WO2024039216A1 - Method for culturing animal cells or animal cell line - Google Patents

Abstract

The present invention relates to: an animal cell culture medium composition; a method for culturing animal cells using same; a method for preparing a non-human animal cell line; a cell line prepared using same; and a method for culturing the cell line. An animal cell culture medium composition containing a yeast extract or peptone according to an aspect has the effects of allowing the growth and proliferation of animal cells even in a medium not containing serum, and promoting the growth and proliferation of animal cells. An animal cell culture medium composition containing a basic medium and a yeast extract according to an aspect has the effects of allowing the effective proliferation of cells even in a basic medium not containing vitamins and organic acids, and reducing the price of a cell culture medium by minimizing culture medium components. A method for preparing a cell line according to an aspect, the method including a step for separating cells from non-human animals and a step for suspension-culturing the separated cells, has the effect of making it possible to effectively obtain a cell line from non-human animals.

Description

Cultivation method of animal cells or animal cell lines

The present invention relates to a medium composition for culturing animal cells, a method of cultivating animal cells using the same, a method of producing a non-human animal cell line, a cell line produced thereby, and a method of cultivating the cell line.

With the continuous increase in population, problems with environmental pollution are emerging, and in particular, problems with marine environmental pollution are intensifying to a serious level. Since the 1960s, environmental pollution and indiscriminate fishing have led some species to reach unsustainable levels. Marine creatures can suffocate by getting caught in trash, but a more serious problem is that plastic trash floating in the ocean is directly exposed to ultraviolet rays, creating microplastics. The microplastics produced in this way gradually accumulate in the bodies of marine life, causing various diseases in the living body, threatening the survival of marine life. In addition, it is also being taken seriously that microplastics are accumulating in humans who consume marine organisms with accumulated microplastics. In addition, the temperature of sea water is gradually increasing due to rapid climate change due to warming, further encouraging the decline in marine species.

Despite the destruction of the marine ecosystem, the demand for marine resources is increasing due to population growth, and the demand for crustaceans such as shrimp, crabs, and lobsters continues to increase. In order to supply this increasing demand, aquaculture has increased, but this has also brought with it various side effects. However, with the increase in aquaculture, carbon gas emissions, a major cause of global warming, have worsened, and the supply method to meet demand is ultimately causing new environmental pollution. Additionally, while creating space for aquaculture, it destroys ecosystem resources such as coastal ecosystems that function to absorb carbon, one of the causes of warming. In addition, there is an inherent risk that group mortality may occur due to contamination by increased waste in nearby waters and viral infection due to group inhabitation in crowded spaces.

Cultured meat is being proposed as a solution to curb environmental pollution and meet demand. Cultured meat can produce multiple individuals by extracting and proliferating cells from one individual, thus reducing carbon gas emissions and showing high production relative to area. In addition, cultured meat has the advantage of providing safe food that is not contaminated with heavy metals and microplastics, unlike marine products caught in the sea, as cells are proliferated in a laboratory incubator, and it is sustainable and does not destroy the marine environment. There is an advantage to not having it.

In order to develop and produce cultured meat, cell extraction technology, cell proliferation technology, and customized cell culture medium must be combined to create it, and for commercialization, reducing the cost of the culture medium is known to be the biggest problem. In general, cells are cultured by adding 5 to 20% of fetal bovine serum (FBS), which contains proteins, amino acids, and hormones. Fetal bovine serum is considered essential in cell culture, and is important for cell growth and Although it is very effective in proliferation, it has the disadvantage of being very unethical and quite expensive since it is extracted directly from the blood of bovine fetuses.

In addition, the most important thing in commercializing cultured meat is reducing the production price of cultured meat, and the thing that has the greatest impact on reducing the price for producing cultured meat is the cell culture medium. Various ingredients are added to the basic culture medium used when cultivating cultured meat. If cultured meat can be produced using a culture medium without the addition of these ingredients, it can greatly help reduce costs.

In addition, the main technology for producing crustacean cultured meat is to establish a technology to separate and culture cells from the tissue of the cultured meat being made, which was previously (George et al., In Vitro Cell.Dev.Biol.-Animal 2010 ), a method of isolating and culturing muscle cells from shrimp was reported, but the proliferative ability was limited, and the cells showed no further growth after culturing for a certain period of time (<7 days).

Accordingly, the present inventors completed the present invention by developing a culture medium in which crustacean cells actively grow and proliferate even without serum or various components, a cell separation method that can be commonly applied to crustaceans, and an effective cell culture method without limitations in proliferative capacity. .

One aspect is to provide a medium composition for culturing animal cells containing yeast extract or peptone.

Another aspect is to provide a medium composition for culturing animal cells including a basic medium and yeast extract.

Another aspect is to provide a method for cultivating animal cells, comprising culturing cells in a medium composition for culturing animal cells containing yeast extract or peptone.

Another aspect is to provide a method for cultivating animal cells, comprising culturing cells in a medium composition for culturing animal cells containing a basic medium and a yeast extract.

Another aspect includes isolating cells from a non-human animal; and

Suspension culturing of the separated cells

To provide a method for producing a cell line, including.

Another aspect is to provide a cell line produced according to the above production method.

Another aspect is to provide a method for cultivating a cell line, including performing suspension culture to proliferate the cell line.

One aspect provides a medium composition for culturing animal cells containing yeast extract or peptone.

Another aspect provides a medium composition for culturing animal cells comprising a basic medium and a yeast extract.

Another aspect provides a method of cultivating animal cells, comprising culturing cells in a medium composition for culturing animal cells containing yeast extract or peptone.

Another aspect provides a method of cultivating animal cells, comprising culturing the cells in a medium composition for culturing animal cells comprising a basic medium and a yeast extract.

In one embodiment, the medium composition may contain 0.01 to 20, 0.01 to 10, 0.6 to 10, or 0.6 to 2% (w/v) of the yeast extract or peptone.

Peptone is a general term for products produced when various proteins are enzymatically decomposed or hydrolyzed, and is a mixture of components such as polypeptides, amino acids, and minerals.

In one embodiment, the peptone may be animal peptone, bacterial peptone, or vegetable peptone, preferably vegetable peptone, but is not limited thereto.

The animal peptone may be meat and/or casein.

The bacterial peptone may be Bacto peptone.

In one embodiment, the vegetable peptone is soy peptone, wheat peptone, broadbean peptone, potato peptone, pea peptone, papaic soy peptone ( It may be one or more selected from the group consisting of papic soy peptone and lupine peptone, and preferably soy peptone, but is not limited thereto.

