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 applications thereof

Abstract

The application provides a proliferation culture medium for long-term stable passage of bovine cells, a method for promoting long-term stable passage of bovine cells and application. The components of the proliferation culture medium include complete culture medium, cycloastragenol and metformin hydrochloride. Research shows that the proliferation culture medium can make bovine fetal fibroblasts (BFFs) break through the proliferation limit in the process of in-vitro passage culture, form a continuous cell line, and be beneficial to the production of cell culture meat products of excellent quality. The culture medium provided by the application can support the continuous passage of BFFs to 145 generations, and the cell type of the bovine fetal fibroblast cell line passed to 145 generations is unchanged, having the advantage of long-term stable passage. The induction by the proliferation culture medium can make BFFs break through the aging limit, realize long-term stable passage (≥100 generations) and maintain a positive rate of >90% of vimentin, meet the demand of cell culture meat mass production for the number of cells, and have very broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and particularly relates to a proliferation culture medium for long-term stable passage of bovine cells, a method for promoting long-term stable passage of bovine cells, and its application. Background Technology

[0002] Beef is known for its delicious taste and rich nutritional value, containing abundant protein, fat, B vitamins, niacin, calcium, phosphorus, iron, cholesterol, and other components. It is widely favored by consumers for its health benefits, including invigorating the stomach, promoting recovery, and aiding weight loss. However, with increasing demand for beef, the contradiction between declining cattle resources and growing demand is becoming increasingly prominent, necessitating the development of sustainable alternatives.

[0003] Cell-cultured meat technology produces meat by expanding animal cells (i.e., seed cells) in vitro, which can significantly alleviate the environmental burden (such as greenhouse gas emissions and excessive resource consumption) and animal welfare problems caused by traditional animal husbandry. This technology only requires culture medium, specific temperature and humidity, and carbon dioxide to provide the necessary environment for nutrition and growth, without causing environmental pollution. It does not consume the feed and water required in traditional meat production, and it requires less space, saving space costs. Therefore, it represents a major research and development direction for future artificial meat. Thus, developing bovine cell-cultured meat technology to produce bovine-derived cell-cultured meat is an effective means of addressing the decline of bovine resources.

[0004] Currently, the seed cells used for producing bovine cell-cultured meat mainly include bovine embryonic fibroblasts (BEFs), bovine embryonic stem cells (ESCs), bovine pluripotent stem cells (iPSCs), and immortalized / gene-edited cell lines. While ESCs and iPSCs possess unlimited proliferative potential, their culture relies on feeder layers or complex combinations of factors (such as bFGF, Activin A, LIF, and multiple pathway inhibitors), resulting in high costs and a tendency for spontaneous differentiation, making them unsuitable for large-scale production of cell-cultured meat. Immortalized / gene-edited cell lines (such as highly efficient CRISPR-Cas9-edited transformation cell lines) require the introduction of exogenous genes, facing regulatory barriers to genetically modified foods (such as the EU EFSA requiring a 5-10 year safety assessment) and insufficient consumer acceptance (studies show that over 60% of consumers prefer non-GMO cell-cultured meat). In contrast, BEFs, due to their easy availability, non-GMO nature, and strong proliferative capacity, are seed cells with greater commercial potential. However, there are still some problems when using BEFs as seed cells to produce cultured meat. For example, BEFs gradually age during traditional in vitro passage, and their proliferation capacity usually decreases significantly after the 12th generation, making it difficult to meet the cell quantity requirements for large-scale production of cultured meat. Under traditional culture conditions, they cannot break through the proliferation limit and cannot meet the mass production needs of cultured meat.

