WO2024039216A1 - Procédé de culture de cellules animales ou d'une lignée de cellules animales - Google Patents

Procédé de culture de cellules animales ou d'une lignée de cellules animales Download PDF

Info

Publication number
WO2024039216A1
WO2024039216A1 PCT/KR2023/012255 KR2023012255W WO2024039216A1 WO 2024039216 A1 WO2024039216 A1 WO 2024039216A1 KR 2023012255 W KR2023012255 W KR 2023012255W WO 2024039216 A1 WO2024039216 A1 WO 2024039216A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
medium
animal
cell
culture
Prior art date
Application number
PCT/KR2023/012255
Other languages
English (en)
Korean (ko)
Inventor
박길준
김희정
이민수
차지민
최하연
박수용
하수빈
이슬기
Original Assignee
셀미트주식회사
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020220121026A external-priority patent/KR102636615B1/ko
Priority claimed from KR1020220121025A external-priority patent/KR102626827B1/ko
Application filed by 셀미트주식회사 filed Critical 셀미트주식회사
Publication of WO2024039216A1 publication Critical patent/WO2024039216A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • 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.
  • 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.
  • FBS fetal bovine serum
  • 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.
  • cultured meat 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • soy peptone refers to a decomposition product of protein obtained from plants.
  • 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.
  • 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.
  • 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.
  • the yeast extract may be usable in food.
  • Yeast extract is known to contain minerals, vitamins, nucleic acids, peptides, and amino acids.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the animal cells may refer to animal-derived cells made by isolating cells from animal tissues and culturing them in vitro.
  • the animal cells may be crustacean cells.
  • the animal cells may be cells derived from shrimp, crab, or lobster.
  • culture media refers to a material that supports the growth and survival of cells, including stem cells, in vitro.
  • 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.
  • 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), ⁇ -MEM ( ⁇ -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.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Base Medium Eagle
  • any medium used in the industry is sufficient, and the medium may contain amino acids, vitamins, inorganic salts, and other ingredients.
  • non-essential amino acids and L-glutamine vitamins include vitamin B12
  • inorganic salts include trace components (CuSO45H2O, Fe(NO3)39H2O, ZnSO4), phosphoenol pyruvate, and monoethanol.
  • Amines Momoethanolamine
  • other ingredients include D-glucose, Linoleic acid, Lipoic acid and Sodium pyruvate
  • hypoxantine sodium putrescine hydrochloride (Putrescine HCl), and polyamine solution.
  • the medium may be a serum-free medium.
  • 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.
  • 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).
  • mammals e.g., pigs, horses, cows, goats, sheep, and dogs
  • FBS fetal bovine serum. serum
  • 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.
  • the culture may be a suspension culture.
  • cell culture refers to the process of artificially growing living cells in vitro under controlled conditions.
  • 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.
  • suspension culture refers to culturing cells to be cultured in a floating state in a culture medium without being fixed to a substrate.
  • SR serum replacement
  • the culture 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 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.
  • 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.
  • 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).
  • cell surface labeling e.g., staining cells with tissue-specific or cell-label specific antibodies
  • changes in cell morphology e.g., using techniques such as flow cytometry or immunocytochemistry.
  • PCR polymerase chain reaction
  • the medium composition may further include an amino acid mixture or trace elements.
  • amino acids included in the amino acid mixture 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.
  • 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.
  • 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): Va
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the basic medium may not contain vitamins.
  • the basic medium may not contain one or more organic acids selected from the group consisting of fumaric acid, malic acid, and succinic acid.
  • the basic medium may not contain organic acids.
  • 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.
  • the method for producing the cell line may not include the step of treating biological or chemical exogenous immortalization factors.
  • 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.
  • 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.
  • 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.
  • the method for producing the cell line may not include the step of treating albumin or exogenous albumin.
  • 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.
  • the non-human animal can be cattle, sheep, pigs, poultry, crustaceans, or fish.
  • the crustacean may be shrimp, crab, or lobster.
  • 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.
  • 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.
  • 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.
  • the suspension culture may be performed in a serum-free medium.
  • the suspension culture may be performed in a medium containing serum.
  • suspension culture refers to culturing cells to be cultured in a floating state in a culture medium without being fixed to a substrate.
  • SR serum replacement
  • the culture 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.
  • 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), ⁇ -MEM ( ⁇ -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.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential
  • any medium used in the industry is sufficient, and the medium may contain amino acids, vitamins, inorganic salts, and other ingredients.
  • non-essential amino acids and L-glutamine vitamins include vitamin B12
  • inorganic salts include trace components (CuSO45H2O, Fe(NO3)39H2O, ZnSO4), phosphoenol pyruvate, and monoethanol.
  • Amines Momoethanolamine
  • other ingredients include D-glucose, Linoleic acid, Lipoic acid and Sodium pyruvate
  • hypoxantine sodium putrescine hydrochloride (Putrescine HCl), and polyamine solution.
  • 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.
  • 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.
  • 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).
  • mammals e.g., pigs, horses, cows, goats, sheep, and dogs
  • FBS fetal bovine serum. serum
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m 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.
  • fat in liquid form 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.
  • 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.
  • FD&C Blue No. 1 brilliant blue FCF
  • FD&C Blue No. 2 ingotine
  • cochineal extract, curcumin, lutein, carotenoids, monascin, paprika, riboflavin, saffron, turmeric, and combinations thereof can be used, but are not particularly limited thereto.
  • 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.
  • 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.
  • a medium composition for culturing animal cells containing yeast extract or peptone 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.
  • 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;
  • 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.
  • a and/or B means A or B, or A and B.
  • 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.
  • DMEM-F12, L15 or Grace's insect media commonly used basic media
  • FBS Fetal Bovine Serum
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a base medium Gibce's insect media, serum free
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • electrolytic sodium hypochlorite NaOCl
  • 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.
  • PBS 200 mg/L KCl, 200 mg/L KH2PO4, 8000 mg/L NaCl, 2160 mg/L Na2HPO4, pH 7.3
  • 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 ⁇ m cell strainers. After centrifugation at 800 g for 5 minutes, the supernatant was removed.
  • FBS fetal bovine serum
  • 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.
  • 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.
  • Cellbanker2 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.
  • 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.
  • electrolytic sodium hypochlorite NaOCl
  • 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).
  • the static culture method was mainly used.
  • 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°C250 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.
  • suspension culture showed higher cell growth than stationary culture.
  • 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.
  • 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.
  • culturing cells in suspension using the previously known static culture method can increase the growth rate of crustacean cells.
  • 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.

