A serum-free culture medium and its application in the preparation of cultured meat from large yellow croaker cells.

Abstract

This invention belongs to the field of bioengineering technology and relates to a serum-free culture medium and its application in the preparation of cell-cultured meat from large yellow croaker. This invention provides a serum-free culture medium comprising a basal medium and exogenous additives; the basal medium is DMEM high-glucose medium, DMEM / F12 medium, or F10 medium; the exogenous additives include L-carnitine, linoleic acid, cholesterol, vitamin E, folic acid, dexamethasone, progesterone, lipoic acid, methylethanolamine, TGFβ1, inositol, bovine serum albumin, and HEPES. The serum-free culture medium of this invention significantly reduces costs and provides a feasible product and method for the large-scale production of cell-cultured meat.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a serum-free culture medium and its application in the preparation of cell-cultured meat from large yellow croaker. Background Technology

[0002] With economic and social development, human demand for meat is increasing, but traditional meat production methods are no longer sufficient to meet this demand. Furthermore, traditional meat production methods consume large amounts of grain and water resources and cause serious environmental pollution. Cultured meat involves extracting highly proliferating animal stem cells or tissues and placing them in a culture dish to multiply, eventually differentiating into primitive muscle tissue fibers. Cultured meat production involves the isolation and purification of stem cells, cell proliferation and differentiation, and requires only culture medium, specific temperature and humidity, and carbon dioxide to provide the necessary nutrients and growth environment. It does not cause environmental pollution, does not require the feed and water needed in traditional meat production, and requires less space, saving space costs. Therefore, it represents a major research and development direction for future artificial meat.

[0003] In traditional cell culture, a certain amount of fetal bovine serum is required to provide the nutrients and biological factors needed for cell adhesion, proliferation and growth. However, fetal bovine serum has certain disadvantages, mainly: (1) it is expensive; (2) the composition of substances in the serum is uncertain; (3) it may contain fungi, bacteria, viruses, mycoplasma and other risk contaminants; (4) there are large batch-to-batch differences, and the serum obtained at different times and under different conditions is inconsistent, which causes instability in the quality of culture products.

[0004] Serum-free culture medium is a synthetic culture medium that can maintain cell growth and proliferation in vitro for a long time without the addition of serum. For example, patent document CN 112210525 A discloses a serum-free culture medium in which all exogenous added components are chemically or biosynthesized. Since the artificially synthesized components are clearly defined, the product is stable between batches and has high reproducibility of cell culture. At the same time, it reduces the risk of microbial contamination such as endogenous and exogenous viruses, bacteria and mycoplasma caused by the use of animal serum. Moreover, industrial production ensures sufficient supply and is also conducive to the downstream purification of biological products.

[0005] Although commercially available serum-free culture media exist, their prices are very high, making them unsuitable for large-scale industrial applications. Therefore, reducing costs during large-scale culture is a pressing issue that needs to be addressed. Summary of the Invention

[0006] The purpose of this invention is to provide a serum-free culture medium and its application in the preparation of cultured meat from large yellow croaker cells. The serum-free culture medium of this invention significantly reduces costs and provides a feasible product and method for the subsequent large-scale production of cultured meat.

[0007] This invention provides a serum-free culture medium, which includes a basal medium and exogenous additives; the basal medium is DMEM high glucose medium, DMEM / F12 medium or F10 medium; the exogenous additives include L-carnitine, linoleic acid, cholesterol, vitamin E, folic acid, dexamethasone, progesterone, lipoic acid, methanolamine, TGFβ1, inositol, bovine serum albumin and HEPES.

[0008] Preferably, the mass concentrations of the exogenous additives in the serum-free culture medium are as follows: L-carnitine 0.1–30 μg / mL, linoleic acid 0.05–15 μg / mL, cholesterol 0.1–33 μg / mL, vitamin E 2.5–750 μg / mL, folic acid 0.2–50 μg / mL, dexamethasone 0.2–50 ng / mL, progesterone 1–100 ng / mL, lipoic acid 3.5–700 ng / mL, methylethanolamine 0.1–50 μg / mL, TGFβ 1 0.05–15 ng / mL, inositol 0.5–200 μg / mL, bovine serum albumin 0.1–20 mg / mL, and HEPES 1–500 mg / mL.

