 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 enrichment.
[16] A cell proliferation promoter for cultured meat production, comprising egg white or dried egg white.
[17] The cell proliferation promoting agent 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 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 a cow, a pig, or a chicken.

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

FIG. 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. FIG. 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. FIG. 3 shows the cell proliferation rate relative to the cell number in an FBS-supplemented medium when porcine fibroblasts were cultured in a serum-free medium containing dried egg white. FIG. 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) were cultured in serum-free medium. FIG. 5 shows the results of molecular weight analysis of the freeze-dried egg white sample.

The present invention relates to a medium for cell growth, 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 a step of culturing cells in a medium for cell growth, 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 also relates to a cell growth promoter for cultured meat production, comprising egg white or dried egg white.

[Cell growth medium]
The cell growth medium according to the present invention includes 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 ingredient, the cells cultured in the medium according to the present invention are highly safe as a food. Furthermore, since egg white is an inexpensive ingredient, the medium according to 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. 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, cell growth activity can be enhanced. By incorporating at a low concentration, aggregation is suppressed and medium preparation becomes easier. Since the cells cultured in such a medium are highly safe as a food, they can be used in cultured meat production. The medium according to the present invention relates to a serum-free medium that includes egg white or dried egg white but does not include animal-derived serum.

Animal-derived serum refers to serum produced from the blood of an animal. The supernatant obtained by clotting the collected blood is called serum. The animal-derived serum may be serum derived from any animal, for example, cow, horse, goat, donkey, rabbit, chicken, etc., 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, and further contains hormones, cytokines, growth factors, etc. Fetal serum in particular is rich in components required for cell growth, and is generally added to culture media in research and the pharmaceutical field. A culture 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 recombinant proteins of serum-derived components.

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 called a cell culture supplement). The cell proliferation promoter of the present invention can be used in cell culture for the production of cultured meat, and can be added to an animal-derived serum-free medium.

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 viewpoint of mass availability, poultry such as chickens, quails, turkeys, ducks, geese, and ostriches are preferred. Egg white may be isolated from eggs or may be a dried product of the isolate. The dried product is usually provided as a dry powder. Dried egg white is preferred because it is expected to reduce transportation costs. The dried product may be prepared using any drying method, but is more preferably prepared by freeze-drying. Dried egg white may be commercially available or may be prepared by drying egg white. Egg white or dried egg white can be dissolved in a medium and then filtered using a filter with an appropriate pore size to remove insoluble matter. For example, it can be filtered using a filter with a pore size of 0.3 to 0.5 μM, particularly 0.45 μM. 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. As an example, it is added to the basal medium so that it is 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 promotion effect reaching a plateau or suppressing a decrease, it 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 the dry matter.

　Egg white is mainly composed of various proteins, such as 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. The average molecular weight varies depending on the treatment of the egg white, but from the viewpoint of ensuring that no hydrolysis treatment, particularly enzymatic hydrolysis treatment or chemical hydrolysis treatment, has been performed, it is particularly preferable for the average molecular weight to be 10,000 Da or more, 15,000 Da or more, or 18,000 Da or more.

Basal medium is a medium for cell culture, and refers to a medium that contains the minimum components necessary for the maintenance and proliferation of cells. By seeding cells in the basal medium, the cells can be maintained without dying, and may be capable of proliferation. 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. Monosaccharides such as glucose, disaccharides such as sucrose, oligosaccharides, and polysaccharides may be added as carbon sources. Typically, cell culture media can be prepared by adding additives such as serum to the basal medium. As the basal medium, any basal medium known in the art can be used, 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 modified medium, and Fisher's medium.

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 called 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 known components added to serum-free media in this technical field. Additives added to serum-free media in this technical field 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 of these may be used. Examples of cytokines include IL-1α and IL-1β, which may be added to a concentration of 0.1 to 1000 ng/mL, for example. Examples of serum-derived proteins include fetuin, fibronectin, albumin, globulin, and the like, which may be added to a concentration of 0.0001 to 1%. Examples of antibiotics include penicillin and streptomycin, which may be added to a concentration 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 media and low-serum media, may also be added to the serum-free medium containing egg white or dried egg white of the present invention. The amount of the added agent may be, for example, 0.1 to 5% of a 100-fold concentrated premix solution.