[0005] Therefore, developing a novel culture system that allows for the long-term stable passage of seed cells without genetic manipulation and at low cost to promote the industrialization of bovine cell-cultured meat is an urgent problem to be solved. Summary of the Invention

[0006] This invention provides a proliferation medium for the long-term stable passage of bovine embryonic fibroblasts (BEFs). This medium supports continuous passage of BEFs up to 145 generations, maintaining the same cell type throughout the 145 passages, thus offering the advantage of long-term stable passage. This medium allows BEFs to achieve stable passages of ≥100 generations in vitro, enhancing their proliferative capacity and meeting the cell quantity requirements for large-scale production of cultured meat, thus promoting the production of high-quality cultured meat products. Induction culture using this medium significantly extends the in vitro replication lifespan of BEFs, greatly delaying replicative senescence, achieving stable passage (≥100 generations) and maintaining a >90% vimentin positivity rate, meeting the demands of mass production of cultured meat. Using this medium to culture BEFs allows for more than 100 consecutive passages, providing a new approach to establishing bovine embryonic fibroblast lines and demonstrating broad application prospects.

[0007] This invention also provides a method for promoting long-term stable passage of bovine cells, using the aforementioned proliferation medium to culture bovine cells. Studies have shown that this proliferation medium enables bovine embryonic fibroblasts (BEFs) to achieve stable passages of ≥100 generations during in vitro passage culture, giving them stronger proliferative capacity. Induction culture using this proliferation medium significantly prolongs the in vitro replication lifespan of bovine embryonic fibroblasts, greatly delays replicative senescence, achieves stable passage (≥100 generations), and maintains a vimentin positivity rate of >90%. This method allows for continuous passage of BEFs more than 100 times, and is characterized by ease of operation, stability, and reproducibility, providing a new approach for establishing bovine embryonic fibroblast cell lines.

[0008] This invention also provides the application of the above-mentioned proliferation medium for long-term stable passage of bovine cells in the preparation of cultured meat. Studies have shown that this proliferation medium enables bovine embryonic fibroblasts (BEFs) to achieve stable passage ≥100 generations during in vitro passage culture, giving them stronger proliferative capacity. Induction culture using this proliferation medium can significantly extend the in vitro replication lifespan of bovine embryonic fibroblasts, greatly delay replicative senescence, achieve stable passage (≥100 generations), and maintain a vimentin positivity rate of >90%. Therefore, in the preparation of cultured meat, this proliferation medium can be used to culture seed cells (bovine cells), enabling them to achieve stable passage ≥100 generations during in vitro passage culture, significantly delaying replicative senescence, achieving long-term stable passage, and meeting the cell quantity requirements for large-scale production of cultured meat.

[0009] A first aspect of the present invention provides a proliferation medium for long-term stable passage of bovine cells, wherein the proliferation medium comprises a complete culture medium, cycloastragalol, and metformin hydrochloride.

[0010] 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.

[0011] The proliferation medium for long-term stable passage of bovine cells as described above, based on the final concentration, contains cycloastragalool at a concentration of 0.1–20 μmol / L and metformin hydrochloride at a concentration of 5–100 μmol / L.

[0012] In the proliferation medium for long-term stable passage of bovine cells as described above, the concentration of cycloastragalool is 1–10 μmol / L, and the concentration of metformin hydrochloride is 5–20 μmol / L.

[0013] The proliferation medium for long-term stable passage of bovine 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 medium for long-term stable passage of bovine cells as described above comprises, by volume percentage: 85-94 vol% basal medium, 5-10 vol% fetal bovine serum, and 1-2 vol% penicillin-streptomycin solution.

[0015] A second aspect of the present invention provides a method for promoting long-term stable passage of bovine cells, wherein bovine cells are cultured using the aforementioned proliferation medium for long-term stable passage of bovine cells.

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

[0017] 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.

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

[0019] In the method for promoting long-term stable passage of bovine cells as described above, the specific inoculation density is 0.3 × 10⁻⁶. 5 ~2×10 5 Cells / mL.

[0020] A third aspect of the present invention provides the application of the proliferation medium described above for long-term stable passage of bovine cells in the preparation of cultured meat.

