Proliferation culture medium for long-term stable passage of bovine-derived cells, method for promoting long-term stable passage of bovine-derived cells and application

Abstract

The invention provides a proliferation culture medium for long-term stable passage of bovine-derived cells, a method for promoting long-term stable passage of bovine-derived cells and application of the proliferation culture medium. The proliferation culture medium comprises the following components: a complete culture medium, cycloastragenol and metformin hydrochloride. Researches show that the proliferation culture medium can make bovine embryo fibroblasts (BFFs) break through the proliferation limit in the in-vitro subculture process, a continuous cell line is formed, and production of cell culture meat products with excellent quality is facilitated. The culture medium provided by the invention can support continuous passage of BFFs to 145 generations, and the cell type of the bovine embryo fibroblast cell line subjected to passage to 145 generations is unchanged, so that the culture medium has the advantage of long-term stable passage. Through induction of the proliferation culture medium, the BFFs can break through aging limitation, long-term stable passage (more than or equal to 100 generations) is realized, the waveform protein positive rate of more than 90% is maintained, the requirement of mass production of cell culture meat on the cell number is met, and the proliferation culture medium has a very wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell culture, and particularly relates to a proliferation culture medium for long-term stable passage of bovine-derived cells, a method for promoting long-term stable passage of bovine-derived cells, and application. BACKGROUND

[0002] Beef has the characteristics of delicious meat and rich nutrition, contains rich protein, fat, vitamin B group, nicotinic acid, calcium, phosphorus, iron, cholesterols and other ingredients, has the effects of benefiting qi and nourishing stomach, promoting recovery and weight loss, and is favored by consumers. With the increasing demand for beef consumption, the contradiction between the decline of cattle resources and the growth of beef demand is becoming increasingly prominent, and it is urgent to develop a sustainable alternative.

[0003] Cell culture meat technology produces meat by in vitro expansion of animal cells (i.e. seed cells), which can significantly alleviate the environmental burden (such as greenhouse gas emissions, excessive consumption of resources) and animal welfare problems caused by traditional animal husbandry. This technology only needs culture medium, certain temperature and humidity, carbon dioxide and other nutrients to provide the environment needed for growth, does not cause environmental pollution, does not need to consume feed and water needed in traditional meat production, and occupies small area, saving space cost. It is the main research and development direction of future artificial meat. Therefore, developing bovine cell culture meat technology to produce bovine cell culture meat is an effective means to solve the decline of cattle resources.

[0004] At present, the seed cells for producing bovine cell culture meat mainly include bovine fetal fibroblasts (BFFs), bovine embryonic stem cells (ESCs), bovine pluripotent stem cells (iPSCs) and immortalized / gene edited cell lines. Among them, although ESCs or iPSCs have unlimited proliferation potential, their culture depends on feeder layers or complex factor combinations (such as bFGF, Activin A, LIF and various pathway inhibitors), which are costly and prone to spontaneous differentiation, making it difficult to meet the needs of large-scale production of cell culture meat; immortalized / gene edited cell lines (such as CRISPR-Cas9 edited high-efficiency transformation cell lines) need to introduce exogenous genes, which face regulatory barriers of genetically modified food (such as the requirement of 5-10 years of safety evaluation by the European Union EFSA) and insufficient consumer acceptance (research shows that more than 60% of consumers prefer non-genetically modified cell meat). In contrast, BFFs are more commercially potential seed cells due to their easy availability, non-genetic modification characteristics and strong proliferation capacity. However, there are still some problems in producing cell culture meat with BFFs as seed cells, for example, BFFs gradually age during traditional in vitro passage, and their proliferation capacity significantly decreases after the 12th passage, making it difficult to meet the demand for cell number in large-scale production of cell culture meat, and unable to break through the proliferation limit under traditional culture conditions, which cannot meet the demand for mass production of cell culture meat.

[0005] Therefore, it is an urgent problem to develop a new culture system for long-term stable passage of seed cells by non-genetic manipulation and low cost to promote the industrialization process of bovine cell culture meat. SUMMARY

[0006] The application provides a proliferation culture medium for long-term stable passage of bovine cells. The proliferation culture medium supports continuous passage of BFFs to 145 generations, and the cell type of the bovine embryonic fibroblast cell line is unchanged after being passed to 145 generations, which has the advantage of long-term stable passage. The proliferation culture medium can make bovine embryonic fibroblasts (BFFs) break through the proliferation limit and form a continuous cell line during in vitro subculture, so that the bovine embryonic fibroblasts have stronger proliferation capacity, meet the demand for cell number in large-scale production of cell culture meat, and are beneficial to the production of cell culture meat products with excellent quality. The bovine embryonic fibroblasts (BFFs) break through the aging limit and realize long-term stable passage (≥100 generations) and maintain a vimentin positive rate of >90% by induction culture with the proliferation culture medium, which meets the demand for mass production of cell culture meat. The bovine embryonic fibroblasts (BFFs) can be subcultured more than 100 times by using the proliferation culture medium, which provides a new idea for the establishment method of bovine embryonic fibroblast cell lines and has a very broad application prospect.

