CN114574433A - Culture medium with specific chemical components for myogenic cell in-vitro proliferation - Google Patents

Culture medium with specific chemical components for myogenic cell in-vitro proliferation Download PDF

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CN114574433A
CN114574433A CN202210105530.7A CN202210105530A CN114574433A CN 114574433 A CN114574433 A CN 114574433A CN 202210105530 A CN202210105530 A CN 202210105530A CN 114574433 A CN114574433 A CN 114574433A
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cell
myogenic
medium
proliferation
culture medium
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CN114574433B (en
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周光宏
吴中元
丁世杰
唐长波
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Nanjing Zhouzi Future Food Technology Co ltd
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Nanjing Zhouzi Future Food Technology Co ltd
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Abstract

The invention provides a culture medium with definite chemical components for myogenic cell proliferation in vitro, which is characterized in that a series of problems of serum are avoided by adding cell culture supplementary factors to replace the function of the serum in the myogenic cell proliferation process, the myogenic cell can maintain the proliferation capacity of about 3 times of proliferation of each generation in the passage process of at least 3 generations (9 days), and the proportion of living cells is maintained at about 90%. On the molecular level, the culture medium with definite chemical components for myogenic cell in-vitro proliferation can obviously improve the expression of myogenic genes and the expression of extracellular matrix protein genes. In addition, an immunofluorescence result shows that the culture medium with definite chemical components for myogenic cell in-vitro proliferation maintains the positive cell ratio of myogenic protein, extracellular matrix protein and cell specific protein expression in the passage process, and maintains the cell population unchanged.

Description

Culture medium with specific chemical components for myogenic cell in-vitro proliferation
Technical Field
The invention belongs to the technical field of stem cell and animal cell culture meat, and particularly relates to a culture medium with definite chemical components for myogenic cell in-vitro proliferation.
Background
Meat is one of the most important foods for human because of rich nutritional ingredients such as protein, carbohydrate, fat, mineral substances, vitamins and the like, but the traditional meat production mode by animal husbandry has the problems of resource waste, easy spread of food-borne diseases, threat of animal welfare, difficulty in improving meat quality and the like, and is difficult to become a green and sustainable meat production mode. The cell culture meat technology serving as a novel alternative protein production technology has the advantages of low carbon, environmental protection, pathogen transmission reduction, antibiotic use reduction, avoidance of animal welfare problems and the like, and provides a new approach for meat production.
The cell culture meat is obtained by culturing stem cells in vitro according to the principle of animal muscle growth and repair, and the technology does not need animal culture but directly produces meat in a cell factory way. The in vitro production technology of cell culture meat mainly comprises seed cell obtaining and amplification, and induced differentiation after cell enrichment, so as to promote most cells to differentiate to form muscle tissues. It is therefore important to select a chemically defined medium that promotes the long-term proliferation of myogenic cells in vitro.
The proliferation culture medium of myogenic cells commonly used in the prior art is a basic culture medium added with 10-20 vol% of fetal calf serum and 1 vol% of double antibody. Although the culture medium can help myogenic cells to maintain a certain dryness and quickly proliferate, the addition of fetal calf serum has the problems of undefined chemical components of the culture medium, unstable components among culture medium batches, easy pathogen pollution, high cost, no contribution to animal welfare and the like. Therefore, it is important to develop a chemically defined medium for the in vitro proliferation of myogenic cells.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a myogenic cell proliferation culture medium with definite chemical components and an application method thereof to replace serum components in the traditional myogenic cell proliferation culture medium.
The first purpose of the invention is to provide a cell proliferation culture medium with definite chemical components, which is a cell culture medium without serum components. The cell culture medium without serum components refers to a culture medium formula without any animal serum components such as fetal calf serum, horse serum, human serum and the like. The culture medium with definite chemical components for myogenic cell in-vitro proliferation realizes excellent proliferation culture effect by adding cell culture supplement factors, has definite components, is easy for quality control, and well replaces the traditional myogenic cell proliferation culture medium.
The technical scheme of the invention is as follows:
the first purpose of the invention is to provide a culture medium with definite chemical components for the in vitro proliferation of myogenic cells, which comprises cell culture supplementary factors and a cell proliferation culture medium, wherein the culture medium does not contain serum components.