[0021] The solution of the present invention has at least the following effects:

[0022] The proliferation medium provided by this invention for the long-term stable passage of bovine embryonic fibroblasts (BEFs) comprises a complete medium, cycloastragalool, and metformin hydrochloride. Studies have shown that this proliferation medium supports continuous passage of BEFs up to 145 generations, and the cell type of the bovine embryonic fibroblast cell line remains unchanged after 145 passages, demonstrating the advantage of long-term stable passage. This proliferation medium enables bovine embryonic fibroblasts (BEFs) to achieve stable passage ≥100 generations during in vitro passage culture, giving them stronger proliferative capacity, meeting the cell quantity requirements for large-scale production of cultured meat, and facilitating the production of high-quality cultured meat products. Induction culture using this proliferation medium can significantly extend the in vitro replication lifespan of bovine embryonic fibroblasts, greatly delay replicative senescence, achieve stable passage (≥100 generations), and maintain a vimentin positivity rate of >90%, meeting the needs of mass production of cultured meat. Using this proliferation medium to culture BEFs allows for continuous passage more than 100 times, providing a new approach to the establishment of bovine embryonic fibroblast cell lines and possessing very broad application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 These are microscope images of primary BEFs and P1 generation BEFs from the experimental examples of this invention, wherein... Figure 1 'a' is a microscopic image of primary BEFs. Figure 1 b is a microscope image of P1 generation BEFs;

[0025] Figure 2 This invention illustrates the effects of different proliferation culture media on the senescence of bovine embryonic fibroblasts in experimental examples. Figure 2 a is a microscopic image of P12 generation BEFs after subculturing using culture medium A in Example 1 according to the above method; Figure 2 b is a microscopic image of BEFs from generation P12, which were cultured using culture medium B in Comparative Example 1 according to the above method.

[0026] 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 after β-galactosidase staining.

[0027] Figure 4 This illustrates the effect of different proliferation culture media on the doubling time of bovine embryonic fibroblast populations in the experimental examples of this invention.

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

[0029] Figure 6 The effect of different proliferation culture media on the expression level of bovine embryonic fibroblast tumor protein p53 (P53) gene;

[0030] Figure 7 This is a microscope image of bovine embryonic fibroblasts (BEFs) cultured in culture medium A in Example 1 of the present invention, at passage P145.

[0031] Figure 8 This is a microscopic image of P145 generation BEFs after being stained with vimentin immunofluorescence assay using bovine embryonic fibroblasts cultured in culture medium A as described in Example 1 of the present invention. Figure 7 a represents the staining results of vimentin, a fibroblast marker, in P145 generation BEFs. Figure 7b represents the staining results of the nuclei of P145 generation BEFs. Figure 7 c is a combined diagram of the vimentin and nucleus of P145 generation BEFs. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0033] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0034] Those skilled in the art will understand that the numerical ranges in the embodiments of the present invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] As mentioned in the background section, bovine embryonic fibroblasts (BEFs) gradually age during traditional in vitro passage, and their proliferative capacity usually declines significantly after the 12th generation, making it difficult to meet the cell quantity requirements for large-scale production of cell-cultured meat. Under traditional culture conditions, they cannot break through the proliferation limit and thus cannot meet the demand for mass production of cell-cultured meat.

[0037] Based on this, in a first aspect, the present invention provides a proliferation medium for long-term stable passage of bovine cells, wherein the components of the proliferation medium include complete medium, cycloastragalol and metformin hydrochloride.

[0038] 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.

[0039] In this invention, the combination of components in the proliferation culture medium enables bovine cells (in this embodiment, bovine embryonic fibroblasts (BEFs)) to achieve stable passage ≥100 times during in vitro passage culture, thereby giving them stronger proliferative capacity.