[0007] The application also provides a method for promoting long-term stable passage of bovine cells, which uses the above-mentioned proliferation culture medium to culture bovine cells. Research shows that the proliferation culture medium can make bovine embryonic fibroblasts (BFFs) break through the proliferation limit and form a continuous cell line during in vitro subculture, so that the bovine embryonic fibroblasts have stronger proliferation capacity. The bovine embryonic fibroblasts (BFFs) break through the aging limit and realize long-term stable passage (≥100 generations) and maintain a vimentin positive rate of >90% by induction culture with the above-mentioned proliferation culture medium. The bovine embryonic fibroblasts (BFFs) can be subcultured more than 100 times by using the method, which has the characteristics of easy operation, stability and easy repetition, and provides a new idea for the establishment method of bovine embryonic fibroblast cell lines.

[0008] The application also provides the use of the above-mentioned proliferation culture medium for long-term stable passage of bovine cells in the preparation of cell culture meat. Research shows that the proliferation culture medium can make bovine embryonic fibroblasts (BFFs) break through the proliferation limit and form a continuous cell line during in vitro subculture, so that the bovine embryonic fibroblasts have stronger proliferation capacity. The bovine embryonic fibroblasts (BFFs) break through the aging limit and realize long-term stable passage (≥100 generations) and maintain a vimentin positive rate of >90% by induction culture with the above-mentioned proliferation culture medium. Therefore, in the process of preparing cell culture meat, the proliferation culture medium can be used to culture seed cells (bovine cells), so that the seed cells break through the proliferation limit and form a continuous cell line during in vitro subculture, break through the aging limit, realize long-term stable passage, meet the demand for cell number in large-scale production of cell culture meat, and meet the demand for mass production of cell culture meat.

[0009] The first aspect of the present application provides a proliferation culture medium for long-term stable passage of bovine-derived cells, the components of the proliferation culture medium comprising complete culture medium, cycloastragenol and metformin hydrochloride;

[0010] The complete culture medium comprises, in terms of volume percentage, 79-94 vol% of basal medium, 5-20 vol% of fetal bovine serum and 1-3 vol% of penicillin-streptomycin double-antibiotic solution.

[0011] The proliferation culture medium for long-term stable passage of bovine-derived cells as described above comprises, in terms of final concentration, 0.1-20 μmol / L of cycloastragenol and 5-100 μmol / L of metformin hydrochloride.

[0012] The proliferation culture medium for long-term stable passage of bovine-derived cells as described above comprises 1-10 μmol / L of cycloastragenol and 5-20 μmol / L of metformin hydrochloride.

[0013] The proliferation culture medium for long-term stable passage of bovine-derived cells as described above, wherein the basal medium is any one of DMEM medium, MEM medium, DMEM / F12 medium, Ham's F-12K medium and Leibovitz L-15 medium.

[0014] The proliferation culture medium for long-term stable passage of bovine-derived cells as described above, wherein the complete culture medium comprises, in terms of volume percentage, 85-94 vol% of basal medium, 5-10 vol% of fetal bovine serum and 1-2 vol% of penicillin-streptomycin double-antibiotic solution.

[0015] The second aspect of the present application provides a method for promoting long-term stable passage of bovine-derived cells, which uses the proliferation culture medium for long-term stable passage of bovine-derived cells to culture bovine-derived cells.

[0016] The method for promoting long-term stable passage of bovine-derived cells as described above comprises the following steps:

[0017] The bovine-derived cells are inoculated into the proliferation culture medium at a certain inoculation density, and the culture medium is replaced once every 1-3 days; when the cell confluence reaches 80%-90%, the cells are subcultured in the proliferation culture medium at a ratio of 1:3.

[0018] The method for promoting long-term stable passage of bovine-derived cells as described above, wherein the bovine-derived cells comprise bovine embryonic fibroblasts.

[0019] The method for promoting long-term stable passage of bovine-derived cells as described above, wherein the certain inoculation density is 0.3×10 5 -2×105 about 1 x 106cells / mL.

[0020] The third aspect of the present application provides a use of the proliferation culture medium for long-term stable passage of bovine-derived cells in the preparation of cell culture meat.