Further, the cell proliferation culture medium comprises a myogenic cell basic culture medium and a penicillin-streptomycin double-resistant solution;
preferably, the volume ratio of the myogenic cell basic culture medium to the penicillin-streptomycin double-antibody solution is 99: 1;
preferably, the myogenic cell basic medium is selected from one of a DMEM medium, a MEM medium, a DMEM/F12 medium, and a F10 medium.
Preferably, in the penicillin-streptomycin double-resistant solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10 mg/ml.
Further, the cell culture supplement factors include a plurality of non-essential amino acid supplements, lipids, antioxidants, recombinant proteins, signal pathway modulators, insulin, transferrin, ethanolamine, sodium selenite, dexamethasone, albumin, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, cortisol, sodium bicarbonate.
Further, the nonessential amino acid supplement is selected from one or more of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine;
the lipid is selected from one or more of Tween 80, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, Pluronic F68, stearic acid, and sodium salt of oleoyl lysophosphatidic acid;
the antioxidant is selected from one or more of ascorbic acid, ascorbic acid trisodium phosphate salt, and water-soluble vitamin E, N-acetyl-L-cysteine;
the recombinant protein is selected from one or more of insulin-like growth factor, epidermal growth factor, basic fibroblast growth factor, leukemia inhibitory factor, hepatocyte growth factor and platelet-derived growth factor;
the signal path regulator is selected from one or more of Rock inhibitor, forskolin, P38 inhibitor and Rho inhibitor.
In certain specific embodiments, the Rock inhibitor is ZINC00881524, Y-276322 HCl, Thiazovivin; the P38 inhibitor is Adezmapimod, SB202190, Ralimetinib; the Rho inhibitor is GSK429286A, RKI-1447.
Further, in the culture medium for the in vitro proliferation of the myogenic cells, the total concentration of the cell culture supplementary factors added is in the range of 0.03-50 mg/mL.
Preferably, in the culture medium for the in vitro proliferation of the myogenic cells, the total concentration of the cell culture supplementary factors added is in the range of 0.5-30 mg/mL.
Further preferably, in the culture medium for the in vitro proliferation of the myogenic cells, the total adding concentration of the cell culture supplementary factors is 1-4 mg/mL.
Further, in the culture medium for the in vitro proliferation of the myogenic cells, the concentration of any added cell culture supplementary factor is 0.5ng/mL-30 mg/mL.
Preferably, the culture medium for the in vitro proliferation of the myogenic cells has the concentration of any added cell culture supplementary factor of 1ng/mL-10 mg/mL.
Further preferably, the concentration of any added cell culture supplementary factor in the culture medium for the in vitro proliferation of the myogenic cells is 1ng/mL-1 mg/mL.
The second purpose of the invention is to provide the application of the culture medium for myogenic cell in-vitro proliferation with definite chemical components in myogenic cell in-vitro proliferation culture, and the culture medium for myogenic cell in-vitro proliferation is adopted for in-vitro proliferation culture.
The application comprises the following steps:
1) separating to obtain myogenic cells;
2) inoculating the myogenic cells into the culture medium which is prepared from the specific chemical components and used for the in-vitro proliferation of the myogenic cells, changing the culture medium the next day after inoculation, digesting the cells with pancreatin the 3 rd day after inoculation, counting the obtained cell suspension, and then subculturing the cells into a fresh culture medium again.
3) And (3) carrying out cytomorphological observation and photographing on the cells every day, and collecting samples for blood cell counting, real-time quantitative PCR detection and immunofluorescence detection every time of passage.
Furthermore, the myogenic cells are all cell types of muscle origin including muscle stem cells, fibroblasts and smooth muscle cells.
Furthermore, the cell culture needs to be carried out in a culture dish pre-plated with type I rat tail collagen, and the specific plating method comprises the following steps: dissolving type I rat tail collagen with 0.02mol/L acetic acid solution to final concentration of 0.5mg/mL, spreading the solution on the surface of a cell culture dish, placing at 37 deg.C for at least 4 hr, washing with PBS for 2 times, and air drying.
Further, the cell culture is maintained at 5% CO2Concentration, 37 ℃ in carbon dioxide incubator environment.
Further, the culture medium with definite chemical components for the in vitro proliferation of the myogenic cells can maintain the normal cell morphology of myogenic cells in the proliferation process.