[0009] Preferably, the mass concentrations of the exogenous additives in the serum-free culture medium are as follows: L-carnitine 1.9 μg / mL, linoleic acid 0.5 μg / mL, cholesterol 3.3 μg / mL, vitamin E 100 μg / mL, folic acid 5 μg / mL, dexamethasone 2 ng / mL, progesterone 16.82 ng / mL, lipoic acid 70 ng / mL, methylethanolamine 0.2 μg / mL, TGFβ 11.5 ng / mL, inositol 5 μg / mL, bovine serum albumin 9 mg / mL, and HEPES 4.766 mg / mL.

[0010] The present invention also provides the application of the serum-free culture medium described in the above technical solution in the in vitro culture of animal stem cells or animal muscle cells.

[0011] The present invention also provides the application of the serum-free culture medium described in the above technical solution in the preparation of cultured meat from animal cells.

[0012] Preferably, the animal includes a fish; the fish includes the large yellow croaker.

[0013] Preferably, the large yellow croaker includes adult large yellow croaker or juvenile large yellow croaker.

[0014] This invention also provides a method for preparing large yellow croaker cell culture meat based on the serum-free culture medium described above, comprising the following steps:

[0015] Large yellow croaker muscle satellite cells were inoculated into the serum-free culture medium described in the above technical solution and cultured.

[0016] Preferably, the inoculum size of the large yellow croaker muscle satellite cells during culture is 1×10⁻⁶. 4 ~1×10 6 Serum-free culture medium (number per mL)

[0017] Preferably, the culture temperature is 25℃~27℃; and the culture time is 24~72h.

[0018] This invention provides a serum-free culture medium. The serum-free culture medium of this invention can significantly reduce costs, providing a feasible product and method for the subsequent large-scale production of cell-cultured meat. Experimental results show that when the serum-free culture medium of this invention is used for the culture of large yellow croaker muscle satellite cells, it can achieve more than 85% of the normal cell growth rate within the growth cycle, and the obtained cells have basically normal morphology and maintain cell stemness, meeting the requirements of cell culture. Compared with serum-containing culture medium (DMEM / F12, 100 μL / mL FBS), the serum-free culture medium provided by this invention saves culture costs and is suitable for large-scale industrial application. Specifically, the serum-free culture medium provided by this invention adds 13 substances that promote cell growth to the basic culture medium, replacing fetal bovine serum. This not only saves costs, but also, compared with the general muscle satellite cell culture medium (DMEM high glucose, with 100 μL / mL FBS), the serum-free culture medium provided by this invention has a similar ability to maintain and proliferate cell stem cells, indicating that the serum-free culture medium of this invention is more suitable for the large-scale industrial production of cell-cultured meat. Compared to existing serum-free culture media, the serum-free exogenous additives provided by this invention are lower in cost, fewer in number, and most of them are simple, readily available, and common compounds. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0020] Figure 1Figure 1 shows the preliminary results of the investigation of exogenous added components in the serum-free culture medium provided by the present invention; (A) is a typical electron microscope image of cells cultured in the 8th group of the optimal serum-free culture medium; (B) is an electron microscope image of cells cultured in general normal serum; (C) is a figure showing the comparison of cell viability of cells cultured in each serum-free experimental group after 24 hours and 48 hours.

[0021] Figure 2 The diagram shows the optimization results of exogenous added components in the serum-free culture medium provided by the present invention; wherein (A) is the cell viability result of each group after 24 hours of serum-free cell culture; (B) is the cell viability result of each group after 48 hours of serum-free cell culture; and (C) is the cell viability result of each group after 72 hours of serum-free cell culture.

[0022] Figure 3 The figure shows the further optimization results of the exogenous added components in the serum-free culture medium provided by the present invention; (A) is a typical electron microscope image of cells cultured in the 4th group of serum-free culture medium; (B) is a typical electron microscope image of cells cultured in normal serum; (C) is the cell viability of cells after 72 hours under each serum-free culture condition.

[0023] Figure 4 The figure shows the effect of different concentrations of bovine serum albumin on cell proliferation in the serum-free culture medium provided by this invention.

[0024] Figure 5 The following figures illustrate the effect of serum-free culture medium on cell stemness provided by this invention: (A) is an immunofluorescence staining image of desmin, a cell differentiation marker protein, cultured in serum-free culture medium; (B) is an immunofluorescence staining image of desmin, a cell differentiation marker protein, cultured in normal serum culture medium; and (C) is a comparison of cell fusion index between serum-free and serum-containing culture media. Detailed Implementation

[0025] This invention provides a serum-free culture medium, which includes a basal medium and exogenous additives; the basal medium is DMEM high glucose medium, DMEM / F12 medium or F10 medium; the exogenous additives include L-carnitine, linoleic acid, cholesterol, vitamin E, folic acid, dexamethasone, progesterone, lipoic acid, methanolamine, TGFβ1, inositol, bovine serum albumin and HEPES.