[0021] The scheme of the present application has at least the following effects:

[0022] The proliferation culture medium for long-term stable passage of bovine-derived cells provided by the present application comprises complete culture medium, cycloastragenol and metformin hydrochloride. Research shows that the proliferation culture medium supports BFFs to be continuously passaged to 145 generations, and the cell type of the bovine embryonic fibroblast cell line passaged to 145 generations is unchanged, having the advantage of long-term stable passage. The proliferation culture medium can make bovine embryonic fibroblasts (BFFs) break through the proliferation limit and form a continuous cell line during in vitro passage culture, so that the BFFs have stronger proliferation capacity, meet the demand of cell culture meat for large-scale production for the number of cells, and are conducive to the production of cell culture meat products with excellent quality; the BFFs induced and cultured by the proliferation culture medium break through the aging limit, realize long-term stable passage (≥100 generations) and maintain a positive rate of >90% of vimentin, and meet the demand of cell culture meat for mass production; the BFFs cultured by the proliferation culture medium can be continuously passaged more than 100 times, which provides a new idea for the establishment method of bovine embryonic fibroblast cell lines, and has very broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] Figure 1 are microscope images of primary BEFs and P1 BEFs in the test example of the present application, wherein, Figure 1 a of is a microscope image of primary BEFs, Figure 1 b of is a microscope image of P1 BEFs;

[0025] Figure 2 are the effects of different proliferation culture media on the aging of bovine embryonic fibroblasts in the test example of the present application, wherein, Figure 2 a of is a microscope image of P12 BEFs passaged and cultured according to the above method using the culture medium A in Example 1; Figure 2 b of is a microscope image of P12 BEFs passaged and cultured according to the above method using the culture medium B in Comparative Example 1;

[0026] Figure 3 Effects of different proliferation media on the senescence of bovine embryonic fibroblasts in the test example of the present application, wherein, Figure 3 a is a microscope image of P12 generation BEFs after β-galactosidase staining by subculture using medium A in Example 1 according to the above method; Figure 3 b is a microscope image of P12 generation BEFs after β-galactosidase staining by subculture using medium B in Comparative Example 1 according to the above method;

[0027] Figure 4 Effects of different proliferation media on the population doubling time of bovine embryonic fibroblasts in the test example of the present application;

[0028] Figure 5 Effects of different proliferation media on the expression level of telomerase reverse transcriptase (TERT) gene of bovine embryonic fibroblasts;

[0029] Figure 6 Effects of different proliferation media on the expression level of tumor protein p53 (P53) gene of bovine embryonic fibroblasts;

[0030] Figure 7 A microscope image of P145 generation BEFs by culturing bovine embryonic fibroblasts with medium A in Example 1 in the test example of the present application;

[0031] Figure 8 A microscope image of P145 generation BEFs after staining P145 generation BEFs by using a vimentin immunofluorescence method by culturing bovine embryonic fibroblasts with medium A in Example 1 in the test example of the present application, wherein, Figure 7 a is a staining result of fibroblast marker vimentin of P145 generation BEFs, Figure 7 b is a staining result of the nucleus of P145 generation BEFs, Figure 7 c is a combined image of vimentin and the nucleus of P145 generation BEFs. DETAILED DESCRIPTION

[0032] In order to make the objectives, technical solutions and advantages of the present application clearer, the following will combine embodiments of the present application to make a clear and complete description of the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application. If a specific technology or condition is not specified in the embodiments, it is performed according to the technology or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained from the market.

[0033] Those skilled in the art should understand that, in the following description of the embodiments of the present application, the order of the serial numbers does not mean the order of execution, and some or all steps can be executed in parallel or in sequence, and the execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0034] Those skilled in the art should understand that the numerical ranges in the embodiments of the present application should be understood as also specifically disclosing each intermediate value between the upper limit and the lower limit of the range. Each smaller range within any stated range or within a stated range of intermediates, as well as any other stated or intermediate range, is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0035] Unless otherwise specified, the technical / scientific terms used herein have the same meaning as generally understood by those of ordinary skill in the art to which the present application belongs.

[0036] Based on the bovine fetal fibroblasts (BFFs) indicated in the background art, the bovine fetal fibroblasts gradually age during the traditional in vitro subculture process, and the proliferation ability significantly decreases after the 12th generation, which is difficult to meet the demand for the number of cells for large-scale production of cell culture meat, and cannot break through the proliferation limit under traditional culture conditions, which cannot meet the demand for mass production of cell culture meat.

[0037] Based on this, in a first aspect, the present application provides a proliferation culture medium for long-term stable subculture of bovine cells, the components of the proliferation culture medium comprising complete culture medium, cycloastragenol and metformin hydrochloride.

[0038] The complete culture medium comprises, by volume percentage: 79-94 vol% of basal medium, 5-20 vol% of fetal bovine serum, and 1-3 vol% of penicillin-streptomycin double-antibiotic solution.