Furthermore, the culture medium with definite chemical components for the in-vitro proliferation of the myogenic cells can maintain the normal expansion fold and the viable cell rate of the myogenic cells in the culture process.
Further, the myogenic cell in-vitro proliferation culture medium can maintain normal adherence and proliferation capacity of the myogenic cells, and/or maintain normal cell morphology, and/or maintain the proliferation capacity of the myogenic cells which is 3 times of that of each generation in the passage process of at least 3 generations (9 days), and/or the proportion of living cells is not lower than 80%.
Furthermore, the culture medium for the in vitro proliferation of the myogenic cells can improve the expression of myogenic genes and/or extracellular matrix genes of the myogenic cells. The myogenic cell in-vitro proliferation culture medium can improve the expression of myogenic genes of myogenic cells and also obviously improve the expression of extracellular matrix protein genes.
Furthermore, the culture medium with definite chemical components for the myogenic cell in-vitro proliferation can maintain the expression of myogenic proteins, extracellular matrix proteins and/or cell specific marker proteins of the myogenic cells and maintain the purity of the cells.
The existing myogenic cell in vitro proliferation culture medium widely used is mostly a culture medium added with fetal calf serum, and has great limitations in aspects of biochemistry, medicine, food science, development and industrialization of cell culture meat and the like. The culture medium for myogenic cell in-vitro proliferation with clear chemical components can avoid a series of problems caused by serum, maintain the proliferation capacity of myogenic cells about 3 times of each generation in the passage process, and maintain the proportion of living cells at about 90 percent. In the aspect of gene expression, the culture medium for myogenic cell in-vitro proliferation with clear chemical components can obviously improve the expression of myogenic genes and the expression of extracellular matrix protein genes. In addition, immunofluorescence detection of cell-specific proteins shows that the culture medium for myogenic cell in-vitro proliferation with definite chemical components maintains the positive cell ratio of myogenic proteins, extracellular matrix proteins and cell-specific proteins in the passage process, and maintains the cell population unchanged.
The technical scheme of the invention has the following beneficial effects:
the amplification culture of myogenic cells is one of the most critical links in the production of cell culture meat, and the traditional cell proliferation culture medium is a serum-containing culture medium at present, has the problems of undefined chemical components, unstable components among culture medium batches, easy pathogen pollution, high cost, harm to animal welfare and the like, and seriously hinders the promotion and industrialized development of the cell culture meat technology.
The culture medium with definite chemical components for the in-vitro proliferation of the myogenic cells is based on a commercially available basic culture medium, and replaces the function of fetal calf serum in the myogenic cell proliferation process by adding cell culture supplement factors to avoid a series of problems of serum, so that the myogenic cells can maintain the proliferation capacity of about 3 times of proliferation of each generation in the passage process of at least 3 generations (9 days), and the proportion of living cells is maintained to be not less than 80%. On the molecular level, the culture medium with definite chemical components for myogenic cell in-vitro proliferation can obviously improve the expression of myogenic genes and the expression of extracellular matrix protein genes. In addition, an immunofluorescence result shows that the culture medium with definite chemical components for myogenic cell in-vitro proliferation maintains the positive cell proportion of myogenic protein, extracellular matrix protein and cell specific protein in the passage process, and maintains the cell population unchanged. The chemical components are clear, the components of the culture medium among batches are stable, the condition of pathogen pollution is avoided, the quality control is easy, and the cost is reduced compared with a serum-containing culture medium. Provides a new culture medium selection for the expanded culture of myogenic cells and supplements a safe and cheap cell amplification mode for the cell culture meat technology.
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FIG. 1 is a statistical chart of the detection of proliferation-promoting effects of various cell culture supplements.
FIG. 2 is a photograph showing the results of bright field photography of muscle stem cells cultured continuously for 9 days (3 passages) in a proliferation medium having a definite chemical composition, in which only 99 vol% DMEM/F12 basal medium +1 vol% penicillin-streptomycin double antibody was used as a control.
FIG. 3 is a statistical graph of the cell number and the viable cell rate of muscle stem cells cultured continuously in 2 proliferation media with well-defined chemical compositions for 9 days (3 generations).