[0026] In this invention, the basal culture medium is DMEM high-glucose medium, DMEM / F12 medium, or F10 medium, preferably DMEM / F12 medium. Compared to DMEM high-glucose medium and F10 medium, the combination of DMEM / F12 medium and the above-mentioned exogenous additives is more conducive to the proliferation of large yellow croaker muscle satellite cells.

[0027] The exogenous additives described in this invention can supplement the nutrients and biological factors required for cell adhesion, proliferation, and growth maintenance, achieving effects essentially the same as those in normal serum-containing culture media. This invention does not specifically limit the source of the above-mentioned components; conventional commercially available products well-known to those skilled in the art can be used.

[0028] In this invention, the serum-free culture medium preferably contains 0.1–30 μg / mL of L-carnitine, more preferably 1.9 μg / mL. In this invention, L-carnitine can promote fatty acid oxidation for energy.

[0029] In this invention, the serum-free culture medium preferably contains 0.05–15 μg / mL of linoleic acid, more preferably 0.5 μg / mL. In this invention, linoleic acid is an essential fatty acid that can lower blood cholesterol; cholesterol must bind with linoleic acid to be properly transported and metabolized in the body.

[0030] In this invention, the serum-free culture medium preferably contains 0.1–33 μg / mL of cholesterol, more preferably 3.3 μg / mL. In this invention, cholesterol participates in the composition of cell membranes in vivo and is also an important raw material for the synthesis of steroid hormones, which regulate the metabolism of carbohydrates, fats, proteins, water, and electrolytes.

[0031] In this invention, the serum-free culture medium preferably contains 2.5–750 μg / mL of vitamin E, more preferably 100 μg / mL. In this invention, vitamin E is an essential fat-soluble vitamin for the human body and a powerful antioxidant. It can protect cell membrane stability by interrupting the chain reaction of free radicals, maintain the stability of genetic material, prevent chromosomal structural variations, and regulate the orderly conduct of metabolic activities.

[0032] In this invention, the serum-free culture medium preferably contains 0.2–50 μg / mL of folic acid, more preferably 5 μg / mL. In this invention, folic acid is a B vitamin that acts as a coenzyme in the one-carbon unit transferase system during biochemical reactions in the body, serving as a one-carbon unit carrier. It participates in the synthesis of purines and thymine, further synthesizing DNA and RNA, and participating in amino acid metabolism. It acts as a one-carbon unit carrier in the interconversion between glycine and serine, histidine and glutamate, and homocysteine ​​and methionine. It also participates in the synthesis of hemoglobin and methyl compounds such as adrenaline, choline, and creatine. Folic acid plays an important role in cell division and growth, as well as the synthesis of nucleic acids, amino acids, and proteins.

[0033] In this invention, the serum-free culture medium preferably contains 0.2–50 ng / mL of dexamethasone, more preferably 2 ng / mL. In this invention, dexamethasone is an adrenocortical hormone extracted from the adrenal cortex that has the strongest effect on carbohydrate metabolism.

[0034] In this invention, the serum-free culture medium preferably contains 1-100 ng / mL of progesterone, more preferably 16.82 ng / mL. In this invention, progesterone promotes cell proliferation and migration.

[0035] In this invention, the serum-free culture medium preferably contains 3.5–700 ng / mL of lipoic acid, more preferably 70 ng / mL. In this invention, lipoic acid can act as a coenzyme to participate in acyl transfer during metabolism in the body, and can eliminate free radicals that lead to accelerated aging and disease.

[0036] In this invention, the serum-free culture medium preferably contains 0.1–50 μg / mL of methanolamine, more preferably 0.2 μg / mL. In this invention, methanolamine is a carbon dioxide gas absorbent, a nonionic surfactant, and a lipid substance that can protect the integrity of the cell membrane.

[0037] In this invention, the serum-free culture medium preferably contains 0.05–15 ng / mL of TGFβ, more preferably 1.5 ng / mL. In this invention, TGFβ-1 belongs to a newly discovered TGF-β superfamily that regulates cell growth and differentiation.

[0038] In this invention, the serum-free culture medium preferably contains 0.5–200 μg / mL of inositol, more preferably 5 μg / mL. In this invention, inositol is a growth factor for animals and microorganisms.