[0040] In this invention, cycloastragenol is a telomerase activator (TAT2), which promotes telomere DNA synthesis by activating telomerase reverse transcriptase (TERT) transcription. Adding cycloastragenol to the culture medium can maintain telomere length and delay replicative senescence. It can also simultaneously activate the PI3K / Akt signaling pathway, inhibit the expression of the pro-apoptotic factor BAX (BCL2-associated X protein), and reduce the apoptosis rate. Metformin hydrochloride can induce the expression of autophagy-related genes (LC3-II / Beclin1), clear senescence-related β-galactosidase, and inhibit the generation of advanced glycation end products (AGEs), blocking their binding to receptors (RAGEs), thus reducing intracellular reactive oxygen species (ROS) levels and protecting telomeres from oxidative damage.

[0041] This invention uses cycloastragalool and metformin hydrochloride to prepare a proliferation medium, which can achieve the following: (1) dynamic maintenance of telomeres: cycloastragalool prolongs telomeres, and metformin reduces ROS erosion of telomeres, thus increasing telomere stability; (2) metabolic-epigenetic coupling: metformin hydrochloride inhibits the mTORC1 (mammalian target of rapamycin complex 1) pathway, reducing the burden of ribosome biosynthesis and reducing protein misfolding. Cycloastragalool enhances TERT activity through the Akt pathway, thus increasing TERT expression under the inhibition of mTOR (mammalian target of rapamycin), which helps maintain telomere length; (3) stress resistance balance: metformin hydrochloride can induce protective autophagy, while cycloastragalool inhibits excessive activation of autophagy, thus optimizing autophagy flux homeostasis.

[0042] Previous studies (Metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase 7, DOI: 10.1111 / acel.12765) have shown that metformin hydrochloride can prolong the finite replication lifespan of cells, but does not change the essential property of "finite replication". This invention uses the aforementioned proliferation medium containing cycloastragaloyl alcohol and metformin hydrochloride to support continuous passage of BEFs to 145 generations, and the cell type of the bovine embryonic fibroblast cell line remains unchanged after 145 generations, demonstrating the advantage of long-term stable passage. This proliferation medium enables bovine embryonic fibroblasts (BEFs) to achieve stable passages of ≥100 generations during in vitro culture, giving them stronger proliferative capacity and meeting the cell quantity requirements for large-scale production of cultured meat. This is beneficial for producing high-quality cultured meat products. Induction culture using this proliferation medium can significantly extend the in vitro replication lifespan of bovine embryonic fibroblasts, greatly delay replication senescence, achieve stable passages (≥100 generations), and maintain a vimentin positivity rate of >90%, meeting the needs of mass production of cultured meat. Using this proliferation medium to culture BEFs, BEFs can be passaged more than 100 times continuously, providing a new approach to the establishment of bovine embryonic fibroblast cell lines and showing great application prospects.

[0043] In some embodiments, 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, based on the final concentration.

[0044] When the concentrations of cycloastragalool and metformin hydrochloride in the proliferation medium are within the above range, bovine cells (in this embodiment, bovine embryonic fibroblasts (BEFs)) can have strong proliferative capacity and achieve long-term stable passage.

[0045] Furthermore, the concentration of cycloastragalool is 1–10 μmol / L, and the concentration of metformin hydrochloride is 5–20 μmol / L. Using these concentrations of cycloastragalool and metformin hydrochloride can enhance the proliferative capacity of bovine cells (in this invention, bovine embryonic fibroblasts (BEFs)) and achieve long-term stable passage.

[0046] In some embodiments, 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.

[0047] In some embodiments, 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.