FIG. 4 is a statistical chart of dry and myogenic gene expression of muscle stem cells cultured continuously for 9 days (3 generations) in 2 proliferation media with well-defined chemical compositions, and the control was 84 vol% DMEM/F12 basal medium, 15 vol% serum, and 1 vol% penicillin-streptomycin double antibody. FIG. A is a statistical chart of the expression of PAX7 gene for characterization of the sternness of muscle stem cells; FIG. B is a statistical chart of MyoD gene expression profile characterizing myogenic levels of muscle stem cells; FIG. C is a statistical chart of MyoG gene expression that characterizes the myogenic differentiation level of the muscle stem cells; panel D is a statistical chart of MyHC gene expression characterizing the level of differentiation and maturation of muscle stem cells.
FIG. 5 shows immunofluorescence staining results of dry and myogenic proteins of muscle stem cells cultured in proliferation medium with defined chemical composition for 9 days (3 generations).
FIG. 6 is a photograph showing the results of continuously culturing fibroblasts in a proliferation medium with a definite chemical composition for 9 days (3 passages) in a bright field, in comparison with a medium containing only 99 vol% of DMEM/F12 basal medium plus 1 vol% of penicillin-streptomycin double antibody.
FIG. 7 is a statistical graph of the number of cells and the viable cell rate of fibroblasts cultured continuously in 2 proliferation media with well-defined chemical composition for 9 days (3 generations).
FIG. 8 is a statistical chart of extracellular matrix protein gene expression of fibroblasts cultured continuously for 9 days (3 passages) in 2 proliferation media with well-defined chemical compositions, and 89 vol% DMEM/F12 basic medium +10 vol% serum +1 vol% penicillin-streptomycin double antibody medium is used as a control. FIG. A is a statistical chart of the expression of the Collagen I gene; FIG. B is a statistical chart of the expression of the Collagen III gene; FIG. C is a statistic chart of Laminin gene expression; and the figure D is a statistical chart of the expression condition of the Elastin gene.
FIG. 9 shows the results of immunofluorescent staining of extracellular matrix proteins of fibroblasts in a proliferation medium with defined chemical composition for 9 days (3 passages).
FIG. 10 is a photograph showing the results of bright field photography of smooth muscle cells cultured continuously for 9 days (3 passages) in a proliferation medium with a definite chemical composition, in which only 99 vol% DMEM/F12 basal medium +1 vol% penicillin-streptomycin double antibody was used as a control.
FIG. 11 is a statistical chart of the number of cells and the viable cell rate of smooth muscle cells cultured continuously in 2 growth media with defined chemical compositions for 9 days (3 generations).
FIG. 12 is a statistical chart of extracellular matrix protein gene expression of smooth muscle cells cultured continuously for 9 days (3 passages) in 2 proliferation media with defined chemical compositions, as compared with a medium consisting of 84 vol% DMEM/F12 basal medium, 15 vol% serum and 1 vol% penicillin-streptomycin double antibody. FIG. A is a statistical chart of the expression of the Collagen I gene; FIG. B is a statistical chart of the expression of the Collagen III gene; FIG. C is a statistic chart of Laminin gene expression; FIG. D is a statistical chart of the expression of Elastin gene.
FIG. 13 shows immunofluorescence staining results of extracellular matrix proteins of smooth muscle cells cultured continuously for 9 days (3 passages) in a chemically defined proliferation medium.
Detailed Description
The culture medium for myogenic cell in-vitro proliferation with definite chemical components is a cell proliferation culture medium added with cell culture supplementary factors, and the conditions in other aspects are consistent with the normal myogenic cell in-vitro culture method.
The cells used in the following examples are myogenic cells, and further, one of muscle stem cells, fibroblasts, and smooth muscle cells, which are all adherent cells.
The culture conditions used in the following examples are all CO2Culturing at 37 deg.C in incubator with CO2All concentrations of (a) were 5% (v/v).
The statistical analysis methods of the data in the following examples are t-test, P <0.05, P <0.01, P <0.001, P < 0.0001.
The detection methods employed in the following examples are, unless otherwise indicated, experimental methods, detection methods and preparation methods disclosed in the art.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The detection methods employed in the following examples are, unless otherwise indicated, experimental methods, detection methods and preparation methods disclosed in the art. See in particular Shijie Ding et al in the literature "Maintaining bone mineral cells step through p38 path" (DOI: 10.1038/s41598-018-
EXAMPLE 1 detection of Proliferative Effect of additional addition of cell culture supplements
1) Type i rat tail collagen pre-plating: dissolving type I rat tail collagen with 0.02mol/L acetic acid solution to final concentration of 0.5mg/mL, adding the solution into 96-well plate at volume of 100uL per well, standing at 37 deg.C for at least 4 hr, washing with PBS for 2 times, and air drying.