[0039] In this invention, the serum-free culture medium preferably contains 0.1–20 mg / mL of bovine serum albumin, more preferably 5–9 mg / mL, and most preferably 9 mg / mL. In this invention, bovine serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions, and many therapeutic molecules.

[0040] In this invention, the serum-free culture medium preferably contains 1-500 mg / mL of HEPES, more preferably 4.766 mg / mL. In this invention, HEPES is an important buffer that can maintain the stability of the solution pH.

[0041] This invention also provides the application of the serum-free culture medium described above in the in vitro culture of animal stem cells or animal muscle cells. In this invention, the animal stem cells preferably include animal muscle stem cells.

[0042] This invention also provides the application of the serum-free culture medium described in the above-mentioned technical solution in the preparation of cultured meat from animal cells. In this invention, the cultured meat is preferably obtained by inoculating and culturing animal muscle stem cells.

[0043] In this invention, the animal preferably includes fish; the fish preferably includes large yellow croaker. In this invention, the fish can preferably be other marine or freshwater fish. In this invention, the large yellow croaker includes adult or juvenile large yellow croaker. In this invention, during the preparation of cell-cultured meat, the muscle satellite cells of large yellow croaker are preferably cultured, i.e., the serum-free culture medium is preferably used as the culture medium for large yellow croaker muscle satellite cells. In this invention, the large yellow croaker muscle satellite cells are preferably isolated from the axial muscle of large yellow croaker. Large yellow croaker (Larimichthys crocea) is a fish belonging to the family Sciaenidae and the genus Larimichthys. Large yellow croaker has high economic value, tender flesh, and is rich in protein, making it a prized fresh food. However, due to overfishing, its resources are rapidly declining. Therefore, developing cell culture technology for large yellow croaker to produce cell-cultured meat to meet human needs is a means to address resource depletion, but currently, there is a lack of a low-cost culture medium that allows for the rapid proliferation of large yellow croaker muscle satellite cells. The serum-free culture medium described in this invention, when used for culturing large yellow croaker muscle satellite cells, can achieve over 85% of the normal cell growth rate within the growth cycle, and the obtained cells exhibit essentially normal morphology and maintain cell stemness, meeting the requirements for cell culture. Compared to serum-containing culture media, the serum-free culture medium provided by this invention saves on culture costs and is suitable for large-scale industrial applications. Compared to existing serum-free culture media, the serum-free exogenous additives provided by this invention are lower in cost, require fewer exogenous additives, and most are simple, readily available, and common compounds.

[0044] This invention also provides a method for preparing large yellow croaker cell culture meat based on the serum-free culture medium described above, comprising the following steps:

[0045] Large yellow croaker muscle satellite cells are inoculated into the serum-free culture medium described in the above-mentioned technical solution for culture. In this invention, the preferred inoculation amount of large yellow croaker muscle satellite cells during culture is 1×10⁻⁶. 4 ~1×10 6 Serum-free medium at a concentration of cells / mL. In this invention, the culture temperature is preferably 25℃~27℃; the culture time is preferably 24~72h. In this invention, the culture does not require medium change. The serum-free medium of this invention enables rapid proliferation of large yellow croaker muscle satellite cells.

[0046] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a serum-free culture medium provided by the present invention and its application in the preparation of cultured meat from large yellow croaker cells. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0048] DMEM / F12 medium ( PM150312); FBS fetal bovine serum ( 086-150); Vitamin E ( 59-02-9); HEPES 7365-45-9); Linoleic acid ( 60-33-3); Cholesterol ( 57-88-5); L-carnitine ( 541-15-1); Progesterone ( 57-83-0); Thioctic acid ( 62-46-4); Folic acid ( 59-30-3); Dexamethasone ( ST1258-50mg); Methanolamine ( 141-43-5); Inositol ( 87-89-8); TGFβ1 ( BIO-000006) and bovine serum albumin ( BIO-000001).

[0049] Primary satellite cells of large yellow croaker muscle were prepared in the laboratory using standard methods. Muscle tissue was isolated from the axial region of the muscle of juvenile large yellow croaker, and then digested with 0.1% type IV collagenase solution and 0.1% trypsin solution, respectively. The digested tissues were filtered through 70 μm and 40 μm cell sieves, centrifuged at 300g for 5 min, and the cell pellet was resuspended in complete culture medium. Then, the cells were processed at 1×10⁻⁶ cells / mL. 6 Cells were seeded at a concentration of 10 cells / mL in 6-well plates and cultured in an incubator at 27°C and 5% CO2.