As used herein, the term “vegetable peptone” refers to a decomposition product of protein obtained from plants. For example, soy peptone refers to a product obtained by decomposing total protein obtained from soybeans. Decomposition of the vegetable protein can be performed by partial digest. Decomposition of the protein may be performed through acid treatment, base treatment, enzyme treatment, high pressure treatment, heat treatment, or physical treatment. The physical treatment is, for example, grinding.

In the present invention, vegetable peptone is a partial decomposition product of vegetable protein, and may be in the form of a mixture containing not only single molecule amino acids, but also peptides composed of several to dozens of amino acids and intact protein molecules.

The plant source from which the vegetable peptone of the present invention is obtained is not particularly limited, and peptone derived from any plant is included as long as it has the ability to promote cell proliferation.

The medium composition of the present invention may include a microbial extract, and the microorganisms may be edible.

The microorganism may be, but is not limited to, bacteria, archaea, or yeast.

In this specification, the term "yeast extract" is a natural product that does not cause the problem of producing harmful substances such as acid-decomposed HVP (Hydrolyzed vegetable protein) because it is produced through autodigestion using NaCl, ethanol, or enzyme treatment after culturing yeast. It refers to a biological material and is used in microbial fermentation media or natural seasoning.

The yeast extract can be prepared by adding edible enzymes to edible yeast and hydrolyzing polypeptides of the edible yeast, and can be used for food.

In one embodiment, the yeast extract may be usable in food.

Yeast extract is known to contain minerals, vitamins, nucleic acids, peptides, and amino acids.

In one embodiment, the yeast is from the genus Saccharomyces ( Saccharomyces sp.), Wicherhamomyces sp., Pichia sp., and Hanseniaspora sp. It may be one or more types selected from the group consisting of.

The vegetable peptone and the yeast extract may be ingredients derived from plants or microorganisms that replace animal ingredients. The plant- or microbial-derived ingredients are very suitable for use in biopharmaceutical and food applications.

In one embodiment, the animal cell may be one or more types selected from the group consisting of muscle stem cells, muscle cells, and progenitor cells thereof.

The muscle stem cells may be satellite cells or myoblasts, for example, but are not limited thereto.

As used herein, the term “muscle stem cell” refers to a cell having the characteristics of a muscle stem cell, including proliferation without transformation, unlimited proliferation, self-reproduction ability, and the ability to differentiate into muscle. Any cell that shows self-reproduction ability, unlimited proliferation ability, or muscle differentiation ability can be included without limitation. The self-reproduction ability and muscle differentiation ability can be confirmed using markers. Additionally, the muscle stem cells may be, for example, cells that exhibit self-renewal ability and expression of CD29, CD56, Oct4, Nanog, or Pax7. The type and origin of the muscle stem cells are not limited as long as they have differentiation ability and self-renewal ability. The muscle stem cells may be derived from, for example, mammals, humans, monkeys, pigs, horses, cows, chickens, ducks, sheep, dogs, cats, mice, or rabbits. In one embodiment, the muscle stem cells may be bovine or chicken muscle stem cells.

The muscle stem cells may be cells that express Pax7 or MyoD in the process of being induced into myoblasts.

As used herein, the term "myoblast" refers to a precursor cell in the process of differentiating into a muscle cell, a cell that proliferates like a myogenic cell and can differentiate into a muscle cell (myogenic cell, myocyte) through fusion between cells. it means.

As used herein, the term “muscle cell” refers to cells capable of forming muscle tissue generated from myoblasts through the differentiation process of myoblasts.

The muscle cells may be myocytes or myotubes, for example, myocytes, but are not limited thereto.

The progenitor cells refer to cells capable of generating cells differentiated into multiple lineages, such as myoblasts, fibroblasts, adipocytes, stromal cells, pericytes, smooth muscle cells, and endothelial cells. Progenitor cells differ from stem cells in that they typically do not have extensive self-replication capabilities.

The animal cells may further include fat cells (fat cells or adipocytes) and/or their progenitor cells, stromal cells (connective tissue) and/or their progenitor cells, or endothelial cells (blood vessels) and/or their progenitor cells. , but is not limited to this.

In one embodiment, the animal may be one or more species selected from the group consisting of cattle, sheep, pigs, poultry, crustaceans, and fish, but is not limited thereto.

In one embodiment, the animal cells may refer to animal-derived cells made by isolating cells from animal tissues and culturing them in vitro.

In one embodiment, the animal cells may be crustacean cells.

In one embodiment, the animal cells may be cells derived from shrimp, crab, or lobster.

As used herein, the term “culture media” refers to a material that supports the growth and survival of cells, including stem cells, in vitro.

As used herein, the term “basic medium” refers to a medium containing basic components for culturing cells. In general, when cells are cultured using only basic media, the cells often do not proliferate smoothly, so various ingredients are added for optimal cell proliferation.

In one embodiment, the medium or basic medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture) F-10), DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium) ), Knockout DMEM, E8 (Essential 8 Medium), SF-DMEM (serum free-Dulbecco Modified Eagle Medium), L15 Medium, and Grace's Insect Medium, but is not limited to this.

In addition to the above medium, any medium used in the industry is sufficient, and the medium may contain amino acids, vitamins, inorganic salts, and other ingredients. For example, non-essential amino acids and L-glutamine, vitamins include vitamin B12, and inorganic salts include trace components (CuSO45H2O, Fe(NO3)39H2O, ZnSO4), phosphoenol pyruvate, and monoethanol. Amines (Momoethanolamine), Sodium selenite and Sodium bicarbonate, other ingredients include D-glucose, Linoleic acid, Lipoic acid and Sodium pyruvate ), hypoxantine sodium, putrescine hydrochloride (Putrescine HCl), and polyamine solution.

In one embodiment, the medium may be a serum-free medium.

In one embodiment, the medium composition for culturing animal cells may not contain serum.

Serum-free medium refers to a cell culture medium that does not use serum, which has unethical, unenvironmental, and high cost problems. It refers to a cell culture medium that does not require the use of serum.

In one embodiment, the medium may be a medium containing serum.

The serum may be, but is not limited to, animal blood, and is preferably serum derived from the blood of mammals (e.g., pigs, horses, cows, goats, sheep, and dogs), and more preferably fetal bovine serum. serum, FBS).

In one embodiment, the serum is fetal bovine serum (FBS), human platelet lysate (hPL), human serum albumin (HSA), and human serum (HS). ), Platelet-Rich Plasma (PRP), platelet poor plasma (PPP), calf serum, horse serum, porcine serum and sheep serum. It may be one or more types selected from the group consisting of, but is not limited thereto.

In one embodiment, the culture may be a suspension culture.

As used herein, the term “cell culture” refers to the process of artificially growing living cells in vitro under controlled conditions. In addition, a portion of an individual's tissue is aseptically removed, the intercellular connecting material is decomposed with enzymes, and the resulting suspension is spread on the flat bottom of a culture dish such as a bottle or Petri dish to grow and proliferate cells.