2) Cell inoculation and adherence: muscle stem cells before the high purity P5 passage were seeded at a density of 1200 cells/well into 96-well plates pre-plated with type i rat tail collagen, cultured in serum-containing medium: DMEM/F12+ 15% fetal bovine serum + 1% penicillin-streptomycin double antibody culture medium formula overnight adherent culture.
3) Preparation of each group of culture medium with definite chemical components: the culture medium for the in vitro proliferation of the myogenic cells comprises cell culture supplementary factors and a cell proliferation culture medium.
All cell proliferation culture media are prepared by adding 1% vol penicillin-streptomycin double antibody into 99 vol% DMEM/F12 basal culture media and then respectively adding cell culture supplementary factors, wherein in the penicillin-streptomycin double antibody solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10 mg/ml.
Cell culture supplement factor types and concentrations are shown in table 1:
Figure BDA0003493373340000071
Figure BDA0003493373340000081
the components of the culture medium for the in vitro proliferation of myogenic cells of each experimental group are set as follows:
group 1: a cell proliferation medium plus common cell culture auxiliary factors of components 1-21 shown in table 1;
group 2: on the basis of the group 1, a component 22 (leukemia inhibitory factor, LIF) shown in the table 1 is added; group 3: on the basis of the group 1, a component 23 (ascorbic acid, VC) shown in the table 1 is added;
group 4: group 1 was supplemented with component 24 (ascorbic acid trisodium phosphate, 2PAA) shown in table 1;
group 5: group 1 was supplemented with component 25(Rock inhibitor, Y-276322 HCl) shown in Table 1;
group 6: the component 26 (N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid, HEPES) shown in the table 1 is added on the basis of the group 1;
group 7: group 1 was supplemented with component 27 (cortisol) shown in table 1;
group 8: adding component 28 (water-soluble vitamin E, Trolox) shown in Table 1 on the basis of group 1;
group 9: on the basis of the group 1, a component 29 (Forskolin ) shown in the table 1 is added;
group 10: group 1 was supplemented with component 30 (hepatocyte growth factor, HGF) shown in table 1;
group 11: group 1 was supplemented with component 31 (N-acetyl-L-cysteine, NAC) shown in table 1;
group 12: on the basis of the group 1, a component 32 (oleoyl lysophosphatidic acid sodium salt, LPA) shown in the table 1 is added;
group 13: group 1 was supplemented with a component 33 (nonessential amino acid supplement, NEAA) as shown in table 1 selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine mixtures;
group 14: on the basis of the group 1, a complete cell proliferation culture medium with definite chemical components of the components 22-33 is added.
4) Culture medium exchange culture of myogenic cells in vitro proliferation with clear chemical components: after the cells are basically attached to the wall, the serum-containing culture medium in the 96-well plate is sucked, the 96-well plate is cleaned once by using the culture medium which is prepared in advance and is provided with different cell culture supplement factors and has definite chemical components, the myogenic cell in-vitro proliferation culture medium corresponding to each group is added for continuously culturing the cells, and the liquid is changed after 48 hours.
5) And (3) cell staining counting: after culturing for 4 days with myogenic cell in-vitro proliferation culture medium with clear chemical components, sucking out culture solution in a 96-well plate, and performing in a proportion of 1: and (3) diluting the Hoechst dye by 500(v/v), adding the Hoechst dye into a 96-well plate, incubating the plate for 30min at 37 ℃, washing the plate for 2 times, and then placing the plate in a high-content high-throughput imaging system for photographing and counting.
6) The results show that: the first data column in fig. 1 is the culture medium of group 1, which contains the currently common cell culture supplement factors, and groups 1 to 14 can satisfy the proliferation requirement and can be used as a proliferation medium; on the basis of the above, other cell culture supplementary factors are additionally added in groups 2 to 14, except for Rock inhibitor (group 5), the cell proliferation number can be obviously increased, but the culture medium of group 5 can still meet the proliferation requirement and can be used as a proliferation culture medium; in particular, the complete chemically defined cell proliferation medium of group 14 was significantly superior to other formulations that added only a portion of the common cell culture supplements in promoting cell proliferation.