When egg white or dried egg white is added to the basal medium as a cell proliferation promoter, additional food ingredient components may be added. Any ingredient may 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. As an example, an ingredient that has a 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, and microorganisms, such as chlorella, may be added. These food-derived ingredients may be added as extracts, or dried powders may be added and insoluble ingredients may be removed by a filter. These food ingredients are added to the basal medium so that the concentration is 0.0025% to 1.0% by mass, and 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 egg white or dried egg white to other food ingredient 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.

[cell]
The cell culture medium according to the present invention can culture any animal cells. From the viewpoint of producing cultured meat, cells derived from livestock such as cows, pigs, goats, sheep, rabbits, chickens, ostriches, and ducks can be used. In particular, it is preferable to use cells from cows, pigs, and chickens. The cell culture medium according to the present invention can also culture tissues in which cells are aggregated. The animal cells may be primary cells obtained from animals, passage cells passaged from primary cells, or established cell lines. Primary cells can be obtained by chopping animal tissue in the medium. The cells may be cells differentiated from stem cells such as somatic stem cells, embryonic stem cells, and induced pluripotent stem cells. From the viewpoint 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.

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 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 texture and flavor.

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

Fat cells are cells that have lipid droplets in the cytoplasm, and are broadly divided into white fat cells and brown fat cells, as well as their precursor cells, adipose stem cells. Fat cells mature and enlarge by biosynthesizing fatty acids from glucose and taking up fatty acids via the fatty acid transport pathway. When producing cultured meat, flavor can be improved by accumulating fat cells along with fibroblasts and muscle tissue-derived cells.

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 was over 50% compared to when 10% FBS was used, which was increased compared to the negative control without FBS, and the relative proliferation rate was higher than when soybeans, bonito flakes, and wheat were used (Figure 1A). When myoblasts were cultured, the relative proliferation rate was 34% compared to when 10% FBS was used, which was increased compared to the negative control without FBS, and the relative proliferation rate was higher than when soybeans, bonito flakes, and wheat were used (Figure 1B). It has been disclosed that an enzyme-treated egg white preparation is used instead of FBS for culturing bovine myoblasts (Non-Patent Document 4). The present invention can be said to be a simple method in that bovine fibroblasts and myoblasts can be proliferated even when egg white is used as is or only dried. When culturing bovine fibroblasts and myoblasts, ITS may be added in addition to egg white or dried egg white.

When chicken cells are used, egg white or dried egg white can be used, particularly for the purpose of culturing fibroblasts and myoblasts. When fibroblasts are cultured, the relative proliferation rate is at least 80% compared to when 10% FBS is used, and depending on the concentration used, the relative proliferation rate is equal to or higher than when 10% FBS is added. By using egg white or dried egg white, the proliferation rate is significantly increased compared to the negative control without FBS, and the relative proliferation rate is higher than when wheat, rice, corn, and wheat protein are used (Figure 2A). In addition, when chicken fibroblasts are cultured using soybeans or chlorella, the relative proliferation rate is equal to when 10% FBS is added, so these components can also be used as cell proliferation promoters. In addition, when myoblasts are cultured using egg white or dried egg white, the relative proliferation rate is more than 70% compared to when 10% FBS is used. This relative proliferation rate was increased compared to the relative proliferation rate of the negative control without added ingredients, and was shown to be higher than the relative proliferation rates of wheat, rice bran, chickpea, bonito, fish gelatin, shrimp, whey, porcine gelatin, and the negative control without added ingredients (Figure 2B). The use of egg white/egg yolk fractions instead of FBS in the culture of chicken skin cells has been disclosed (Non-Patent Document 5: Jpn J Exp Med. (1985). The present invention is characterized by the proliferation of fibroblasts and/or myoblasts in order to produce cultured meat. In the culture of chicken myoblasts, while FBS is usually a poor substitute, egg white or dried egg white has a sufficient proliferation-promoting effect as a substitute for FBS.

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 results in a significant increase compared to the unsupplemented negative control (Figure 3).