As used herein, the term “suspension culture” refers to culturing cells to be cultured in a floating state in a culture medium without being fixed to a substrate.

In the above culture process, SR (serum replacement) may be added to the medium to create a suspended state of the cultured cells, or the culture may be performed using a low-adhesion culture dish or bioreactor, but is not limited to this, and various suspended cultures used in the industry method can be used.

The culture may be growth and proliferation.

As used herein, the terms “growth and proliferation” mean an increase in the number of cells. The culture may be undifferentiated proliferation. The undifferentiated proliferation means that stem cells proliferate into cells with the same properties as the original cells, that is, with potency and self-renewal ability, without being differentiated into specific cells. . In this specification, the term “differentiation” refers to the phenomenon in which cells become specialized in structure or function while they divide and grow, that is, the cells, tissues, etc. of living organisms change their form or function to perform a given task. Means that. Measuring or determining the degree of differentiation into a specific cell type can be performed by methods well known in the art. Additionally, the differentiation can be characterized by cell surface labeling (e.g., staining cells with tissue-specific or cell-label specific antibodies) and changes in cell morphology (e.g., using techniques such as flow cytometry or immunocytochemistry). By examining cell morphology using light or confocal microscopy, measuring (for example, nuclear/cytoplasmic ratio), or by analyzing the cell's morphology, such as polymerase chain reaction (PCR) and gene-expression profiling. It can be confirmed by measuring changes in gene expression using well-known techniques.

In one embodiment, the medium composition may further include an amino acid mixture or trace elements.

There is no limitation on the types of amino acids included in the amino acid mixture, and include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, It may be one or more of serine, threonine, selenocysteine, valine, tryptophan, and tyrosine.

The amino acid mixture may include one or more selected from the group consisting of serine, aspartic acid, glutamic acid, alanine, glutamine, proline, tyrosine, valine, and leucine.

In one embodiment, the medium composition contains an amino acid mixture of 100 to 5000 mg/L, 100 to 4500 mg/L, 100 to 4000 mg/L, 100 to 3500 mg/L, 500 to 5000 mg/L, 1000 to 1000 mg/L. 5000 mg/L, 1500 to 5000 mg/L, 2000 to 5000 mg/L, 2500 to 5000 mg/L, 500 to 4500 mg/L, 1000 to 4000 mg/L, 1500 to 3500 mg/L or 2000 to 3000 It can be included in mg/L concentration.

As used herein, the single letter (triple letter) of amino acids refers to the following amino acids according to standard abbreviation conventions in the field of biochemistry: A (Ala): alanine; C(Cys): Cysteine; D(Asp): Aspartic acid; E(Glu): glutamic acid; F(Phe): Phenylalanine; G(Gly): glycine; H(His): histidine; I(IIe): Isoleucine; K(Lys): Lysine; L(Leu): leucine; M(Met): methionine; N(Asn): Asparagine; O(Ply): pyrrolysine; P(Pro): Proline; Q(Gln): Glutamine; R(Arg): arginine; S(Ser): Serine; T(Thr): threonine; U(Sec): Selenocysteine, V(Val): Valine; W(Trp): tryptophan; Y(Tyr): Tyrosine.

The trace elements refer to substances that are required in very small amounts among the substances essential for the growth of living things, and are essential chemical elements that play an important role in the growth, development and physiology of living things.

In one embodiment, the trace elements include calcium (Ca), potassium (K), sodium (Na), iron (Fe), chlorine (Cl), boron (B), manganese (Mn), zinc (Zn), It may be one or more types selected from the group consisting of copper (Cu) and molybdenum (Mo).

In one embodiment, the medium composition contains 10 to 500 mg/L, 10 to 450 mg/L, 10 to 400 mg/L, 10 to 350 mg/L, 50 to 500 mg/L, 100 to 100 mg/L. 500 mg/L, 150 to 500 mg/L, 200 to 500 mg/L, 250 to 500 mg/L, 50 to 450 mg/L, 100 to 400 mg/L, 150 to 350 mg/L or 200 to 300 It can be included in mg/L concentration.

In one embodiment, the medium composition contains the amino acid mixture or trace elements at 100 to 5000 mg/L, 100 to 4500 mg/L, 100 to 4000 mg/L, 100 to 3500 mg/L, 500 to 5000 mg/L. L, 1000 to 5000 mg/L, 1500 to 5000 mg/L, 2000 to 5000 mg/L, 2500 to 5000 mg/L, 500 to 4500 mg/L, 1000 to 4000 mg/L, 1500 to 3500 mg/L Alternatively, it may be contained at 2000 to 3000 mg/L.

In one embodiment, the basic medium contains biotin, D-Ca pantothenate, a-ketoglutaric acid, choline chloride, and folic acid ( Folic acid, i-inositol, nicotinic acid, pyridoxine-HCl, riboflavin, thiamine-HCl, and penta-amino benzoic acid. It may not contain one or more vitamins selected from the group consisting of benzoic acid.

In one embodiment, the basic medium may not contain vitamins.

In one embodiment, the basic medium may not contain one or more organic acids selected from the group consisting of fumaric acid, malic acid, and succinic acid.

In one embodiment, the basic medium may not contain organic acids.

In the case of vitamins and organic acids, if excessive amounts are applied to cells, they may actually inhibit cell proliferation.

Another aspect includes isolating cells from a non-human animal; And providing a method for producing a cell line, including the step of suspension culturing the separated cells.

In one embodiment, the method for producing the cell line may not include the step of treating biological or chemical exogenous immortalization factors. Alternatively, the method for producing the cell line may not include the step of inducing immortalization of cells by treating biological or chemical exogenous factors.

The biological exogenous immortalization factor or biological exogenous factor may be a vector into which muscle-specific and/or fat-specific growth genes are integrated.

For example, the genes include MyoD, SMARCD3, Pax3, Pax7, myosin-1, integrin alpha-7, cadherin-15, myogenin, growth hormone, and insulin-like. It may be an insulin-like growth factor, myostatin, growth differentiation factor, hyperglycemic hormone, or myosin heavy chain gene.

The vector may be an adenovirus vector or a lentivirus vector.

Additionally, the biological exogenous immortalization factor or biological exogenous factor may be reprogramming micro RNA and mRNA.

The reprogramming micro RNA may be mir302a-d, mir367, Oct4, Sox2, Klf4, c-Myc or Lin28.

The chemical exogenous immortalization factor or chemical exogenous factor may be a telomerase activator.

The chemical extrinsic agent or chemical extrinsic agent may be Cycloastragenol, Genistein from Glycine Max (soybean) or Resveratrol.