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne : une composition de milieu de culture de cellules animales ; un procédé de culture de cellules animales l'utilisant ; un procédé de préparation d'une lignée cellulaire animale non humaine ; une lignée cellulaire préparée à l'aide de celle-ci ; et un procédé de culture de la lignée cellulaire. Une composition de milieu de culture de cellules animales contenant un extrait de levure ou une peptone selon un aspect présente les effets de permettre la croissance et la prolifération de cellules animales même dans un milieu sans sérum, et de favoriser la croissance et la prolifération de cellules animales. Une composition de milieu de culture de cellules animales contenant un milieu basique et un extrait de levure selon un aspect présente les effets de permettre la prolifération efficace de cellules même dans un milieu basique ne contenant pas de vitamines et d'acides organiques, et de réduire le prix d'un milieu de culture cellulaire en minimisant les composants du milieu de culture. La présente invention concerne également un procédé de préparation d'une lignée cellulaire selon un aspect, le procédé comprenant une étape de séparation des cellules d'animaux non humains et une étape de culture en suspension des cellules séparées, et présente l'avantage de permettre l'obtention efficace d'une lignée cellulaire à partir d'animaux non humains.
PCT/KR2023/012255 2022-08-19 2023-08-18 Procédé de culture de cellules animales ou d'une lignée de cellules animales WO2024039216A1 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
KR10-2022-0104288 2022-08-19
KR10-2022-0104306 2022-08-19
KR20220104306 2022-08-19
KR20220104288 2022-08-19
KR10-2022-0121025 2022-09-23
KR1020220121026A KR102636615B1 (ko) 2022-08-19 2022-09-23 비-인간 동물 세포주의 제조방법, 이에 따라 제조된 세포주 및 세포주의 배양방법
KR1020220121025A KR102626827B1 (ko) 2022-08-19 2022-09-23 동물세포 배양용 배지 조성물 및 이를 이용한 동물세포의 배양방법
KR10-2022-0121026 2022-09-23
KR1020230020437A KR102608766B1 (ko) 2022-08-19 2023-02-16 동물세포 배양용 배지 조성물 및 이를 이용한 동물세포의 배양방법
KR10-2023-0020437 2023-02-16

Publications (1)

Publication Number Publication Date
WO2024039216A1 true WO2024039216A1 (fr) 2024-02-22

Family

ID=89942027

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2023/012255 WO2024039216A1 (fr) 2022-08-19 2023-08-18 Procédé de culture de cellules animales ou d'une lignée de cellules animales

Country Status (1)