[Cultured meat]
Cultured meat refers to meat produced through cell culture. In the present invention, "for producing cultured meat" refers to the method used to produce cultured meat, which is required to be acceptable in terms of food hygiene. In terms of food hygiene, it is preferable to avoid the use of animal-derived serum, hormones, and genetically modified proteins. In general, 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 a culture of fibroblasts, muscle tissue-derived cells, or fat cells. It is more preferable for cultured meat to contain a culture of multiple types of cells. In addition to cultured cells of fibroblasts or muscle tissue-derived cells, cultured meat may contain an extracellular matrix. The method for producing cultured meat can be, for example, as follows:
The method includes the steps of: culturing at least one cultured cell selected from the group consisting of fibroblasts or muscle tissue-derived cells; and recovering and enriching the cultured cells. The method for producing cultured meat may further include a differentiation induction step and a culturing step after enrichment. The present invention also relates to cultured meat comprising cells cultured in the cell proliferation medium of the present invention.

The cells are cultured by seeding the cells in the cell growth medium according to the present invention, i.e., a medium containing a basal medium and egg white or dried egg white as a cell growth promoter. The cells are cultured under conditions well known in the art, for example, in a 37°C _CO2 incubator^. The culture may be plate culture or suspension culture. The grown cells can be collected as a culture by trypsin treatment or the like, and may be subcultured after collection. The cells can also be cultured by seeding the cells on a detachable structure. The structure to which the grown cells are attached can be collected as a culture. Such a structure can be constructed of an extracellular matrix such as collagen, elastin, fibronectin, laminin, entactin, etc., and the cultured meat may be formed by accumulating the structures to which the cells are attached.

The accumulation step includes shaping the culture of one or more types of cells that have been collected. The culture formed in the accumulation step may be a piece of meat such as a steak, a carcass, or minced meat. The accumulation step includes accumulating the cell culture together 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 proliferation medium of the present invention. As an example, muscle tissue-derived cells cultured in the cell proliferation medium of the present invention may be accumulated with adipose tissue-derived cells and/or fibroblasts cultured in the cell proliferation medium of the present invention. Co-culture may also be performed after accumulation. As an example, the culture of one or more types of cells that have been collected may be mixed and seeded on an extracellular matrix for co-culture. As the extracellular matrix, collagen, elastin, fibronectin, laminin, entactin, etc. may be used. The cell proliferation medium of the present invention may also be used as the medium in this case.

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. As an example, blood, fat tissue, muscle tissue, etc. separated during meat processing may be accumulated with the culture to produce cultured meat.

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 myotube cells and further mature as muscle fibers, or to accumulate lipid droplets in the cytoplasm. Differentiation induction may be performed by a method known in the art, and one example is a method of culturing under a high carbon dioxide concentration. For example, differentiation into myotube cells can be promoted by culturing under a 5 to 10% (v/v) _CO2 atmosphere.

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

All documents referred to 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 are not intended to 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, may be made without departing from the spirit of the present invention.

Example 1: Preparation of cells (1) Harvesting of bovine myoblasts Bovine myoblasts were harvested by the following process. The harvested muscle tissue was washed with ethanol and phosphate buffered saline (PBS) and then finely chopped using scissors in a clean bench. The muscle tissue was digested by shaking culture at 37°C for 1.5 hours in Dulbecco's modified Eagle's (DMEM) medium supplemented with 0.2% collagenase II (Worthington). The reaction was stopped by adding 20% FBS to the digested reaction solution. The digestion solution was centrifuged at 80 x g for 3 minutes, the floating tissue was removed with tweezers, and the supernatant was separated. The supernatant obtained by centrifuging again at 80 x 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, 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 for 5 minutes with red blood cell lysis solution 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), seeded on a culture dish, and the proliferated cells were used for the test.

(2) Collection of bovine and porcine fibroblasts Fibroblasts were collected from the skin tissue of domestic cattle and domestic pigs by 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 chopped 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.

(3) Collection of chicken myoblasts Fibroblasts were collected from 18-day-old chicken fertilized eggs by the following process. After mincing the breast tissue with scissors, it was placed in DMEM medium containing 2 mg/mL collagenase II (Sigma) and subjected to an enzyme 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 filtered suspension was centrifuged at 1500 rpm for 5 minutes, and the precipitate was suspended in DMEM medium containing 20% FBS and 4 ng/mL bFGF. The cell suspension was seeded on a tissue culture dish. Non-adherent cells after overnight culture were seeded on a collagen-coated culture dish and used as myoblasts after two passages.