In one embodiment, the method for producing the cell line may not include the step of treating albumin or exogenous albumin.

In one embodiment, the cells may be one or more types selected from the group consisting of muscle stem cells, muscle cells, and progenitors thereof.

The muscle stem cells may be satellite cells or myoblasts, for example, but are not limited thereto.

The muscle stem cells are as described above.

The muscle stem cells may be cells that express Pax7 or MyoD in the process of being induced into myoblasts.

The myoblasts are as described above.

The muscle cells are as described above.

The muscle cells may be myocytes or myotubes, for example, myocytes, but are not limited thereto.

The progenitor cells refer to cells capable of generating cells differentiated into multiple lineages, such as myoblasts, fibroblasts, adipocytes, stromal cells, pericytes, smooth muscle cells, and endothelial cells. Progenitor cells differ from stem cells in that they typically do not have extensive self-replication capabilities.

The cells may further include fat cells (fat cells or adipocytes) and/or their progenitor cells, stromal cells (connective tissue) and/or their progenitor cells, or endothelial cells (blood vessels) and/or their progenitor cells. It is not limited to this.

In one embodiment, the non-human animal can be cattle, sheep, pigs, poultry, crustaceans, or fish.

In one embodiment, the crustacean may be shrimp, crab, or lobster.

In one embodiment, the suspension culture is performed at 100 to 1000 RPM, 100 to 900 RPM, 100 to 800 RPM, 100 to 700 RPM, 100 to 600 RPM, 100 to 500 RPM, 100 to 400 RPM, 100 to 300 RPM, Can be performed at 200 to 1000 RPM, 300 to 1000 RPM, 200 to 900 RPM, 200 to 800 RPM, 200 to 700 RPM, 200 to 600 RPM, 200 to 500 RPM, 200 to 400 RPM, 250 RPM or 300 RPM there is.

In one embodiment, the suspension culture is performed for 1 to 42 days, 1 to 35 days, 1 to 28 days, 7 to 42 days, 14 to 42 days, 21 to 42 days, or 7 to 35 days. It may be performed daily, 14 to 28 days, 21 to 28 days, or 28 days.

In one embodiment, the suspension culture may be performed at 10 to 50°C, 10 to 40°C, 10 to 30°C, 20 to 50°C, 25 to 50°C, 20 to 40°C, 25 to 30°C, or 28°C.

In one embodiment, the suspension culture may be performed in a serum-free medium.

In one embodiment, the suspension culture may be performed in a medium containing serum.

As used herein, the term “suspension culture” refers to culturing cells to be cultured in a floating state in a culture medium without being fixed to a substrate.

In this specification, the terms “suspension culture” and “suspension culture” are synonymous.

In this specification, the terms “stationary culture” and “attached culture” are synonyms.

In the above culture process, SR (serum replacement) may be added to the medium to create a suspended state of the cultured cells, or the culture may be performed using a low-adhesion culture dish or bioreactor, but is not limited to this, and various suspended cultures used in the industry method can be used.

The culture may be growth and proliferation.

The growth and proliferation are as described above.

In one embodiment, the medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10) ), DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), Knockout It may be one or more selected from the group consisting of DMEM, E8 (Essential 8 Medium), SF-DMEM (serum free-Dulbecco Modified Eagle Medium), L15 Medium, and Grace's Insect Medium, but is not limited thereto.

In addition to the above medium, any medium used in the industry is sufficient, and the medium may contain amino acids, vitamins, inorganic salts, and other ingredients. For example, non-essential amino acids and L-glutamine, vitamins include vitamin B12, and inorganic salts include trace components (CuSO45H2O, Fe(NO3)39H2O, ZnSO4), phosphoenol pyruvate, and monoethanol. Amines (Momoethanolamine), Sodium selenite and Sodium bicarbonate, other ingredients include D-glucose, Linoleic acid, Lipoic acid and Sodium pyruvate ), hypoxantine sodium, putrescine hydrochloride (Putrescine HCl), and polyamine solution.

In one embodiment, the medium may be a serum-free medium.

Serum-free medium refers to a cell culture medium that does not use serum, which has unethical, unenvironmental, and high cost problems. It refers to a cell culture medium that does not require the use of serum.

Another aspect includes isolating cells from a non-human animal; and providing a cell line prepared according to a method for producing a cell line, which includes the step of culturing the separated cells in suspension.

In one embodiment, the cell line may maintain cell proliferation ability at -200 to -75°C for 1 to 12 months, but is not limited thereto.

Another aspect provides a method for cultivating a cell line including the step of performing suspension culture to proliferate the cell line prepared according to the method for producing the cell line.

The RPM and temperature at which suspension culture for proliferating the cell line can be performed are as described above.

In one embodiment, suspension culture for proliferating the cell line may be performed in a medium containing serum.

The serum may be, but is not limited to, animal blood, and is preferably serum derived from the blood of mammals (e.g., pigs, horses, cows, goats, sheep, and dogs), and more preferably fetal bovine serum. serum, FBS).

In one embodiment, the serum is fetal bovine serum (FBS), human platelet lysate (hPL), human serum albumin (HSA), and human serum (HS). ), Platelet-Rich Plasma (PRP), platelet poor plasma (PPP), calf serum, horse serum, porcine serum and sheep serum. It may be one or more types selected from the group consisting of, but is not limited thereto.

In one embodiment, the serum may be included at 1 to 20% by weight, 3 to 18% by weight, 5 to 16% by weight, 7 to 14% by weight, 9 to 12% by weight, or 10% by weight based on the total weight of the medium. You can.

Another aspect includes isolating cells from a non-human animal; And it provides cultured meat containing a cell line produced according to a cell line production method, which includes the step of suspension culturing the separated cells.

In the present invention, the term "cultured meat" is also referred to as clean meat, cell-based meat, or cultivated meat, and refers to food that is suitable or edible for human or non-human animals. it means.

In one embodiment, a food composition containing the cultured meat is provided.

The food may be one or more selected from the group consisting of snacks, dumplings, fried foods, stir-fried foods, steamed foods, sauces, seasonings, powder mixes, breads, processed canned foods, beverages, dried seaweed, processed noodles, and processed foods.

The form in which the cultured meat is added to food may be in the form of pulverization into various particle sizes depending on the purpose of use in food. It can be ground uniformly or unevenly in the range of 1 ㎛ to 10 cm and added to food. Alternatively, it can be added in dried powder form depending on the purpose for which it is used in food.

The food composition may further include fat and/or coloring agent. The fat may be injected into fat cells during the production of cultured meat and co-cultured during the proliferation of muscle cells.

Alternatively, it may be included by adding fat in liquid form. In this case, it can be good for your health because you can replace the saturated fatty acids contained in meat with beneficial fats.