Country Link
WO (1) WO2024039216A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070073930A (ko) * 2004-10-29 2007-07-10 백스터 인터내셔널 인코포레이티드 세포 배양용의 동물성 단백질이 없는 배지
KR101242090B1 (ko) * 2003-03-03 2013-03-08 글락소스미스클라인 바이오로지칼즈 에스.에이. 비동물성 세포 배양법
KR20210090632A (ko) * 2018-11-15 2021-07-20 알레프 팜스 리미티드 고품질 배양육, 이를 생산하기 위한 조성물 및 방법
KR20210116551A (ko) * 2019-01-15 2021-09-27 시오크 미트 피티이. 엘티디. 갑각류로부터의 근육 및 지방 세포들의 단리와 배양
KR102420229B1 (ko) * 2021-12-07 2022-07-14 셀미트주식회사 바이오 셀룰로오스를 포함하는 배양육 제조용 스캐폴드 및 이의 제조 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101242090B1 (ko) * 2003-03-03 2013-03-08 글락소스미스클라인 바이오로지칼즈 에스.에이. 비동물성 세포 배양법
KR20070073930A (ko) * 2004-10-29 2007-07-10 백스터 인터내셔널 인코포레이티드 세포 배양용의 동물성 단백질이 없는 배지
KR20210090632A (ko) * 2018-11-15 2021-07-20 알레프 팜스 리미티드 고품질 배양육, 이를 생산하기 위한 조성물 및 방법
KR20210116551A (ko) * 2019-01-15 2021-09-27 시오크 미트 피티이. 엘티디. 갑각류로부터의 근육 및 지방 세포들의 단리와 배양
KR102420229B1 (ko) * 2021-12-07 2022-07-14 셀미트주식회사 바이오 셀룰로오스를 포함하는 배양육 제조용 스캐폴드 및 이의 제조 방법

Similar Documents

Publication Publication Date Title
Rubio et al. Cell-based fish: a novel approach to seafood production and an opportunity for cellular agriculture
Mummery et al. Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells
WO2013009100A2 (fr) Procédé de fabrication d'un extrait de cordon ombilical et son utilisation
Fraser et al. Studies on primary cell cultures derived from ovarian tissue of Penaeus monodon
WO2017213469A2 (fr) Milieu de culture cellulaire contenant un extrait de spiruline (algue bleue-verte), son procédé de préparation et procédé de culture cellulaire l'utilisant
Lemos et al. Ontogenetic variation in digestive proteinase activity, RNA and DNA content of larval and postlarval white shrimp Litopenaeus schmitti
WO2024039216A1 (fr) Procédé de culture de cellules animales ou d'une lignée de cellules animales
Li et al. Characterization of a primary cell culture from lymphoid organ of Litopenaeus vannamei and use for studies on WSSV replication
Lakra et al. A new fibroblast like cell line from the fry of golden mahseer Tor putitora (Ham)
CA3206832A1 (fr) Procedes et processus de culture de cellules
WO2022211461A1 (fr) Composition de milieu pour la culture de cellules souches musculaires comprenant de la curcumine longa, de la glysine ou de l'insuline pour la prolifération de cellules souches musculaires
Azhar et al. Cell-based meat: The molecular aspect
KR102626827B1 (ko) 동물세포 배양용 배지 조성물 및 이를 이용한 동물세포의 배양방법
EP4321609A1 (fr) Composition pour culture cellulaire comprenant un extrait de chlorella
KR102636615B1 (ko) 비-인간 동물 세포주의 제조방법, 이에 따라 제조된 세포주 및 세포주의 배양방법
Chotikachinda et al. Tuna viscera hydrolysate products prepared by different enzyme preparations improve the feed intake and growth of Asian seabass, Lates calcarifer, fed total fishmeal replacement diets.
Lin et al. The anti-inflammatory effect of lutein in broilers is mediated by regulating Toll-like receptor 4/myeloid-differentiation-factor 88 signaling pathway
Fyhn et al. Tissue cultures of cirripeds
Greani et al. Ultrastructural study of vitellogenesis and oogenesis of Metadena depressa (Stossich, 1883) Linton, 1910 (Digenea, Cryptogonimidae), intestinal parasite of Dentex dentex (Pisces, Teleostei)
KR102280199B1 (ko) 중간엽 줄기세포의 면역조절 활성 증진용 조성물
WO2024128564A1 (fr) Composition de culture cellulaire contenant un extrait de microalgues et son utilisation
KR20220135214A (ko) 근육줄기세포 증식을 위한 강황을 포함하는 근육줄기세포 배양용 배지 조성물
Iwanaga In vitro cultivation of cells from ovotestis tissue of pigmented Biomphalaria glabrata
Vega-Villasante et al. Biochemical characterization of crude extract from Pleuroncodes planipes (Crustacea: Galatheidae) as potential feed additive, considerations for a new fishery along the Mexico Pacific coast
Lester Primary cell cultures from Cod (Gadus morhua) and Atlantic Halibut (Hippoglossus hippoglossus).

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23855182

Country of ref document: EP

Kind code of ref document: A1