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 ingredients that promote proliferation in serum-free medium. The serum-free medium used was a DMEM medium supplemented with 1% penicillin-streptomycin (PS) solution, 1% ITS liquid medium supplement (insulin-transferrin-sodium selenite), 2 ng/mL bFGF, and lipid additive for cell culture (sigma). Egg white, soybeans, wheat flour, and dried bonito flakes (all dried powders) were used as food raw materials. Various food ingredients were 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, which was used for the test. Myoblasts were seeded at approximately 5×10 ³ cells/cm ³ on a collagen-coated culture dish and cultured for 3 days. The positive control was a DMEM medium supplemented with 10% FBS. Fibroblast cells were seeded on a tissue culture dish at 1 x 10 ⁴ cells/cm ³ and cultured for 3 to 4 days. The positive control was a DMEM medium supplemented with 10% FBS. After culturing in a CO ₂ incubator set at 37°C and CO ₂ concentration 5%, the number of live cells obtained by trypsin treatment was counted, and the ratio of the number of cells when each culture medium was used to the number when a serum-containing medium was used (relative proliferation rate (%)) was calculated. The results are shown in Figure 1. When bovine fibroblast cells were cultured (A), the negative control without addition was 30%, while when each food component was added, it increased to 59% (egg white), 48% (soybean), 40% (bonito flakes), and 37% (wheat flour), and a high cell proliferation rate was achieved, especially when egg white was used. When bovine myoblasts were cultured (B), the cell proliferation rate was 20% in the unsupplemented negative control, whereas when each food component was added, the rate was 34% (egg white), 24% (soybean), and 19% (wheat); a particularly high cell proliferation rate was achieved when egg white was used.

(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 to which 4 μM insulin, 1% penicillin-streptomycin (PS) solution, 0.2% BSA, and 1% lipid additive for cell culture were added. The serum-containing medium used was DMEM medium to which 10% FBS was added as a positive control. Wheat flour, rice flour, corn flour, soybean, egg white, and chlorella (all dried powders) were used as food raw material components. Various food components were dissolved in serum-free medium at 0.5%, and the supernatant after centrifugation was filtered through a 0.45 μm filter to remove insoluble components, which was then diluted to 0.1, 0.02, and 0.01% in serum-free medium and used in the test.
Approximately 6 x ¹⁰⁴ cells/ ^cm3 were seeded and cultured in a _CO2 incubator set at 37°C and _CO2 concentration 5%. After 3 days of culture, 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 with a plate reader, and the absorbance when each culture medium was used was calculated relative to the absorbance when a serum-containing medium was used. Various food ingredients were added at 0.1% to 0.01% to culture chicken fibroblast cells, and the effect on cell proliferation was evaluated. The results are shown in Figure 2 (A). When 0.1% was added, cell proliferation comparable to that when serum was added was confirmed in soybeans, chlorella, and egg white, but when 0.01% was added, cell proliferation activity was confirmed only 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 higher growth-promoting effect than soybeans and chlorella, which have been reported as serum replacements. In addition, Food Funct., (2020) 11, 2477-2488 (Non-Patent Document 4) reports that egg white hydrolysates have a slight growth-promoting effect, but it was found that the amount of egg white added can be reduced compared to that report. It was also found that growth was equivalent to that in serum-containing media without the use of growth factors.

(3) Chicken myoblasts Chicken myoblasts were used to search for components that promote proliferation in serum-free media. The serum-free media used were DMEM media 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 a serum-containing medium, DMEM media supplemented with 10% FBS and 5 ng/mL bFGF was used as a positive control. As food raw material components, wheat flour, rice bran, chickpeas, bonito flakes, fish gelatin, shrimp powder, egg white, and porcine gelatin (all dried powders) were used. Various food components were dissolved in serum-free media at 0.1% (egg white only 0.025%), and the supernatant after centrifugation was filtered through a 0.45 μm filter and used for the test. As a positive control, serum-containing medium was prepared by adding 10% FBS and 5 ng/mL bFGF to DMEM medium. Myoblast cells were seeded on a collagen-coated plate at about 2×10 ⁴ cells/cm ³ , and after 3 days of culture, the number of live cells obtained by trypsin treatment was counted, and the ratio of the number of cells when each culture medium was used to the number when serum-containing medium was used was calculated. The results are shown in FIG. 2(B). Chicken myoblast cells also showed the highest growth promotion effect when egg white was added, with 73% growth when serum was added.