In one embodiment, the fat is soybean oil, corn oil, canola oil, rice bran oil, sesame oil, extracted sesame oil, perilla oil, extracted perilla oil, safflower oil, sunflower oil, cottonseed oil, peanut oil, olive oil, palm oil, palm oil, and red pepper seed oil. Animal oils such as vegetable oil, edible beef tallow, edible pork, raw beef tallow, raw pork fat, fish oil, etc., and edible oil and processed products such as mixed cooking oil, flavored oil, processed fat, shortening, margarine, imitation cheese, and vegetable cream can be used.

Colorants refer to compounds that give color to foods. Artificial colorants, natural colorants, and natural extracts (e.g., beet root extract, pomegranate fruit extract, cherry) are used to reproduce the red color of beef or pork. extract, carrot extract, red cabbage extract, red seaweed extract), modified natural extract, natural juice (e.g. beetroot juice, pomegranate juice, cherry juice, carrot juice, red cabbage juice, red seaweed juice), Modified natural juice, FD&C (Food Drug & Cosmetics) Red No. 3 (erythrosine), FD&C Green No. 3 (fast green FCF), FD&C Red No. 40 (allura red AC), FD&C Yellow No. 5 (tartazine), FD&C Yellow No. 6 (sunset yellow FCF), FD&C Blue No. 1 (brilliant blue FCF), FD&C Blue No. 2 (indigotine) , titanium oxide, annatto, anthocyanin, betanin, beta-APE 8 carotene, beta-carotene, black currant, burnt sugar, canthaxanthin, caramel, carmine/carminic acid. , cochineal extract, curcumin, lutein, carotenoids, monascin, paprika, riboflavin, saffron, turmeric, and combinations thereof can be used, but are not particularly limited thereto. Additionally, a coloring agent such as nitrite and ascorbic acid, erysobic acid, or salts thereof that promote color development of the nitrite may be further added as a coloring aid.

In addition, antioxidants, emulsifier salts, etc. may be added to stabilize the protein to prevent rancidity, color change, or separation of fat. The antioxidants, emulsifier salts, etc. can be used without limitation as long as they are widely used in the industry.

According to a medium composition for culturing animal cells containing yeast extract or peptone according to one aspect, growth and proliferation of animal cells are possible even in a medium that does not contain serum, and the growth and proliferation of animal cells are promoted. According to a medium composition for animal cell culture containing a basic medium and a yeast extract according to one aspect, cells can be effectively proliferated even in a basic medium that does not contain vitamins and organic acids, and the culture medium components are minimized to form a cell culture medium. It has the effect of reducing cost, and according to one aspect, isolating cells from a non-human animal; According to the method for producing a cell line, which includes the step of culturing the separated cells in suspension, it is possible to effectively obtain a cell line from a non-human animal.

Figure 1 shows the results of confirming the difference in cell proliferation according to different culture media when culturing animal cells.

Figures 2 to 4 show the results of culturing animal cells in a medium containing peptone and confirming the difference in proliferation of animal cells compared to a medium with or without the addition of fetal bovine serum.

Figures 5 to 7 show the results of culturing animal cells in a medium containing yeast extract and confirming the difference in proliferation of animal cells compared to a medium with or without the addition of fetal bovine serum.

Figure 8 shows the measurement of amino acid consumption over time when animal cells are cultured in a medium containing yeast extract.

Figures 9 to 14 show the results of observing the proliferation of animal cells by additionally adding an amino acid mixture to a medium containing yeast extract or peptone.

Figures 15 to 20 show the results of observing the proliferation of animal cells by additionally adding trace elements to a medium containing yeast extract or peptone.

Figure 21 shows the results of confirming the difference in cell proliferation depending on the presence or absence of vitamins in the basic medium.

Figure 22 shows the results of confirming the difference in cell proliferation depending on the presence or absence of organic acids in the basic medium.

Figure 23 shows the results of confirming the difference in cell proliferation depending on the presence or absence of vitamins and organic acids in the basic medium.

Figures 24 and 25 are schematic diagrams showing the process of establishing cell lines from crustaceans.

Figure 26 shows the results of culturing cells isolated from crustaceans and observing them under a microscope.

Figures 27 to 29 show the results of confirming cell proliferation by culturing cells isolated from crustaceans under different culture conditions.

Figure 30 shows the results confirming that cells isolated from crustaceans were established as cell lines and had sustained proliferation ability even after long-term culture.

Figure 31 shows the results confirming that cells isolated from crustaceans were established as cell lines and had continued proliferative ability even after long-term storage.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

The terms or words used in the specification and claims of the present invention are not to be construed as limited to their ordinary or dictionary meanings, and the inventor may appropriately define the concept of terms to explain his or her invention in the best way. It must be interpreted as meaning and concept consistent with the technical idea of the present invention based on principles.

Throughout the specification of the present invention, when a part is said to "include" a certain component, this means that it does not exclude other components but may further include other components, unless specifically stated to the contrary. .

Throughout the specification of the present invention, “A and/or B” means A or B, or A and B.

Example 1. Confirmation of cell proliferation by medium

To determine whether there was a difference in cell proliferation when the culture medium was changed during cell culture, crustacean-derived cells isolated from shrimp were cultured in commonly used basic media (DMEM-F12, L15 or Grace's insect media) at 0, 5 or 10. Culture was performed using medium supplemented with % (v/v) Fetal Bovine Serum (FBS), and the degree of cell proliferation was confirmed.

Specifically, crustacean cells were initially inoculated at ^1.5 To confirm the approximate tendency of cell proliferation, absorbance was measured at OD600, and the exact number of cultured cells was confirmed using a hemocytometer. The results are shown in Figure 1.

As shown in Figure 1, as a result of absorbance measurement and cell count confirmation using a hemocytometer, the highest cell proliferation was confirmed in Grace's insect media. Additionally, higher cell proliferation was observed in the culture medium containing fetal bovine serum than in the culture medium without fetal bovine serum. From the above results, it was confirmed that even if fetal bovine serum was included, the proliferation rate of crustacean cells was significantly different depending on the type of basic medium. Although Grace's insect media was suitable for the proliferation of crustacean cells, fetal bovine serum could be added to other basic culture media. It was found that even if added, cell proliferation occurred.

Example 2. Cell proliferation in medium containing Peptone

To determine whether peptone can be used as an ingredient that can replace fetal bovine serum, which has a cell proliferation effect in animal cell culture, the cell proliferation effect of peptone, which is not used in general animal cell culture, was examined. was confirmed. Peptone is a general term for products produced when various proteins are enzymatically decomposed or hydrolyzed, and is a mixture of components such as polypeptides, amino acids, and minerals. Specifically, peptone was added to three types of basic media (DMEM-F12, L15, or Grace's insect media) to a concentration of 0.01, 0.6, 1, or 10% (w/v) to prepare a culture medium, and fetal bovine serum was added. The proliferation of crustacean-derived cells isolated from shrimp, crab, or lobster was confirmed in the same manner as in Example 1, compared to the culture medium with or without the addition. The results are shown in Figures 2 to 4.

