CN112210525A - Serum-free culture system and application thereof in cell culture of meat - Google Patents
Serum-free culture system and application thereof in cell culture of meat Download PDFInfo
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- 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
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
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- 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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
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- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12N2500/30—Organic components
- C12N2500/36—Lipids
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- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
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Abstract
The invention discloses a serum-free culture system and application thereof in cell culture of meat, belonging to the technical field of biological engineering. The invention can reduce the dependence of the animal cell culture process on serum and reduce the influence of uncertain factors in the serum on the animal cell culture safety. In addition, the exogenous additive components except the basic culture medium are all synthesized completely, so that the cost of the serum-free culture medium can be effectively reduced, and the large-scale application of the technology is facilitated.
Description
Technical Field
The invention relates to a serum-free culture system and application thereof in cell culture of meat, belonging to the technical field of biological engineering.
Background
Animal serum is used in the traditional cell culture stage to provide nutrients and biological factors required by cell adhesion, proliferation and growth maintenance, and the possibility of contaminating exogenous microorganisms and pathogenic factors exists in the process of carrying, collecting and processing animal serum used in batches from natural organisms, so that the biological safety risk exists. In addition, due to factors such as complex serum components, poor batch-to-batch stability, limited supply and high purification difficulty, a serum-containing culture system cannot realize large-scale industrial application.
The serum-free culture medium is a synthetic culture medium which can maintain cells to grow and proliferate in vitro for a long time without adding serum, and has the advantages of definite artificial synthetic components, stable product batches, high cell culture repeatability, reduction of the risk of microbial contamination such as internal and external viruses, bacteria, mycoplasma and the like caused by using animal serum, sufficient supply guarantee in industrial production and contribution to downstream purification of biological products.
Serum-free media are chemically defined serum-free media that are completely serum-free, protein-free, or very low protein-containing media. Compared with the traditional animal serum culture medium, the culture medium has incomparable superiority and safety, has definite components, reduces the cost of a serum-free culture system because all the components of the supplement culture medium are chemical or biological synthesis, is applied to the production process of animal cell culture meat, and is easy to simplify the purification and downstream processing flow of biological products. At present, serum-free culture media are reported, but generally applied to the fields of medicine and pharmacy, have higher cost and are not suitable for large-scale culture, so that the serum-free culture system which is low in cost, safe and suitable for large-scale industrial application has important value for cell culture.
In recent years, cell culture meat analogue (cut meat) attracts more extensive attention due to the characteristics that the source of the meat analogue is traceable, the meat analogue is green and safe, the taste of the meat analogue is closer to that of traditional meat and the like. Researchers extract animal stem cells or tissues capable of being efficiently proliferated and put the animal stem cells or tissues into a culture dish for breeding, and then differentiate the animal stem cells or tissues into original fibers of muscle tissues. The cell culture meat does not consume feed and water in the production process, does not need garbage waste treatment, meets the requirements of human beings on meat, and simultaneously solves the social environment problems brought by the traditional breeding industry, including hormone-free antibiotics, environmental sustainability, whether animals are treated morally or not and the like. The cell culture meat is closer to real meat products in the aspects of nutrition, mouthfeel and flavor, and is the main research and development direction of artificial meat in the future, but a plurality of challenges exist in the aspects of theory and technology, particularly the aspects of low-cost acquisition and food conversion of large-scale muscle cells. The problem that cells cultured in vitro in large scale are polluted by microorganisms or affected by self metabolites is solved, so that metabolic wastes generated by the cells need to be removed in time during in vitro cell culture, and a sterile and nontoxic living environment is provided for the in vitro cultured cells. The traditional cell culture stage uses animal serum to provide nutrients and biological factors required by cell adherence, proliferation and differentiation, and often needs to add antibiotics or antimitotic agents, which cannot meet the requirement of food safety. Therefore, how to optimize the culture conditions is an important influencing factor for realizing safe and large-scale in vitro animal cell tissue culture. With the development of animal cell serum-free culture technology, serum-free culture has been widely applied in the fields of cell biology, pharmacology, oncology and cell engineering, but still has the problems of high cost and low safety.
Disclosure of Invention
The first purpose of the invention is to provide a serum-free culture system, which comprises a basic culture medium and an externally added supplementary component, wherein the basic culture medium comprises the following components: 125-135 mM NaCl, 2.0-4.0 mM KCl, 4.0-6.0 mM D-glucose, NaHCO3 10～14mM，NaH2PO4 0.3～0.7mM，MgCl2 0.5～1.5mM，CaCl21.0-3.0 mM, and 0.3-0.7 mM of butanediamine hydrochloride; the contained exogenous additive supplementary components comprise the following components: 100-300 mM of cell growth factor, 2.0-6.0 mM of phospholipid growth factor, 0.08-1.02 g/L of transferrin, 0.3-0.7 mg/L of lipid, 4-6 g/L of amino acid, 4-6 mM of biotin, 124-6 mM of vitamin B, 68-12 mM of vitamin B and 0.1-0.3 mM of vitamin C.