[0048] In this invention, the basal culture medium is the fundamental guarantee for cell growth, containing a variety of essential nutrients such as amino acids, vitamins, and carbohydrates necessary for cell survival and proliferation, providing core substrates for cell metabolism; fetal bovine serum can supplement a variety of natural growth factors, hormones, minerals, and adhesion factors, which helps promote cell proliferation, maintain normal cell morphology and physiological function, and enhance the cell's ability to adapt to environmental changes; penicillin-streptomycin dual antibiotic solution can effectively inhibit the contamination of Gram-positive and Gram-negative bacteria and other microorganisms that may exist in the culture 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 invention provides a method for promoting long-term stable passage of bovine cells, wherein the bovine cells are cultured using the aforementioned proliferation medium for long-term stable passage of bovine cells. The inventors' research shows that this proliferation medium enables bovine embryonic fibroblasts (BEFs) to achieve stable passage ≥100 generations during in vitro passage culture; induced culture using the above-mentioned proliferation medium can significantly prolong the in vitro replication lifespan of bovine embryonic fibroblasts, greatly delay replicative senescence, achieve stable passage (≥100 generations), and maintain a vimentin positivity rate >90%. Therefore, the above-mentioned proliferation medium can be used to culture bovine cells.

[0050] The above-mentioned method for promoting long-term stable passage of bovine cells includes the following steps:

[0051] 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.

[0052] In some embodiments, the bovine cells include bovine embryonic fibroblasts.

[0053] In some embodiments, the specified inoculation density is 0.3 × 10⁻⁶. 5 ~2×10 5 Cells / mL.

[0054] When the seeding density is within the above range, it has the following effects: (1) Ensure cell survival rate: At this density, the distance between cells is moderate, which can avoid the cells from being unable to survive and adhere due to the lack of paracrine factors secreted by adjacent cells due to low density (especially for adherent cells that depend on intercellular interactions), and can also reduce early cell apoptosis caused by excessive initial nutrient competition and rapid accumulation of metabolic waste due to high density; (2) Promote cell proliferation and phenotypic stability: At this density, cells can gradually reach the confluence state during the culture process. During this period, cells can regulate the growth rhythm through normal contact inhibition and maintain a stable biological phenotype (such as morphological and functional marker expression); if the density is too low, the cell proliferation cycle will be prolonged, or even growth arrest will occur; if the density is too high, cells are prone to stacking, abnormal differentiation, or functional disorders; (3) Improve experimental repeatability and efficiency: At this density, cells can reach the growth state required for the experiment within the conventional culture cycle (such as the logarithmic growth phase), and the growth curves of cells cultured in different batches are highly consistent, reducing experimental errors caused by initial density differences and improving the reliability of subsequent detection results.

[0055] In a third aspect, this invention provides the application of the aforementioned proliferation medium for long-term stable passage of bovine cells in the preparation of cultured meat. Studies have shown that this proliferation medium enables bovine embryonic fibroblasts (BEFs) to achieve stable passages of ≥100 generations during in vitro passage culture, giving them stronger proliferative capacity. Induction culture using the aforementioned proliferation medium can significantly extend the in vitro replication lifespan of bovine embryonic fibroblasts, greatly delay replicative senescence, achieve stable passage (≥100 generations), and maintain a vimentin positivity rate of >90%. Therefore, in the preparation of cultured meat, this proliferation medium can be used to culture seed cells (bovine cells), enabling the seed cells to achieve stable passages of ≥100 generations during in vitro passage culture, greatly delaying replicative senescence, achieving long-term stable passage, and meeting the cell quantity requirements for large-scale production of cultured meat.

[0056] The present invention will be further described below through specific embodiments.

[0057] In the following examples, DMEM culture medium was purchased from Gibco (catalog number 31600083); fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology (catalog number 11011-8611); and penicillin-streptomycin antibiotic solution was purchased from Wuhan Pronosai (catalog number PB180120).

[0058] Example 1

[0059] The proliferation medium for long-term stable passage of bovine cells provided in this embodiment consists of the following components: complete medium, cycloastragalool 5 μmol / L, and metformin hydrochloride 10 μmol / L.