[0039] In the present application, the combination of each component in the proliferation culture medium can make bovine cells (the present application is bovine embryonic fibroblasts (BFFs)) break through the proliferation limit and form a continuous cell line during in vitro subculture, so that it has stronger proliferation capacity, breaks through the aging limit, and realizes long-term stable subculture.

[0040] In the present application, Cycloastragenol is a telomerase activator (TAT2), which promotes telomere DNA synthesis by activating telomerase reverse transcriptase (TERT) transcription. After adding Cycloastragenol in the culture medium, it can maintain telomere length and delay replicative senescence, and can simultaneously activate the PI3K / Akt signaling pathway, inhibit the expression of pro-apoptotic factor BAX (BCL2-associated X protein), and reduce the apoptosis rate of cells; Metformin hydrochloride can induce the expression of autophagy-related genes (LC3-II / Beclin1), clear senescence-related beta-galactosidase, and inhibit the generation of advanced glycosylation end products (AGEs), block their binding to receptors (RAGE), and reduce the level of intracellular reactive oxygen species (ROS), thereby protecting telomeres from oxidative damage.

[0041] In the present application, Cycloastragenol and Metformin hydrochloride are used together to prepare a proliferation culture medium. Through the proliferation culture medium, the following can be achieved: (1) telomere dynamic maintenance: Cycloastragenol extends the telomere, and Metformin hydrochloride reduces the erosion of ROS on the telomere, so that the stability of the telomere is increased; (2) metabolic-epigenetic coupling: Metformin hydrochloride inhibits the mTORC1 (Mammalian Target of Rapamycin Complex 1) pathway, reduces the ribosome biosynthesis burden, and reduces protein misfolding, and Cycloastragenol enhances TERT activity through the Akt pathway, so that the expression of TERT under mTOR (mammalian target of rapamycin) inhibition is improved, which helps to maintain the length of the telomere; (3) stress resistance balance: Metformin hydrochloride can induce protective autophagy, and Cycloastragenol inhibits excessive activation of autophagy, so as to realize the optimization of autophagy flow homeostasis.

[0042] It has been shown that metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase 7, DOI: 10.1111 / acel.12765, metformin hydrochloride can prolong the limited replicative life span of cells, but does not change the essential properties of the limited replication, and does not make the limited cell line into a continuous cell line capable of unlimited proliferation. The proliferation medium containing cycloastragenol and metformin hydrochloride used in the present application supports the continuous passage of BFFs to 145 generations, and the cell type of the bovine embryonic fibroblast cell line passed to 145 generations is unchanged, having the advantage of long-term stable passage. The proliferation medium can make bovine embryonic fibroblasts (BFFs) break through the proliferation limit and form a continuous cell line during in vitro subculture, so that they have stronger proliferative capacity, meet the demand for the number of cells for large-scale production of cell culture meat, and are conducive to the production of cell culture meat products of excellent quality; the proliferation medium induces the culture of BFFs to break through the aging limit, realize long-term stable passage (≥100 generations) and maintain a positive rate of >90% of vimentin, meet the demand for mass production of cell culture meat; the use of the proliferation medium for culturing BFFs can continuously subculture BFFs for more than 100 times, providing a new idea for the establishment method of bovine embryonic fibroblast cell lines, and having very broad application prospects.

[0043] In some embodiments, the concentration of cycloastragenol in the proliferation medium is 0.1-20 μmol / L, and the concentration of metformin hydrochloride is 5-100 μmol / L, based on the final concentration.

[0044] When the concentration of cycloastragenol and the concentration of metformin hydrochloride in the proliferation medium are in the above range, bovine cells (in the present embodiment, bovine embryonic fibroblasts (BFFs)) can break through the proliferation limit, form a continuous cell line, have stronger proliferative capacity, and realize long-term stable passage.

[0045] Further, the concentration of cycloastragenol is 1-10 μmol / L, and the concentration of metformin hydrochloride is 5-20 μmol / L. The use of cycloastragenol and metformin hydrochloride in the above concentrations can further make bovine cells (in the present embodiment, bovine embryonic fibroblasts (BFFs)) break through the proliferation limit, form a continuous cell line, have stronger proliferative capacity, and realize long-term stable passage.

[0046] In some embodiments, the base medium is any one of DMEM medium, MEM medium, DMEM / F12 medium, Ham's F-12K medium, and Leibovitz L-15 medium.

[0047] In some embodiments, the complete medium comprises, in volume percent: basal medium 85-94 vol%, fetal bovine serum 5-10 vol%, penicillin-streptomycin double-antibiotic solution 1-2 vol%.