In one embodiment of the invention, the basal medium comprises the following components: 130mM NaCl, 3.0mM KCl, 5.0mM D-glucose, NaHCO3 12mM，NaH2PO4 0.5mM，MgCl2 1.0mM，CaCl22.0mM, and 0.5mM of butanediamine hydrochloride; the contained exogenous additive supplementary components comprise the following components: cell growth130mM of factor, 3.0mM of phospholipid growth factor, 0.1g/L of transferrin, 0.5mg/L of lipid, 5g/L of amino acid, 5mM of biotin, 125 mM of vitamin B125 mM, 610 mM of vitamin B610 mM and 0.2mM of vitamin C.
In one embodiment of the invention, the cell growth factor, lipid, amino acid or vitamin is produced by microbial fermentation or whole cell transformation methods.
The second purpose of the invention is to provide the application of the serum-free culture system in culturing animal stem cells, animal muscle cells or animal fat cells.
In one embodiment of the invention, the animal stem cell comprises an embryonic stem cell, a myoblast, a mesenchymal stem cell or an adipogenic stem cell.
In one embodiment of the present invention, the amount of cell inoculation is 10 in the culture3-105one/mL serum-free culture system.
In one embodiment of the present invention, the culturing is at 19-24 ℃ for 40-100 h.
In one embodiment of the present invention, the culturing is at 19-24 ℃ for 40-50 h.
The third purpose of the invention is to provide a method for preparing animal cell culture meat, which is to inoculate animal stem cells into the serum-free culture system for culture.
The fourth purpose of the invention is to provide the application of the serum-free culture system in the food field.
The invention has the beneficial effects that:
the serum-free culture medium provided by the invention is an artificially synthesized culture medium, does not need to add exogenous animal serum, is chemically synthesized or biologically synthesized from all exogenous additive components, has definite components, and can effectively avoid pollution caused by the animal serum in the cell culture process. The present invention provides a defined serum-free culture system for culturing animal stem cells, in particular animal embryonic stem cells, muscle stem cells and the like. In addition, the invention also provides a corresponding animal cell tissue culture method, which comprises the incubation of the stem cells and the muscle cell differentiation in a serum-free culture system and is used for producing the artificial meat. The method can effectively reduce the problems of high cost and the like of the traditional serum-containing culture medium.
Detailed Description
(one) obtaining of an exogenously added supplemental ingredient
Microbial recombinant expression of cell growth factor and phospholipid growth factor: growth factor protein (GF) was expressed using the eukaryotic expression vector pGAPZ. alpha.A. The full-length Gene sequences of different animal cell growth factors and phospholipid growth factors are found from Gene bank as target genes. And connecting and transforming the target gene and pGAPZ alpha A plasmid to construct a shuttle vector pGAPZ alpha A-GF, linearizing the shuttle vector after sequencing, and then electrically transforming the shuttle vector into pichia pastoris GS115 for expression. The obtained protein is subjected to affinity chromatography purification, and the biological activity of the GF protein is detected and analyzed by using Western Blot.
Microbial recombinant expression of bovine transferrin: bovine liver tissue is taken as a test material, primers are designed according to a transferrin sequence in a bovine genome, a coding region sequence of a bovine transferrin gene is obtained by cloning by utilizing a PCR technology, a prokaryotic recombinant expression vector of bovine transferrin is constructed by a genetic engineering means, heterologous expression is carried out in E.coli BL21(DE3), and the biological activity of the prokaryotic recombinant expression vector is detected.
Biosynthesis of fatty acids and amino acids: the method is characterized in that saccharomyces cerevisiae is used as a chassis cell, efficient microbial synthesis from glucose to fatty acid and amino acid is realized through integration and synthesis regulation of a fatty acid/amino acid synthesis way, and high-purity fatty acid and amino acid components are obtained through a subsequent biological separation technology and are used as nutrient addition components of a serum-free culture system.
Biosynthesis of vitamin C: the method comprises the steps of taking gluconobacter oxydans as a chassis cell, biologically synthesizing a precursor 2-keto-L-gulonic acid of vitamin C, synthesizing the vitamin C through one-step chemical catalysis, and taking the vitamin C as one of exogenous additive supplementary components of a serum-free culture medium.
Vitamin B6 and vitamin B12 are chemically synthesized.
EXAMPLE 1 serum-free culture System Components
TABLE 1 basic culture Medium
TABLE 2 exogenous addition of supplemental ingredients
The serum-free culture system included a basal medium (table 1) and an exogenously added supplement component (table 2).