[0050] Example 1

[0051] Preliminary Investigation of Exogenous Additions to Serum-Free Culture Media

[0052] Approximately 3×10 4 Myosatellite cells were seeded into each well of a 96-well plate with 100 μL of culture medium and cultured aseptically at 27°C for 72 h. Cell viability was measured using the CCK-8 kit to evaluate the exogenous components added to the serum-free culture medium.

[0053] The basal culture medium was determined to be DMEM / F12, with bovine serum albumin concentrations of 2 mg / mL and HEPES concentrations of 4.766 mg / mL. First, the concentrations of different exogenous additives were set: L-carnitine: 1 μg / mL, 2 μg / mL, 3 μg / mL; linoleic acid: 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL; cholesterol: 1.1 μg / mL, 2.2 μg / mL, 3.3 μg / mL; vitamin E: 25 μg / mL, 50 μg / mL, 75 μg / mL; folic acid: 2 μg / mL, 3.5 μg / mL, 7 μg / mL; dexamethasone... Pine: 2 ng / mL, 3.5 ng / mL, 7 ng / mL; Progesterone: 3 ng / mL, 6 ng / mL, 9 ng / mL; Alpha-lipoic acid: 35 ng / mL, 0 ng / mL, 70 ng / mL; Methanolamine: 1 μg / mL, 2.5 μg / mL, 5 μg / mL; TGFβ1: 0.5 ng / mL, 1 ng / mL, 1.5 ng / mL; Inositol: 5 μg / mL, 12.5 μg / mL, 20 μg / mL. Results are as follows... Figure 1 As shown.

[0054] The different concentrations of each factor were combined using the statistical analysis software SPSS. The specific experimental design is shown in Table 1. Based on the cell viability experiment results and electron micrographs, the factors that play a major role were identified and the design was further optimized, as shown in Table 2.

[0055] Table 1 Experimental Design Table

[0056]

[0057]

[0058] Table 2. Optimized exogenous additives

[0059]

[0060]

[0061] After culturing at 27°C for 48 hours, the cells in group 8 cultured in serum-free medium showed normal morphology. Figure 1 In A), the cell count is 7 × 10⁻⁶. 5 The cell count was approximately 70% of that in control group 2 (DMEM / F12, supplemented with 100 μl / mL FBS), and the cell yield was 10 × 10⁶ cells / mL. 5 cells / mL ( Figure 1 (B) Compared with control group 1, the serum-free culture medium of the present invention can promote cell proliferation by increasing the exogenous added components.

[0062] The results of the cell viability experiment ( Figure 1 In step C), statistical analysis was performed using SPSS software to determine the main contributing factors as ethanolamine, progesterone, vitamin E, and L-carnitine. Therefore, these four factors were selected as the main exogenous additives for serum-free culture medium, and further optimization will be carried out based on this.

[0063] Example 2

[0064] Optimization of exogenous components in serum-free culture medium

[0065] The different concentrations of the four main factors identified in Example 1 were combined using statistical analysis software, totaling approximately 3 × 10⁻⁶. 4 Myosatellite cells were seeded into each well of a 96-well plate with 100 μL of culture medium and cultured aseptically at 27°C for 72 h. Cell morphology was observed by electron microscopy and cell viability was detected by the CCK-8 assay kit to evaluate the effect of serum-free basal medium on myosatellite cell proliferation.

[0066] The basal culture medium was DMEM / F12. The concentrations of exogenous additives were determined to be 0.5 μg / mL linoleic acid, 3.3 μg / mL cholesterol, 5 μg / mL folic acid, 2 ng / mL dexamethasone, 70 ng / mL lipoic acid, 11.5 ng / mL TGFβ and 5 μg / mL inositol. The specific experimental design for the other four major factors is shown in Table 3.

[0067] The results are as follows Figure 2 As shown, the cell viability test results after 48 hours of culture were statistically analyzed using SPSS software, and the optimal combination was found to be 0.2 μg / mL methylethanolamine, 16.82 ng / mL progesterone, 1.9 μg / mL L-carnitine, and 100 μg / mL vitamin E. The cell viability test results after 72 hours of culture were statistically analyzed, and the optimal combination was found to be 0.2 μg / mL methylethanolamine, 17.90 ng / mL progesterone, 1.9 μg / mL L-carnitine, and 90.16 μg / mL vitamin E.