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

[0061] The method for preparing the proliferation medium for long-term stable passage of bovine cells provided in this embodiment includes the following steps:

[0062] On a clean bench, DMEM culture medium, fetal bovine serum and penicillin-streptomycin solution were prepared in a volume ratio of 94 vol%: 5 vol%: 1 vol% to obtain complete culture medium;

[0063] Cycloastragalol was dissolved in dimethyl sulfoxide (DMSO) to prepare a cycloastragalol stock solution with a concentration of 10 mmol / L.

[0064] Metformin hydrochloride was dissolved in deionized water and filtered through a 0.22 μm filter membrane for sterilization to prepare a metformin hydrochloride stock solution with a concentration of 50 mmol / L.

[0065] The cycloastragalool stock solution and metformin hydrochloride stock solution were added to the complete culture medium and mixed evenly to make the final concentration of cycloastragalool 5 μmol / L and the final concentration of metformin hydrochloride 10 μmol / L. The mixture was then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining the proliferation culture medium (denoted as culture medium A) for long-term stable passage of bovine cells.

[0066] Comparative Example 1 (Standard 5% Fetal Bovine Serum Culture Medium)

[0067] The proliferation medium provided in this comparative example was prepared by using 94 vol% DMEM medium, 5 vol% fetal bovine serum, and 1 vol% penicillin-streptomycin solution.

[0068] The method for preparing the proliferation medium provided in this comparative example includes the following steps:

[0069] On a clean bench, DMEM medium, fetal bovine serum and penicillin-streptomycin solution were mixed in a volume ratio of 94 vol%: 5 vol%: 1 vol% to obtain proliferation medium (denoted as medium B).

[0070] Test case

[0071] 1. Effects of different proliferation culture media on senescence of bovine embryonic fibroblasts

[0072] 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 bovine embryonic fibroblast senescence were tested. Specific methods included:

[0073] Primary culture: Using surgical scissors, the head, limbs, and internal organs of the fetal calf (purchased from the slaughterhouse) are removed, leaving the torso. The torso is then cut into 3mm pieces. Take a 1cm sheet, wash it 6 times with saline solution, and then cut it into 1mm pieces. 1mm A 1 mm tissue block, or primary bovine embryonic fibroblasts (primary BEFs), was added to primary culture medium (89 vol% DMEM medium + 10 vol% fetal bovine serum + 1 vol% penicillin-streptomycin solution) and thoroughly mixed. The primary BEFs and primary culture medium were evenly spread in a culture flask and incubated at 37°C in a 5% CO2 incubator. Fresh primary culture medium was replaced every 3 days. When the cells reached 70%–80% confluence, they were passaged at a ratio of 1:3 to the P1 generation to obtain P1 generation bovine embryonic fibroblasts (P1 generation BEFs).

[0074] Passaging: Bovine embryonic fibroblasts (P1 generation BEFs) were cultured at a rate 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 the cells are passaged to the 12th generation (P12), P12 generation BEFs are obtained.

[0075] The morphology of P12 generation BEFs was observed by photographing them under a microscope. Figure 2 As shown.

[0076] To detect the senescence status of P12 generation BEFs, β-galactosidase staining was used for cell senescence characterization analysis. The specific procedure was as follows: The P12 generation BEFs were washed three times with PBS, and then stained according to the instructions of the β-galactosidase staining kit for cell senescence (Beyotime, C0602). After staining, images of the stained cells were acquired using a microscope, as shown below. Figure 3 As shown.

[0077] Figure 1 These are microscope images of primary BEFs and P1 generation BEFs from the experimental examples of this invention, wherein... Figure 1 'a' is a microscopic image of primary BEFs. Figure 1 b is a microscope image of P1 generation BEFs; Figure 2 This invention illustrates the effects of different proliferation culture media on the senescence of bovine embryonic fibroblasts in experimental examples. Figure 2 a is a microscopic image of P12 generation BEFs after subculturing using culture medium A in Example 1 according to the above method; Figure 2b is a microscopic image of BEFs from generation P12, which were cultured using 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 rate 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 the cells are 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, then washed thoroughly with PBS buffer, and the cell count was recorded using a cell counter. The cell population doubling formula is: Td = t × log2 / log (N t / N o (Td represents cell population doubling time, t represents actual culture time, N) o N represents the initial number of cells. 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 from the original] 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 proliferation of the P12 generation BEFs cultured using culture medium B in Comparative Example 1 was stagnant, 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 (BEFs) to achieve stable passage ≥100 generations during in vitro passage culture, 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 rate of 1×10⁻⁶. 5Seed 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 the cells are passaged to the 12th generation (P12), P12 generation BEFs are obtained.