As shown in Figures 2 to 4, very slight cell proliferation was confirmed when fetal bovine serum and peptone were not added to DMEM-F12, L15 or Grace's insect media. However, when cells were cultured with peptone added to three types of basic media, a higher cell proliferation rate was seen than when peptone was added to Grace's insect media. It was confirmed that the highest cell proliferation was observed in this case. In addition, when peptone was added to all basic media and cultured, the cell proliferation rate increased compared to the culture medium containing 10% (v/v) fetal bovine serum, so it was confirmed that peptone was more effective in cell proliferation than fetal bovine serum.

Example 3. Cell proliferation in medium containing yeast extract

In order to determine whether yeast extract can be used as an ingredient that can replace fetal bovine serum, which has a cell proliferation effect in the culture of animal cells, any changes in cell proliferation were examined when yeast extract was added. I checked to see if it was visible. It was confirmed through the same method as Example 2, except that yeast extract was used instead of peptone. The results are shown in Figures 5 to 7.

As shown in Figures 5 to 7, the highest cell proliferation rate was observed when Grace's insect media was used as a base medium and yeast extract was added. From the above results, it was confirmed that adding yeast extract to the medium was more effective in cell proliferation than medium adding fetal bovine serum.

Example 4. Measurement of amino acid consumption during cell culture and cell proliferation in medium supplemented with amino acid mixture

4.1. Measurement of Amino Acid Consumption

To confirm that the culture medium containing yeast extract added to Grace's insect media was effective in cell proliferation, and to find out how to further increase the cell proliferation rate, the consumption of amino acids during the cell culture process was measured using ultra performance liquid chromatography (UPCL). It was measured through. Specifically, shrimp cells were cultured in a culture medium containing 0.6% (w/v) yeast extract in Grace's insect media, and then culture fluids were collected at 3, 6, 10, 12, 24, and 48 hours after cell culture to determine amino acids. Consumption was measured. The results are shown in Figure 8.

As shown in Figure 8, it was confirmed that a total of 9 amino acids, including Serine, Aspartic acid, glutamic acid, alanine, glutamine, proline, tyrosine, valine, and leucine, were consumed by more than 50% within 12 hours.

4.2. Cell proliferation in medium supplemented with amino acid mixture

Assuming that amino acids that are consumed by more than 50% within 12 hours are used in the early stages of cell proliferation and have a close influence on the cell proliferation rate, a mixture of 9 amino acids that are consumed first was added to Grace's insect media containing yeast extract or peptone. Cell proliferation was observed by adding the amino acid mixture. Specifically, Alanine 225mg/L, Aspartic Acid 350mg/L, Glutamic Acid 600mg/L, Glutamine 600mg/L, Leucine 75mg/L, Proline 350mg/L, Serin 1100mg/L, Tyrosine*2Na 75mg/L, Valine 100mg/L. An amino acid mixture was added to achieve a concentration of L, and cell proliferation was observed. The results are shown in Figures 9 to 14.

As shown in Figures 9 to 14, significantly faster cell proliferation was observed in the culture medium with the addition of 9 amino acids than in the culture medium without the addition of 9 amino acids 24 hours after the start of cell culture, and at the start of cell culture Even at 48 hours, it was confirmed that a greater amount of cells proliferated in the culture medium containing the amino acid mixture than in the cell culture medium without the addition of the nine types of amino acids. From the above results, it was found that the addition of amino acids with high initial consumption during cell culture increased the cell proliferation rate and amount.

Example 5. Cell proliferation in medium supplemented with trace elements

Yeast extract mainly consists of amino acids, vitamins, and trace elements. Trace elements refer to substances that are required in very small amounts among the substances essential for the growth of living things, and are essential chemical elements that play an important role in the growth, development and physiology of living things. This includes most heavy metals such as mercury, cadmium, and iodine.

In order to identify substances that can promote cell proliferation in addition to the 9 types of amino acids, the amount of trace elements contained in the yeast extract was analyzed and additional trace elements were added to the culture medium containing the yeast extract or peptone (Grace's insect media + yeast extract + trace elements or Grace's insect media + peptone + trace elements) and a cell proliferation experiment was performed to determine what effect it had on cell proliferation. Specifically, trace elements were added at a concentration of Calcium 5.28mg/L, Magnesium 16.2mg/L, Potassium 258mg/L, Sodium 50.4mg/L, Zinc 0.084mg/L, Copper 0.0414mg/L, and Manganese 0.021mg/L. and observed cell proliferation. The results are shown in Figures 15 to 20.

As shown in Figures 15 to 20, when trace elements were added to the shrimp cell culture medium, it was confirmed that the cell proliferation rate and final proliferation amount increased compared to the control group to which no trace elements were added, and like shrimp cells, crab, Even when trace elements were added to the lobster cell culture medium, it was confirmed that the cell proliferation rate and final proliferation amount increased compared to the control group where trace elements were not added. From the above results, it was confirmed that adding trace elements to the crustacean cell culture medium helps increase the cell proliferation rate and final proliferation amount.

Example 6. Cell proliferation depending on the presence or absence of vitamins in the basic medium

Yeast extract is an extract obtained by extracting the water-soluble components of yeast and removing moisture. It contains amino acids, peptides, carbohydrates, organic acids, vitamins, and minerals. Similar to the components of yeast extract, the basic medium used during cell culture is composed of amino acids, sugars, organic acids, vitamins, organic acids, and minerals. Although the components are similar to those of yeast extract, there is a difference in content.

Yeast extract was added to a basic medium containing or not containing vitamins and the cells were cultured to see if there was a difference in cell proliferation depending on the presence or absence of vitamins contained in the basic medium, and whether the vitamin components of the basic medium were added to the yeast extract. I looked into whether it could be replaced with vitamins.

Specifically, a medium composition in which yeast extract was added at a concentration of 0.6% (w/v) to a base medium (Grace's insect media, serum free) excluding vitamin components, and a yeast extract was added to the base medium at a concentration of 0.6% (w/v). Cell proliferation was compared by preparing medium compositions added at a w/v) concentration. Crustacean cells were initially inoculated at ^1.5 Confirmed. The results are shown in Figure 21.