[0048] In the present application, the basal medium is the basic guarantee for cell growth, and contains various amino acids, vitamins, carbohydrates and other basic nutrients necessary for cell survival and proliferation, providing the core substrate for cell metabolism; fetal bovine serum can supplement various natural growth factors, hormones, minerals and adhesion factors, etc., which helps to promote cell proliferation, maintain normal morphology and physiological function of cells, and also enhances the adaptability of cells to environmental changes; the penicillin-streptomycin double-antibiotic solution can effectively inhibit the contamination of microorganisms such as gram-positive bacteria and negative bacteria that may exist in the medium, thereby creating a sterile and stable environment for cell growth, and improving the success rate and stability of cell culture.

[0049] In a second aspect, the present application provides a method for promoting long-term stable passage of bovine-derived cells, which uses the proliferation medium for long-term stable passage of bovine-derived cells to culture bovine-derived cells. The inventors have found that the proliferation medium can break through the proliferation limit of bovine fetal fibroblasts (BFFs) during in vitro subculture and form a continuous cell line; the BFFs induced by the above-mentioned proliferation medium break through the aging limit and realize long-term stable passage (≥100 passages) with a positive rate of >90% of vimentin. Therefore, the bovine-derived cells can be cultured using the above-mentioned proliferation medium.

[0050] The method for promoting long-term stable passage of bovine-derived cells comprises the following steps:

[0051] The bovine-derived cells are inoculated into the proliferation medium at a certain inoculation density, and the medium is replaced once every 1-3 days; when the cell confluence reaches 80%-90%, the cells are subcultured in the proliferation medium at a ratio of 1:3.

[0052] In some embodiments, the bovine-derived cells comprise bovine fetal fibroblasts.

[0053] In some embodiments, the certain inoculation density is 0.3x10 5 -2x10 5 cells / mL.

[0054] When the inoculation density is in the above range, the following effects are achieved: (1) guaranteeing cell survival rate: at this density, the distance between cells is moderate, which can avoid the difficulty of cell survival and adhesion (especially for adherent cells which rely on cell-cell interaction) due to the lack of paracrine factors secreted by adjacent cells, and can also reduce early cell apoptosis caused by excessive initial nutrient competition and rapid accumulation of metabolic waste due to too high density; (2) promoting cell proliferation and phenotype stability: at this density, cells can gradually reach confluence during culture, and the growth rhythm can be regulated by normal contact inhibition, so that the biological phenotype (such as morphology and functional marker expression) can be maintained stable; if the density is too low, the cell proliferation cycle will be prolonged, and even growth arrest will occur; if the density is too high, the cells are prone to stacking, abnormal differentiation or functional disorder; (3) improving experimental repeatability and efficiency: at this density, the cells can reach the growth state required for experiments (such as logarithmic growth phase) within a conventional culture period, and the growth curves of cells cultured in different batches are highly consistent, which reduces experimental errors caused by differences in initial density and improves the reliability of subsequent detection results.

[0055] In a third aspect, the present application provides a use of the proliferation culture medium for long-term stable passage of bovine-derived cells in the preparation of cell culture meat. Studies have shown that the proliferation culture medium can enable bovine fetal fibroblasts (BFFs) to break through the proliferation limit and form a continuous cell line during in vitro subculture, so that they have stronger proliferation capacity; the BFFs induced and cultured by the above-mentioned proliferation culture medium break through the aging limit and achieve long-term stable passage (≥100 passages) while maintaining a positive rate of >90% of vimentin. Therefore, in the preparation of cell culture meat, the proliferation culture medium can be used to culture seed cells (bovine-derived cells), so that the seed cells break through the proliferation limit and form a continuous cell line during in vitro subculture, break through the aging limit, achieve long-term stable passage, meet the demand for the number of cells for large-scale production of cell culture meat, and meet the demand for mass production of cell culture meat.

[0056] In the following, the present application will be further described through specific examples.

[0057] In the following examples, DMEM medium was purchased from Gibco with the item number 31600083; fetal bovine serum was purchased from Zhejiang Tianhang Biology with the item number 11011-8611; penicillin-streptomycin double-antibiotic solution was purchased from Wuhan Punuo Sai with the item number PB180120

[0058] Example 1

[0059] The proliferation culture medium for long-term stable passage of bovine-derived cells provided in this example is composed of the following components: complete culture medium, cycloastragenol 5 μmol / L, metformin hydrochloride 10 μmol / L;

[0060] The complete culture medium is prepared by DMEM medium at 94 vol%, fetal bovine serum at 5 vol% and penicillin-streptomycin double-antibiotic solution at 1 vol%.