Example 2 in vitro subculture of myogenic cells and detection of cell proliferation fold and survival rate
1) The preparation of the culture medium with definite chemical components for the in vitro proliferation of the myogenic cells comprises the following steps: the cell proliferation culture medium of the culture medium with definite chemical components for the in-vitro proliferation of the myogenic cells is prepared by adding 1% vol penicillin-streptomycin double antibody into 99% vol DMEM/F12 basal medium and respectively adding cell culture supplementary factors. All cell culture supplements and their recommended concentrations that can be used to formulate chemically defined media for myogenic cell proliferation in vitro are shown in table 2, and the examples are divided into two groups: the cell culture supplement factors added to the chemically defined medium 1 are shown in Table 3, and the cell culture supplement factors added to the chemically defined medium 2 are shown in Table 4.
TABLE 2 all cell culture supplements and their recommended concentrations
Figure BDA0003493373340000091
Figure BDA0003493373340000101
TABLE 3 cell culture supplement factor added to chemically defined Medium 1
Figure BDA0003493373340000102
Figure BDA0003493373340000111
TABLE 4 cell culture supplement factor added to chemically defined Medium 2
Figure BDA0003493373340000112
Figure BDA0003493373340000121
2) In vitro subculturing and counting of myogenic cells: respectively adopting culture medium 1 with definite chemical composition and culture medium 2 with definite chemical composition as growth culture medium, and taking myogenic cells at a ratio of 1.5 × 105The density of each disc was inoculated into a 10cm cell culture dish pre-plated with type I collagen, cytomorphological observation and photography were performed every day, the same culture medium was used for changing the medium on day 2 of culture, 0.25% pancreatin was used for digesting the cells on day 3, the cell suspension was centrifuged at 330g and then the cells were re-spun, 10. mu.L of the cell suspension was stained with trypan blue dye, and the cells were counted on a hemocytometerAfter viable cell count, at 1.5X 105Cell density passages per disc, at least 3 passages.
The myogenic cells referred to in this example are muscle stem cells, fibroblasts and smooth muscle cells.
3) The results show that:
for muscle stem cells, cells cultured in the defined formulation of the proliferation medium had normal adherence and cell morphology at passages 4, 5, and 6 and had some proliferation within 3 days, compared to negative controls (cell proliferation medium without added cell culture supplement factors) with few adherent cells (fig. 2). The results of the counting in 3 generations showed that the cells cultured in the medium 1 and the medium 2 each having a definite chemical composition were amplified about 3 times in number per generation and the viable cell rate was maintained at about 90% (FIG. 3).
For fibroblasts, under the condition of negative control (cell proliferation medium without cell culture supplement factors), few cells were observed and could not adhere to the wall, and cells cultured in the proliferation medium formula with clear chemical composition all had normal adherence and cell morphology at the 4 th, 5 th and 6 th generations and had certain proliferation within 3 days (fig. 6). The results of the counting in 3 generations showed that the cells cultured in the medium 1 and the medium 2 each having a definite chemical composition were amplified about 3 times in number per generation and the viable cell rate was maintained at 90% or more (FIG. 7).
For smooth muscle cells, the negative control (cell proliferation medium without added cell culture supplements) had almost no adherent fusiform cells on day 3, and cells cultured in the defined chemical proliferation medium formulation had normal adherence and cell morphology at passages 4, 5, and 6, and had some proliferation within 3 days (fig. 10). The results of the counting in 3 generations showed that the cells cultured in the medium 1 and the medium 2 each having a specific chemical composition were amplified about 3 times in number per generation and the viable cell rate was maintained at 80% or more (FIG. 11).
In summary, the culture medium with definite chemical composition for myogenic cell in vitro proliferation provided by the invention can maintain normal cell morphology and maintain normal proliferation rate and cell viability for 3 myogenic cells, namely myostem cells, fibroblasts and smooth muscle cells, in the in vitro passage process.