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

Example 4: Verification of Effective Concentration Using chicken fibroblast cell line (DF-1) and porcine fibroblast cells, a search was conducted for components that promote proliferation in serum-free medium. As the serum-free medium, DMEM medium to which 4 μM insulin, 1% penicillin-streptomycin (PS) solution, 0.2% BSA, and 1% lipid additive for cell culture were added was used. As the serum-containing medium, DMEM medium to which 10% FBS was added was used as a positive control.
As food raw material components, egg white was dissolved in 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 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 of cells were seeded and cultured for 3 days in a CO ₂ incubator set at 37° C. and CO ₂ concentration 5%, after which a Cell Counting Kit solution (Dojin) was added. A color reaction was carried out in a CO ₂ incubator for 2 hours, and the absorbance at 450 nm was measured. The ratio of the absorbance when each culture solution was used to the absorbance when serum-containing medium was used was calculated. For pig and chicken fibroblasts, proliferation activity was confirmed to be more than 70% of that observed when serum was added at 0.003% or more. 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).

Example 5: Analysis of molecular weight of egg white powder 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 attached to a column oven set at 37°C in a high performance liquid chromatography (HPLC: Shimadzu Corporation), and phosphate buffer (pH = 7.4) containing 280 mM NaCl (hereinafter, eluent) was passed at a flow rate of 0.5 mL/min to equilibrate the flow path. Subsequently, 50 μL of the sample solution and the standard solution were injected, and the absorption wavelength at 214 nm was detected for each elution time, and the molecular weight distribution was confirmed by the obtained peak. The average molecular weight was calculated from a calibration curve created from the retention time for 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 um filter, and then diluting it 10 times with the eluent. The standard solution used was a solution containing 0.5 mg/mL each of ovalbumin, ovomucoid, cytochrome c, aptinin, and (Pro-Pro-Gly) _5. The peak of the egg white solution was detected at a retention time shorter than the retention time of the peak of cytochrome c in the standard solution (27.7 minutes), indicating that the main component of the egg white solution is larger than cytochrome c (105 aa). When calculated using analysis software, the average molecular weight was about 37,000 Da (equivalent to 336 aa). This was a larger molecular weight than the one reported in Food Funct., (2020) 11, 2477-2488 (Non-Patent Document 4) (15 aa or less).

Claims (19)

A medium for cell growth comprising a basal medium and egg white or dried egg white as a cell growth promoter. The medium of claim 1, wherein the expanded cells are cells used in cultured meat production. The medium according to claim 1, wherein the medium does not contain animal-derived serum. The medium according to claim 3, wherein the animal-derived serum is fetal bovine serum (FBS). The medium of claim 1, wherein the cells include fibroblasts or muscle tissue-derived cells. The medium according to any one of claims 1 to 5, wherein the cells are derived from a cow, a pig, or a chicken. 1. A method for preparing cells for cultivated meat production, comprising:
The method comprises a step of culturing cells in a medium containing a basal medium and egg white or dried egg white as a cell growth promoter. The method of claim 7, wherein the medium does not contain animal-derived serum. The method according to claim 8, wherein the animal-derived serum is fetal bovine serum (FBS). The method according to claim 7, wherein the cells are derived from a cow, a pig, or a chicken. The method of claim 10, wherein the cells include at least one cell selected from the group consisting of fibroblasts and muscle tissue-derived cells. 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. The method of 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. The method of claim 13, wherein the other cells are cultured cells or cells obtained from an animal. The method of claim 12, further comprising culturing after enrichment. A cell growth promoter for cultured meat production, containing egg white or dried egg white. The cell growth promoter according to claim 16, which is added to an animal-derived serum-free medium. 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 muscle tissue-derived cells. The cell proliferation promoter according to claim 18, wherein the cells are derived from a cow, a pig, or a chicken.

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