[0061] The preparation method of the proliferation culture medium for long-term stable passage of bovine cells provided in the embodiment comprises the following steps:

[0062] On the clean bench, DMEM medium, fetal bovine serum and penicillin-streptomycin double-antibiotic solution are prepared at a volume ratio of 94 vol%:5 vol%:1 vol% to obtain the complete culture medium.

[0063] Cycloastragenol is dissolved in dimethyl sulfoxide (DMSO) to obtain a cycloastragenol mother liquor with a concentration of 10 mmol / L.

[0064] Metformin hydrochloride is dissolved in deionized water, filtered with a 0.22 μm filter to remove bacteria, and a metformin hydrochloride mother liquor with a concentration of 50 mmol / L is prepared.

[0065] The cycloastragenol mother liquor and the metformin hydrochloride mother liquor are added to the complete culture medium and mixed uniformly, so that the final concentration of cycloastragenol is 5 μmol / L and the final concentration of metformin hydrochloride is 10 μmol / L. The bacteria are removed by filtering with a 0.22 μm filter to obtain the proliferation culture medium for long-term stable passage of bovine cells (denoted as culture medium A).

[0066] Comparative Example 1 (ordinary 5% fetal bovine serum culture medium)

[0067] The proliferation culture medium provided in the comparative example is prepared by DMEM medium at 94 vol%, fetal bovine serum at 5 vol% and penicillin-streptomycin double-antibiotic solution at 1 vol%.

[0068] The preparation method of the proliferation culture medium provided in the comparative example comprises the following steps:

[0069] On the clean bench, DMEM medium, fetal bovine serum and penicillin-streptomycin double-antibiotic solution are prepared at a volume ratio of 94 vol%:5 vol%:1 vol% to obtain the proliferation culture medium (denoted as culture medium B).

[0070] Test Example

[0071] 1. Effect of different proliferation culture media on aging of bovine embryonic fibroblasts

[0072] The culture medium A in the above embodiment 1 and the culture medium B in the comparative example 1 are respectively used as the proliferation culture medium, and the effect of each proliferation culture medium on the aging of bovine embryonic fibroblasts is tested. The specific method comprises:

[0073] Primary culture: the head, limbs, internal organs of fetal calf (purchased at slaughterhouse) are removed to retain the trunk, the trunk is cut into 3mm*1cm pieces, washed with physiological saline for 6 times, after washing, the pieces are cut into 1mm*1mm*1mm tissue blocks, i.e. primary bovine embryonic fibroblasts (primary BEFs), the primary BEFs and the primary culture medium are mixed thoroughly, the primary BEFs and the primary culture medium are evenly spread in the culture bottle, and the culture bottle is placed in a 37℃, 5%CO2 incubator, the fresh primary culture medium is replaced every 3 days, when the cells grow to 70%-80% confluence, the cells are subcultured at a ratio of 1:3 to P1 generation, and P1 generation bovine embryonic fibroblasts (P1 generation BEFs) are obtained.

[0074] Subculture: the P1 generation bovine embryonic fibroblasts (P1 generation BEFs) are inoculated into a 6-well plate at 1×10 5 cells / well, 2mL of proliferation culture medium is added for culture, the culture medium is replaced every 3 days, when the cell confluence reaches 80%-90%, the cells are subcultured in the proliferation culture medium at a ratio of 1:3; when the subculture reaches the 12th generation (P12), P12 generation BEFs are obtained.

[0075] The P12 generation BEFs are photographed and recorded using a microscope, and the morphology of the P12 generation BEFs is observed, as shown in Figure 2 .

[0076] In order to detect the aging state of the P12 generation BEFs, β-galactosidase staining is used for cell aging characterization analysis, and the specific operation is as follows: after the above P12 generation BEFs are washed with PBS for three times, the cells are stained according to the cell aging β-galactosidase staining kit instruction (Biyun Tian, C0602), after staining, the cell images after staining are collected using a microscope, as shown in Figure 3 .

[0077] Figure 1 The microscope images of the primary BEFs and the P1 generation BEFs in the test example of the present application are shown in Figure 1 , wherein, Figure 1 a is the microscope image of the primary BEFs, b is the microscope image of the P1 generation BEFs; Figure 2 The effects of different proliferation culture media on the aging of bovine embryonic fibroblasts in the test example of the present application are shown in Figure 2 , wherein, Figure 2 a is the microscope image of the P12 generation BEFs subcultured using the culture medium A in Example 1 according to the above method; b is the microscope image of the P12 generation BEFs subcultured using the culture medium B in Comparative Example 1 according to the above method.

[0078] Figure 3 This invention illustrates the effects of different proliferation culture media on the senescence of bovine embryonic fibroblasts in experimental examples. Figure 3 a is a microscopic image of P12 generation BEFs after subculturing using culture medium A in Example 1 according to the above method and staining with β-galactosidase. Figure 3 b is a microscopic image of P12 generation BEFs after subculturing using culture medium B in Comparative Example 1 according to the above method and staining with β-galactosidase.