Example 3 detection of Gene expression during in vitro passage of myogenic cells
1) Detection of gene expression level: the 2 culture media with definite chemical compositions in example 2 are used for in vitro culture of myogenic cells, 15% serum-containing culture medium (DMEM/F12+ 15% fetal bovine serum + 1% penicillin-streptomycin double antibody) is used as a control, cells cultured for 3 days in the 4 th, 5 th and 6 th generations are cracked by Trizol to extract RNA, and are reversely transcribed by a reverse transcription kit to obtain cDNA, and myogenic genes PAX7, MyoD, MyoG and MyHC and related extracellular matrix protein genes such as Collagen I, Collagen III, Elastin and Lamin are respectively detected.
2) The results show that: for muscle stem cells, the cell characterization cell dryness gene PAX7 was significantly higher in all 4 th, 5 th, and 6 th passages than cells cultured in medium containing 15% fetal bovine serum when cultured in 2 chemically defined media; the expression level of MyoD gene in the 4 th and 5 th generations is also obviously higher than that of cells cultured in a culture medium containing serum; the MyoG gene was significantly higher in all 4, 5, 6 passages than cells cultured in serum-containing medium; the expression level of the Myosin gene was significantly higher in the 4 th and 5 th generations than in the control group containing serum, and was not different from the control group containing serum in the 6 th generation (fig. 4). For fibroblasts and smooth muscle cells, genes of Collagen I, Collagen III, Elastin and lamin were detected, and the expression level of cells cultured in the medium with clear chemical components was significantly higher in the 4 th, 5 th and 6 th generations than in the medium containing serum (fig. 8 and 12).
Example 4 immunofluorescence detection of specific marker proteins during in vitro passaging of myogenic cells
1) Myogenic cells cultured in the medium 1 with clear chemical composition in example 2 were taken, washed with PBS, fixed with 4% paraformaldehyde, washed three times with PBS, then permeabilized with 0.5% (v/v) TritonX-100 (PBS) for 15min, washed three times with PBS shaker for 5min each time, added with immunofluorescence primary antibody prepared with 1% BSA solution, placed in a wet box, and incubated overnight at 4 ℃. Wash three times with PBS for 5min each time. Adding diluted fluorescent secondary antibody dropwise, incubating in a wet box at 20-37 deg.C for 1h, and washing with PBS for 5min for 2 times. Note that the operation of keeping out of light is required from the start of the dropwise addition of the secondary antibody. Dropping an anti-fluorescence quencher containing DAPI, covering a proper cover glass, and observing and collecting an image under a fluorescence microscope.
2) As a result: for the muscle stem cells, cell specific marker protein, myogenic protein Pax7, myogenic protein MyoD, myogenic protein MyoG and DAPI staining is carried out (fig. 5), and in the continuous passage process of the 4 th generation, the 5 th generation and the 6 th generation, the expression ratios of three myogenic genes of Pax7, MyoD and MyoG are all maintained at higher levels, which shows that the cell purity and the myogenic level are not reduced in the culture process of the culture medium with clear chemical components for the in vitro proliferation of the myogenic cells.
For fibroblasts, myogenic protein alpha-Actin, cell specific marker protein Tcf-4 and DAPI were stained (fig. 9), and the proportion of the fibroblast specific marker protein Tcf-4 was maintained at a level of 95% or more during the serial passages of generations 4, 5 and 6, indicating that the cell purity of the fibroblasts was not decreased during the culture of the chemically defined medium for myogenic cell in vitro proliferation of the present invention.
For smooth muscle cells, extracellular matrix protein Collagen III, myogenic cell myogenic protein, cell specific marker protein SMActin and DAPI staining is carried out (figure 13), and the positive rates of Collagen III and SMActin are improved generation by generation in the continuous passage processes of 4 th, 5 th and 6 th generations, which shows that the continuous passage processes of 4 th, 5 th and 6 th generations help smooth muscle cells to enter a differentiation process generation by generation and promote the secretion of Collagen III.
In conclusion, the culture medium for myogenic cell in-vitro proliferation with clear chemical components maintains the positive cell proportion of myogenic protein, extracellular matrix protein and cell specific protein in the passage process, and maintains the cell population unchanged. Can be used for myogenic cell in-vitro proliferation, provides a new culture medium selection for the expanded culture of myogenic cells, and supplements a safe and cheap cell amplification mode for the cell culture meat technology.
The examples disclosed above are intended to illustrate the disclosed embodiments of the invention, but are not to be construed as limiting the invention, and many different cell culture supplements are listed herein, and many further combinations are possible without departing from the scope and spirit of the invention, and therefore the invention is not limited to the disclosed embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims (10)

1. A culture medium with definite chemical components for the in vitro proliferation of myogenic cells, which is characterized by comprising cell culture supplementary factors and a cell proliferation culture medium, wherein the culture medium does not contain serum components.