As shown in Figure 21, all crustacean cells showed no difference in cell proliferation depending on the presence or absence of vitamins. Through the above results, it was confirmed that only the vitamin components contained in the yeast extract are effective in proliferation of crustacean cells. Therefore, it was experimentally confirmed that the vitamin components of the yeast extract can replace the vitamin components of the basic culture medium (Grace's insect media). .

Example 7. Cell proliferation depending on the presence or absence of organic acids in the basic medium

Yeast extract was added to a basic medium containing or not containing organic acids and the cells were cultured to determine whether there was a difference in cell proliferation depending on the presence or absence of organic acids contained in the basic medium, and whether the organic acid component of the basic medium was added to the yeast extract. I looked into whether it could be replaced with an organic acid.

Specifically, a medium composition containing yeast extract added at a concentration of 0.6% (w/v) to a base medium (Grace's insect media, serum free) and removal of Fumaric acid, Malic acid and Succinic acid from the base medium. And we observed how crustacean cell proliferation changes in a medium composition containing yeast extract at a concentration of 0.6% (w/v). Cell proliferation was compared in the same manner as in Example 6, and the results are shown in Figure 22.

As shown in Figure 22, it was confirmed that the medium composition without the addition of fumaric acid, malic acid, and succinic acid was more effective for cell proliferation than the medium composition with the addition of fumaric acid, malic acid, and succinic acid. Fumaric acid, malic acid, and succinic acid rather promoted crustacean cell proliferation. It was confirmed that it inhibits . Through the above results, it was concluded that the organic acid component contained in the yeast extract alone was effective in cell proliferation, and that the organic acid added as a component of the basic medium actually inhibited cell proliferation.

Example 8. Cell proliferation depending on the presence or absence of vitamins and organic acids in the basic medium

Yeast extract was added to a basic medium containing or not containing vitamins and organic acids and the cells were cultured to determine whether there was a difference in cell proliferation depending on the presence or absence of vitamins and organic acids contained in the basic medium. We investigated whether it could be replaced with the vitamins and organic acids contained in yeast extract.

Specifically, a medium composition in which yeast extract was added at a concentration of 0.6% (w/v) to a base medium (Grace's insect media, serum free), vitamins and organic acids were removed from the base medium, and yeast extract was added to the base medium at a concentration of 0.6% (w/v). A medium composition added at a % (w/v) concentration was prepared and crustacean cells were cultured to confirm cell proliferation. Cell proliferation was compared in the same manner as in Example 6, and the results are shown in Figure 23.

As shown in Figure 23, cells cultured in a medium composition without adding vitamins and organic acids showed faster and higher proliferation than cells cultured in a medium composition with vitamins and organic acids added, and as in the results of Example 4, organic acids It was confirmed that cell proliferation was inhibited.

Taking the above results together, the vitamins contained in the yeast extract can replace the vitamins contained in the basic medium, and if the organic acid components contained in the basic medium are added in addition to the organic acids in the yeast extract, cell proliferation is inhibited. It was confirmed that excluding the vitamins and organic acids contained in the basic medium was effective for crustacean cell proliferation.

Example 9. Isolation of cells from shrimp and establishment of cell lines

Live shrimp were stunned by placing them on ice for 15 minutes before sacrificing them. The body segments were washed with a brush under running tap water and then sterilized by immersing them in electrolytic sodium hypochlorite (NaOCl) three times for 5 minutes. After washing with sterilized distilled water, the head and tail were removed. After breaking the segments of the body, the internal organs were removed by piercing between the segments with a pin, and then all of the skin was removed. It was sterilized by immersing it in electrolytic sodium hypochlorite water for 5 minutes and then washed with sterilized distilled water. The body tissue sections were washed with PBS (200 mg/L KCl, 200 mg/L KH2PO4, 8000 mg/L NaCl, 2160 mg/L Na2HPO4, pH 7.3), and the layers of fat and epidermal tissue were removed.

The muscle tissue of the body was chopped into pieces less than 5 mm in size. Finely chopped muscle tissues were placed in a syringe and a mixing tube was connected. A syringe containing 10 mL of 0.2% collagenase (collagenase type II) preheated at 28°C was inserted on the other side of the mixing tube. Mix gently for 5 minutes until the feeling of foreign matter disappeared. The sufficiently mixed tissue was placed in a 50 mL conical tube. 10 mL of culture medium containing fetal bovine serum (FBS) was added and gently pipetted. After centrifugation at 800 g for 5 minutes, the supernatant was removed and suspended in PBS. Cells were separated from large tissue masses by sequentially filtering using 1000, 100, 70, and 40 μm cell strainers. After centrifugation at 800 g for 5 minutes, the supernatant was removed.

Serum-free medium containing 3% Penicillin/streptomycin, 3% Gentamicin, 3% ZellShield, 1.5% Neomycin, and 0.3% Bacitracin 10 After suspending the cells in mL, 90 mL was added and placed in a 500 mL Erlenmeyer flask. It was placed in a shaking incubator, rotated at 300 RPM, and cultured at 28°C for 7 days.

After 7 days, centrifugation was performed at 800 g for 5 minutes, the supernatant was removed, and explant tissues were removed by washing once with PBS. After centrifugation at 800g for 5 minutes, remove the supernatant and suspend the cells in 10 mL of serum-free medium containing 3% penicillin-streptomycin, 3% gentamicin, 3% Gelshield, 1.5% neomycin, and 0.3% bacitracin. Then, 90 mL was added into a 500 mL Erlenmeyer flask, rotated at 300 RPM in a shaking incubator, and cultured repeatedly at 28°C. At 14 days after cell culture, 1.5x10 ⁷ cells were transferred to an Erlenmeyer flask containing 100 mL of medium containing 0.3% bacitracin to dilute the remaining explant tissue and allow only the cells to proliferate. When the cell density reached 80 to 90%, they were passaged at a 1:3 ratio. Cells were observed under a microscope at 1, 7, 14, and 21 days after repeated culture, and the results are shown in Figure 26. As a result, proliferation of cells with a size of 3 to 10 μm could be observed 7 to 14 days after cell culture. Cell lines were established through subculture for 3 weeks as described above. After centrifuging the cells at 800g for 5 minutes, the supernatant was removed, resuspended in 1ml Cellbanker2 freezing medium (Cellbanker2) at ^1x109 cells/mL, placed in a low-temperature tube, and stored at -80°C for one day, then the next day. It was transferred to a liquid nitrogen tank.

Schematic diagrams of the process of cell isolation and cell line establishment described above are shown in Figures 24 and 25.