[0079] Depend on Figure 2 It can be seen that the P12 generation BEFs cultured using culture medium B in Comparative Example 1 exhibited a senescent morphology, with senescent main axis morphology, increased cell diameter, and accumulation of characteristic vacuoles; while the P12 generation BEFs cultured using culture medium A in Example 1 had clear edges, spindle-shaped cells, tightly arranged cells, clear nuclei, and no senescence was observed, and the cell morphology was significantly better than that of culture medium B.

[0080] As shown in Figure 3, the P12 generation BEFs cultured using culture medium B in Comparative Example 1 exhibited a large amount of senescence marker β-galactosidase blue precipitate, indicating that a large number of cells entered the senescent state; while the P12 generation BEFs cultured using culture medium A in Example 1 did not show β-galactosidase blue precipitate, indicating that the cells did not show senescence, proving that the culture medium A in Example 1 of this invention can resist BEFs senescence and promote BEFs to break through the passage limit.

[0081] 2. Effects of different proliferation media on doubling time of bovine embryonic fibroblast populations

[0082] Using culture medium A from Example 1 and culture medium B from Comparative Example 1 as proliferation media, the effect of each proliferation medium on the doubling time of bovine embryonic fibroblast populations was tested. Specific methods included:

[0083] Bovine embryonic fibroblasts (P1 generation BEFs) were obtained using the primary culture method described above.

[0084] P1 generation bovine embryonic fibroblasts (P1 generation BEFs) were used at a concentration of 1×10⁻⁶. 5 Seed 10 cells / well into a 6-well plate, add 2 mL of proliferation medium and culture. Change the medium every 3 days. When the cell confluence reaches 80% to 90%, place the cells in proliferation medium and passage them at a ratio of 1:3. When passaged to the 12th generation (P12), P12 generation BEFs are obtained.

[0085] At 37°C, P12 generation BEFs were digested with 0.25% trypsin for 5 minutes, followed by thorough elution with PBS buffer. Cell counts were then performed using a cell counter, and the cell population was recorded. The cell population doubling formula is: Td = t × log2 / log(N). t / N0)(Td represents the cell population doubling time, t represents the actual culture time, N0 represents the initial cell number, N t The population doubling time of cells (representing the terminal cell count) is calculated, and the results are as follows: Figure 4 As shown.

[0086] Figure 4 To illustrate the effect of different proliferation culture media on the doubling time of bovine embryonic fibroblast populations in the experimental examples of this invention, [the following text is missing]. Figure 4 It can be seen that the doubling time of the P12 generation BEFs population cultured using culture medium A in Example 1 was (32±1.2) hours; while the doubling time of the P12 generation BEFs population cultured using culture medium B in Comparative Example 1 was (107±4.4) hours. This indicates that the P12 generation BEFs cultured using culture medium B in Comparative Example 1 experienced stunted proliferation, while the P12 generation BEFs cultured using culture medium A in Example 1 still proliferated vigorously. The above results demonstrate that using culture medium A in Example 1 of this invention can enable bovine embryonic fibroblasts (BFFs) to break through the proliferation limit and form a continuous cell line during in vitro passage culture, thus giving them a stronger proliferative capacity.

[0087] 3. Effects of different proliferation media on the expression levels of telomerase reverse transcriptase (TERT) and tumor protein p53 (P53) genes in bovine embryonic fibroblasts.

[0088] Using culture medium A from Example 1 and culture medium B from Comparative Example 1 as proliferation media, the effects of each proliferation medium on the expression levels of TERT and p53 genes in bovine embryonic fibroblasts were tested. Specific methods included:

[0089] Bovine embryonic fibroblasts (P1 generation BEFs) were obtained using the primary culture method described above.

[0090] P1 generation bovine embryonic fibroblasts (P1 generation BEFs) were used at a concentration of 1×10⁻⁶. 5 Seed 10 cells / well into a 6-well plate, add 2 mL of proliferation medium and culture. Change the medium every 3 days. When the cell confluence reaches 80% to 90%, place the cells in proliferation medium and passage them at a ratio of 1:3. When passaged to the 12th generation (P12), P12 generation BEFs are obtained.

[0091] The P12 BEFs were digested with 0.25% trypsin at 37°C for 5 minutes, washed with PBS buffer, centrifuged, and the cells were collected. The total RNA of the collected cells was extracted according to the instructions of the total RNA extraction kit (Tiangen Biotech, DP451), and the RNA was transcribed into cDNA using the reverse transcription kit (Takara, RR047A). Finally, the expression levels of TERT and p53 genes were detected by qPCR (Takara, RR820A), and the results are shown in Figure 5 and Figure 6 .