1 to 5 x 104Individual BHK21 cells (baby hamster kidney cells) were inoculated in 2mL of a serum-free culture system, followed by aseptic culture at 19 to 24 ℃ for 48 hours, and the serum-free culture system was evaluated by counting the cells quantitatively, with the following results: the cell shape is full and stable; normal cell growth numbers can be achieved within the desired growth cycle.
The basic culture medium has unchanged components (see table 1), the content of different components in the exogenous additive supplementary components is adjusted, the culture systems of the groups A, B, C, D and E are set to culture the BHK21 cells, and the growth capacities of the cells of different groups are shown in table 3. Culturing at 19-24 deg.C for 48 hr until the number of cells in group A reaches 6 × 105The number of B group cells reaches 3.6 multiplied by 105The number of the cells in the C group reaches 6 multiplied by 105The number of cells in group D reaches 3.9X 105The number of E group cells reaches 6X 105。
TABLE 3 optimization of exogenously added supplemental ingredients
Relative growth capacity of cells: culturing at 19-24 deg.C for 48h, defining the highest cell growth number as 100%, calculating the ratio of the cell number to the highest value, and calculating the relative growth capacity of the cells.
EXAMPLE 2 use of serum-free culture System in the production of meat analogues
The invention is to addExogenously synthesizing cell growth factor, phospholipid growth factor, transferrin, amino acid, vitamin and other substances, and compounding with the basic culture medium to form the novel serum-free culture medium. The culture system can rapidly proliferate muscle satellite cells (muscle stem cells). Changing the culture solution once in 6-12 hours, and culturing the cells for 48 hours at the temperature of 19-24 ℃. With DMEM + 5% New born bovine serum Medium (Gibco)TMAdvanced DMEM, 12491015) had the same effect. Therefore, the serum-free culture medium can be used for completely replacing a serum-containing culture medium, can culture different animal cells in a large scale at low cost, and is applied to animal cell culture meat.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A serum-free culture system, which is characterized in that the culture system comprises a basic culture medium and an externally added supplementary component, wherein the basic culture medium comprises the following components: 125-135 mM NaCl, 2.0-4.0 mM KCl, 4.0-6.0 mM D-glucose, NaHCO3 10～14mM，NaH2PO4 0.3～0.7mM，MgCl2 0.5～1.5mM，CaCl21.0-3.0 mM, and 0.3-0.7 mM of butanediamine hydrochloride; the contained exogenous additive supplementary components comprise the following components: 100-300 mM of cell growth factor, 2.0-6.0 mM of phospholipid growth factor, 0.08-1.02 g/L of transferrin, 0.3-0.7 mg/L of lipid, 4-6 g/L of amino acid, 4-6 mM of biotin, 124-6 mM of vitamin B, 68-12 mM of vitamin B and 0.1-0.3 mM of vitamin C.
2. The serum-free culture system according to claim 1, wherein the basal medium comprises the following components: 130mM NaCl, 3.0mM KCl, 5.0mM D-glucose, NaHCO3 12mM，NaH2PO4 0.5mM，MgCl2 1.0mM，CaCl22.0mM, and 0.5mM of butanediamine hydrochloride; the contained exogenous additive is supplemented intoComprises the following components: 130mM of cell growth factor, 3.0mM of phospholipid growth factor, 0.1g/L of transferrin, 0.5mg/L of lipid, 5g/L of amino acid, 5mM of biotin, vitamin B125 mM, vitamin B610 mM and 0.2mM of vitamin C.
3. The serum-free culture system according to claim 1 or 2, wherein the cell growth factor, lipid, amino acid or vitamin is produced by microbial fermentation or whole cell transformation.
4. Use of the serum-free culture system according to claim 1 for culturing animal stem cells, animal muscle cells or animal adipocytes.
5. The use of claim 4, wherein the animal stem cells comprise embryonic stem cells, myoblast stem cells, mesenchymal stem cells, or adipogenic stem cells.
6. The use according to claim 4, wherein the amount of cell inoculation in culture is 103-105one/mL serum-free culture system.
7. The use according to claim 4 or 6, wherein the culturing is at 19-24 ℃ for 40-100 h.
8. The use according to claim 7, wherein the culturing is at 19-24 ℃ for 40-50 h.
9. A method for producing a cultured meat product of animal cells, comprising culturing animal stem cells in the serum-free culture system according to claim 1.
10. Use of the serum-free culture system according to claim 1 in the food sector.
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| J. VAN DER VALK ET AL.: "Optimization of chemically defined cell culture media-Replacing fetal bovine serum in mammalian in vitro methods", 《TOXICOLOGY IN VITRO》 * |
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