Example 10. Isolation of cells from crabs and lobsters and establishment of cell lines

Live crabs and lobsters were stunned by placing them on ice for 15 minutes before sacrificing them. The body segments were washed with a brush under running tap water and then sterilized by immersing them in electrolytic sodium hypochlorite (NaOCl) three times for 5 minutes. The front legs of each individual were separated and the skin was removed. Muscle tissue was collected, sterilized by immersing it in electrolytic sodium hypochlorite water for 5 minutes, and then washed with sterilized distilled water. The forelimb tissue sections were washed with PBS (200 mg/L KCl, 200 mg/L KH2PO4, 8000 mg/L NaCl, 2160 mg/L Na2HPO4, pH 7.3), and then the muscle tissue was chopped into pieces of 5 mm or less. A cell line was established through the same process as in Example 9, starting with putting muscle tissue in a mixing tube and treating it with 0.2% collagenase (collagenase type II).

Example 11. Analysis of cell growth according to culture conditions

As a previously known method of isolating and culturing cells from crustaceans, the static culture method was mainly used. In order to determine whether there is a difference in cell growth depending on the culture method, when performing suspension culture and when performing stationary culture using the shrimp, crab, and lobster cell lines obtained in Examples 9 and 10, the cells Proliferation was confirmed and compared. Specifically, for suspension culture, cells were placed in a 100ml Erlenmeyer flask and cultured in a shaking incubator (28℃250 RPM). For stationary culture, cells were placed in a T75 flask and cultured in an incubator (28°C. DMEM/F12, L15 or Grace's insect medium was used as a basic medium, and FBS, fetal bovine serum, was added at a concentration of 0, 5, and 10% to incubate the cells. Growth was confirmed. Cells were inoculated at a cell density of ^1.5 Measurements were made, and the number of cells was directly counted and confirmed using a hemocytometer. The results are shown in Figures 27 to 29.

As shown in Figures 27 to 29, it was confirmed through OD600 and cell counting results that the number of cells increased over time in the culture of shrimp, crab, and lobster cells for up to 48 hours, and as the concentration of FBS increased, the number of cells increased. It was confirmed that the proliferation rate of cells increased.

In comparing cell growth rates between suspension culture and stationary culture methods, as culture time passed, suspension culture showed higher cell growth than stationary culture. In particular, when suspension culture was performed in Grace's insect medium containing 10% FBS, the cell number was found to be the highest, and the cell number increased more than 10 times compared to static culture under the same conditions.

From the above results, it was confirmed that differences in crustacean cell growth rates can occur due to differences in basic media, and it was confirmed that among the three basic media compared, crustacean cells had the highest growth rate in Grace's insect medium. In addition, it was confirmed that cells proliferate even in the absence of fetal bovine serum, but when fetal bovine serum is added, the cell growth rate increases. In addition, it was found that culturing cells in suspension using the previously known static culture method can increase the growth rate of crustacean cells.

Example 12. Confirmation of cell line establishment

It was confirmed whether cells isolated from shrimp, crab, and lobster were established as cell lines with continuous growth. Specifically, as in Examples 9 and 10, three types of cells were isolated from the individual and established through subculture for 3 weeks (early cells) and cells cultured for an additional month thereafter (late cells) )'s growth curve was confirmed. Cells were inoculated into a ^100ml Erlenmeyer flask at a cell density of 1.5 During culture, the number of cells was measured by counting them using a hemocytometer. The results are shown in Figure 30.

As shown in Figure 30, it was confirmed that there was no difference in cell growth between early cells and late cells. Additionally, as a result of observing the cells at each time, it was confirmed that the cells had similar shapes.

It was confirmed whether the established cell line maintained growth even after long-term storage at ultra-low temperature (-196°C). Specifically, shrimp, crab, and lobster cells stored in liquid nitrogen for 1, 3, and 12 months were grown at a cell density of 1.5X10 ⁵ cells/ml ( A total of 10ml) was inoculated into a 100ml Erlenmeyer flask and cultured in suspension in a shaking incubator (28℃250 RPM) for 6, 24, 48, 72, and 96 hours. During incubation, the number of cells was counted using a hemocytometer. Measurements were made and the results are shown in Figure 31.

As shown in Figure 31, it was confirmed that there was little difference in cell growth between cells stored for 1, 3, and 12 months. In addition, it was confirmed that the cell shapes were almost similar between groups.

From the above results, the crustacean cells of shrimp, crab, and lobster obtained according to Examples 9 and 10 show continuous growth without change in their characteristics even when cultured for a long period of time, and cell growth is maintained even after long-term storage, establishing them as a cell line. I could see that it had happened.

Claims (20)

1. A medium composition for animal cell culture containing yeast extract or peptone.
2. A medium composition for animal cell culture comprising a basic medium and yeast extract.
3. According to claim 1 or 2,

   A medium composition containing 0.01 to 10% (w/v) of the yeast extract or peptone.
4. According to claim 1 or 2,

   A medium composition wherein the animal cells are crustacean cells.
5. According to claim 1 or 2,

   A medium composition wherein the animal cells are shrimp, crab or lobster derived cells.
6. According to claim 1 or 2,

   A medium composition wherein the medium is a serum-free medium.
7. According to claim 1 or 2,

   A medium composition wherein the culture is a suspension culture.
8. According to paragraph 2,

   The basic medium contains biotin, D-Ca pantothenate, a-ketoglutaric acid, choline chloride, folic acid, i- A group consisting of inositol, nicotinic acid, pyridoxine-HCl, riboflavin, Thiamine-HCl, and penta-Amino benzoic acid. A medium composition that does not contain one or more vitamins selected from.
9. According to paragraph 2,

   A medium composition wherein the basic medium does not contain vitamins.
10. According to paragraph 2,

    A medium composition wherein the basic medium does not contain one or more organic acids selected from the group consisting of fumaric acid, malic acid, and succinic acid.
11. According to paragraph 2,

    A medium composition wherein the basic medium does not contain organic acids.
12. A method of cultivating animal cells, comprising culturing cells in a medium composition for culturing animal cells containing yeast extract or peptone.
13. A method of cultivating animal cells, comprising culturing cells in a medium composition for culturing animal cells containing a basic medium and yeast extract.
14. isolating cells from non-human animals; and

    Suspension culturing of the separated cells

    Method for producing cell lines, including.
15. According to clause 14,

    The method of claim 1, wherein the non-human animal is a cow, sheep, pig, poultry, crustacean, or fish.
16. According to clause 15,

    A method wherein the crustacean is shrimp, crab or lobster.
17. According to clause 14,

    A method wherein the suspension culture is performed for 1 to 42 days.
18. isolating cells from non-human animals; and

    Suspension culturing of the separated cells

    A cell line manufactured according to a cell line manufacturing method comprising.
19. A cell line culture method comprising the step of performing suspension culture to proliferate the cell line of claim 18.
20. isolating cells from non-human animals; and

    Suspension culturing of the separated cells

    Cultured meat containing a cell line manufactured according to a cell line manufacturing method comprising.

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