[0092] Figure 5 Effect of different proliferation culture media on the expression level of telomerase reverse transcriptase (TERT) gene of bovine embryonic fibroblasts; Figure 6 Effect of different proliferation culture media on the expression level of tumor protein p53 (P53) gene of bovine embryonic fibroblasts.

[0093] As can be seen from Figure 5 and Figure 6 , compared with the medium B in Comparative Example 1, the P12 BEFs cultured with the medium A in Example 1 have a significantly increased expression level of TERT gene and a significantly decreased expression level of p53 gene, which proves that the medium A in Example 1 of the present application enables the BEFs to have long-term division ability.

[0094] 4. Culturing bovine embryonic fibroblasts with the medium A in Example 1

[0095] The medium A in Example 1 above was used as the proliferation culture medium to culture bovine embryonic fibroblasts, and the specific method comprises the following steps:

[0096] The P1 bovine embryonic fibroblasts (P1 BEFs) were obtained according to the primary culture method described above.

[0097] The P1 bovine embryonic fibroblasts (P1 BEFs) were inoculated into a 6-well plate at a concentration of 1×10 5 cells / well, and 2 mL of the proliferation culture medium was added for culture. The culture medium was replaced every 3 days, and when the cell confluence reached 80%-90%, the cells were subcultured in the proliferation culture medium at a ratio of 1:3. When the subculture reached the 145th generation (P145), the P145 BEFs were obtained.

[0098] The P145 BEFs were photographed and recorded using a microscope, and the morphology of the P145 BEFs was observed, as shown in Figure 7 .

[0099] The P145 BEFs were stained by wave protein immunofluorescence method, and the results are shown in Figure 8 .

[0100] Depend on Figure 7 It can be seen that the P145 generation BEFs obtained by culture in culture medium A in Example 1 have clear edges, spindle-shaped cells, are tightly arranged, have clear nuclei, and are normal in morphology.

[0101] Depend on Figure 8 It can be seen that after BEFs cells were cultured for 145 generations using culture medium A in Example 1, the vimentin positivity rate was ≥95%, proving that BEFs cells cultured using culture medium A in Example 1 can maintain the cell type unchanged after long-term passage of BEFs cells, and have the advantage of long-term stable passage.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A proliferation culture medium for long-term stable passage of bovine cells, characterized in that, The proliferation medium consists of complete medium, cycloastragalool, and metformin hydrochloride; The complete culture medium comprises, by volume percentage: 79-94 vol% basal culture medium, 5-20 vol% fetal bovine serum, and 1-3 vol% penicillin-streptomycin solution.

2. The proliferation culture medium for long-term stable passage of bovine cells according to claim 1, characterized in that, Based on the final concentration, the concentration of cycloastragalool in the proliferation medium is 0.1–20 μmol / L, and the concentration of metformin hydrochloride is 5–100 μmol / L.

3. The proliferation culture medium for long-term stable passage of bovine cells according to claim 2, characterized in that, The concentration of cycloastragalool is 1–10 μmol / L, and the concentration of metformin hydrochloride is 5–20 μmol / L.

4. The proliferation culture medium for long-term stable passage of bovine cells according to claim 1, characterized in that, The basal culture medium is any one of DMEM medium, MEM medium, DMEM / F12 medium, Ham's F-12K medium, and Leibovitz L-15 medium.

5. The proliferation culture medium for long-term stable passage of bovine cells according to claim 1, characterized in that, The complete culture medium comprises, by volume percentage: 85-94 vol% basal culture medium, 5-10 vol% fetal bovine serum, and 1-2 vol% penicillin-streptomycin solution.

6. A method for promoting long-term stable passage of bovine cells, characterized in that, Bovine cells are cultured using the proliferation medium for long-term stable passage of bovine cells as described in any one of claims 1-5.

7. The method for promoting long-term stable passage of bovine cells according to claim 6, characterized in that, Includes the following steps: Bovine cells were inoculated into the proliferation medium at a certain inoculation density and cultured, with the medium being changed every 1-3 days. When the cell confluence reached 80%-90%, the cells were placed in the proliferation medium and passaged at a ratio of 1:

3.

8. The method for promoting long-term stable passage of bovine cells according to claim 7, characterized in that, The bovine-derived cells include bovine embryonic fibroblasts.

9. The method for promoting long-term stable passage of bovine cells according to claim 7, characterized in that, The specified inoculation density is 0.3 × 10⁻⁶. 5 ~2×10 5 Cells / mL.

10. The use of the proliferation medium for long-term stable passage of bovine cells as described in any one of claims 1-5 in the preparation of cultured meat.

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