2. The medium for the in vitro proliferation of myogenic cells according to claim 1, wherein the cell proliferation medium comprises a myogenic cell basal medium and a penicillin-streptomycin double antibody solution;
preferably, the volume ratio of the myogenic cell basic culture medium to the penicillin-streptomycin double-antibody solution is 99: 1;
preferably, the myogenic cell basic medium is selected from one of a DMEM medium, a MEM medium, a DMEM/F12 medium, and a F10 medium.
Preferably, in the penicillin-streptomycin double-resistant solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10 mg/ml.
3. The culture medium for the in vitro proliferation of myogenic cells according to claim 1, wherein said cell culture supplementary factors comprise a plurality of non-essential amino acid supplements, lipids, antioxidants, recombinant proteins, signal pathway modulators, insulin, transferrin, ethanolamine, sodium selenite, dexamethasone, albumin, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, cortisol, sodium bicarbonate.
4. The culture medium for the in vitro proliferation of myogenic cells according to claim 3, wherein said non-essential amino acid supplement is selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine in combination with one or more;
the lipid is selected from one or more of Tween 80, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, Pluronic F68, stearic acid, and sodium salt of oleoyl lysophosphatidic acid;
the antioxidant is selected from one or more of ascorbic acid, ascorbic acid trisodium phosphate salt, and water-soluble vitamin E, N-acetyl-L-cysteine;
the recombinant protein is selected from one or more of insulin-like growth factor, epidermal growth factor, basic fibroblast growth factor, leukemia inhibitory factor, hepatocyte growth factor and platelet-derived growth factor;
the signal path regulator is selected from one or more of Rock inhibitor, forskolin, P38 inhibitor and Rho inhibitor.
5. The culture medium for the in vitro proliferation of myogenic cells according to claim 1, wherein the total concentration of cell culture supplement factors added in the culture medium for the in vitro proliferation of myogenic cells is in the range of 0.03-50 mg/mL.
Preferably, in the culture medium for the in vitro proliferation of the myogenic cells, the total adding concentration of the cell culture supplementary factors ranges from 0.5 to 30 mg/mL;
further preferably, in the culture medium for the in vitro proliferation of the myogenic cells, the total adding concentration of the cell culture supplementary factors is 1-4 mg/mL.
6. The culture medium for the in vitro proliferation of myogenic cells according to claim 3, wherein the concentration of any added cell culture supplementary factor in the culture medium for the in vitro proliferation of myogenic cells is 0.5ng/mL-30 mg/mL; preferably, the culture medium for the in vitro proliferation of the myogenic cells has the concentration of any added cell culture supplementary factor of 1ng/mL-10 mg/mL.
Further preferably, the concentration of any added cell culture supplementary factor in the culture medium for the in vitro proliferation of the myogenic cells is 1ng/mL-1 mg/mL.
7. Use of the medium for the in vitro proliferation of myogenic cells according to claim 1 in the culture of the in vitro proliferation of myogenic cells, wherein the medium for the in vitro proliferation of myogenic cells according to claim 1 is used for the culture of the in vitro proliferation of myogenic cells.
8. The use according to claim 7, wherein said culture medium for the in vitro proliferation of myogenic cells is capable of maintaining the normal anchorage and proliferation capacity of myogenic cells, and/or maintaining the normal cell morphology, and/or maintaining the proliferation capacity of myogenic cells at a rate 3 times higher than that of myogenic cells in each passage of at least 3 passages, and/or maintaining the proportion of viable cells at least 80%.
9. The use according to claim 7, wherein said medium for the in vitro proliferation of myogenic cells increases the expression of myogenic genes and/or extracellular matrix genes of myogenic cells.
10. The use according to claim 7, wherein the medium for the in vitro proliferation of myogenic cells can maintain the positive expression rate of myogenic proteins, extracellular matrix proteins and/or cell-specific marker proteins of myogenic cells and maintain the purity of cells.
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CN115927171A (en) * 2022-12-15 2023-04-07 中国海洋大学 Culture medium for rapid differentiation of muscle stem cells of freshwater fishes and application of culture medium

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