[0091] At 37°C, P12 generation BEFs were digested with 0.25% trypsin for 5 minutes. After eluting the cells thoroughly with PBS buffer, they were centrifuged and collected. RNA was then extracted from the collected cells according to the instructions of the Animal Cell Total RNA Extraction Kit (Tiangen Biotech, DP451). The RNA was transcribed into cDNA using a reverse transcription kit (Takara, RR047A). Finally, qPCR (Takara, RR820A) was used to detect the expression levels of TERT and p53 genes, respectively. The results are as follows: Figure 5 and Figure 6 As shown.

[0092] Figure 5 The effect of different proliferation culture media on the expression level of telomerase reverse transcriptase (TERT) gene in bovine embryonic fibroblasts; Figure 6 To investigate the effects of different proliferation culture media on the expression level of the bovine embryonic fibroblast tumor protein p53 (P53) gene.

[0093] Depend on Figure 5 and Figure 6 It can be seen that, compared with culture medium B in Comparative Example 1, the P12 generation BEFs cultured using culture medium A in Example 1 showed a significant increase in TERT gene expression and a significant decrease in p53 gene expression, proving that using culture medium A in Example 1 of this invention enables BEFs to have long-term division ability.

[0094] 4. Bovine embryonic fibroblasts were cultured using culture medium A as described in Example 1.

[0095] Using culture medium A from Example 1 above as a proliferation medium to culture bovine embryonic fibroblasts, the specific method includes:

[0096] Bovine embryonic fibroblasts (P1 generation BEFs) were obtained using the primary culture method described above.

[0097] P1 generation bovine embryonic fibroblasts (P1 generation BEFs) were used at a rate 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 145th generation (P145), P145 generation BEFs are obtained.

[0098] The P145 generation BEFs were photographed and recorded using a microscope to observe their morphology, such as... Figure 7 As shown.

[0099] P145 generation BEFs were stained using vimentin immunofluorescence assay, and the results are as follows: Figure 8 As shown.

[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 embryonic fibroblasts, characterized in that, The proliferation medium comprises a complete medium, cycloastragalool, and metformin hydrochloride; wherein the complete medium comprises, by volume percentage: 79-94 vol% basal medium, 5-20 vol% fetal bovine serum, and 1-3 vol% penicillin-streptomycin solution; the concentration of cycloastragalool in the proliferation medium is 5 μmol / L, and the concentration of metformin hydrochloride in the proliferation medium is 10 μmol / L; the basal medium is any one of DMEM medium, MEM medium, DMEM / F12 medium, Ham's F-12K medium, and Leibovitz L-15 medium.

2. The proliferation culture medium for long-term stable passage of bovine embryonic fibroblasts 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.

3. A method for promoting long-term stable passage of bovine embryonic fibroblasts, characterized in that, Includes the following steps: Bovine embryonic fibroblasts are seeded at a certain seeding density into the proliferation medium described in claim 1 or 2 and cultured, with the medium being changed every 1-3 days; when the cell confluence reaches 80%-90%, the cells are placed in the proliferation medium and passaged at a ratio of 1:

3. The specified inoculation density is 0.3 × 10⁻⁶. 5 ~2×10 5 Cells / mL.

4. The application of the method for promoting long-term stable passage of bovine embryonic fibroblasts as described in claim 3 in the preparation of cultured meat.

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