[0068] Table 3. Optimized Experimental Design

[0069]

[0070]

[0071] Example 3

[0072] Determination of exogenous components added to serum-free culture medium

[0073] Based on the conclusions drawn in Example 2, the exogenous additives for serum-free culture medium were determined, and approximately 3 × 10⁻⁶ components were added. 4Myosatellite cells were seeded into each well of a 96-well plate with 100 μL of culture medium and cultured aseptically at 27°C for 72 h. Cell morphology was observed by electron microscopy and cell viability was detected by the CCK-8 assay kit to evaluate the effect of serum-free basal medium on myosatellite cell proliferation.

[0074] The basal culture medium was DMEM / F12. The exogenous supplements at determined concentrations in Group 1 were linoleic acid (0.5 μg / mL), cholesterol (3.3 μg / mL), folic acid (5 μg / mL), dexamethasone (2 ng / mL), lipoic acid (70 ng / mL), TGFβ (11.5 ng / mL), inositol (5 μg / mL), methylethanolamine (0.2 μg / mL), progesterone (16.82 ng / mL), L-carnitine (1.9 μg / mL), and vitamin E (100 μg / mL). The exogenous supplements at determined concentrations in Group 2 were linoleic acid (0.5 μg / mL), cholesterol (3.3 μg / mL), folic acid (5 μg / mL), dexamethasone (2 ng / mL), lipoic acid (70 ng / mL), TGFβ (11.5 ng / mL), inositol (5 μg / mL), methylethanolamine (0.2 μg / mL), progesterone (17.90 ng / mL), L-carnitine (1.9 μg / mL), and vitamin E. 90.16 μg / mL.

[0075] Based on this, and considering the nutritional needs of cells, the effects of different concentrations of bovine serum albumin on cell proliferation were also investigated. The specific experimental design is shown in Table 4.

[0076] The results are as follows Figure 3 As shown, after aseptic culture at 27℃ for 72 hours, the cells in group 4, prepared with serum-free culture medium, exhibited normal cell morphology, and the harvested cell quantity was 7.8 × 10⁻⁶. 5 The cell count was approximately 87% of that of control group 2 (DMEM / F12, supplemented with 100 μL / mL LFBS), and the harvested cell count from control group 2 was 9.0 × 10⁶ cells / mL. 5 cells / mL. And, according to Figure 3 It can also be seen that the cell proliferation effect is better as the concentration of bovine serum albumin increases.

[0077] Table 4. Further Optimized Experimental Design

[0078]

[0079]

[0080] Example 4

[0081] Further investigation into the effects of different concentrations of bovine serum albumin on cell proliferation.

[0082] Based on the serum-free exogenous additives explored in the above examples, the effects of different concentrations of bovine serum albumin on cell proliferation were further investigated. Approximately 3 × 10⁻⁶ g of bovine serum albumin was used. 4 Myosatellite cells were seeded into each well of a 96-well plate with 100 μL of culture medium and cultured aseptically at 27°C for 72 h. Cell morphology was observed by electron microscopy and cell viability was detected by the CCK-8 assay kit to evaluate the effect of serum-free basal medium on myosatellite cell proliferation. The specific experimental design is shown in Table 5.

[0083] The results are as follows Figure 4 As shown, after aseptic culture at 27℃ for 72 hours, cells with normal morphology were obtained using the serum-free culture medium formulation of group 3, and the harvested cell quantity was 6.3 × 10⁻⁶. 5 The cell count was approximately 95% of that of the control group (DMEM / F12, with 100 μL / mL FBS added). The harvested cell count from control group 2 was 6.6 × 10⁶ cells / mL. 5 Cells / mL. This indicates that the serum-free culture medium formulation described above can achieve normal cell growth within the required growth cycle.

[0084] Table 5. Experimental Design Table of Effects of Different Concentrations of Bovine Serum Albumin on Cell Proliferation

[0085]

[0086]

[0087] Example 5

[0088] Effects of serum-free culture medium on cell stemness

[0089] Based on the final serum-free culture medium formulation obtained in the above embodiments, this invention also investigated the effect of the obtained serum-free culture medium on cell stemness. Approximately 1×10 6 Large yellow croaker myosatellite cells were seeded into each well of a 48-well plate with 500 μL of culture medium and cultured aseptically at 27°C for 24 h. The culture medium was then replaced with differentiation medium. After 5 days of differentiation, desmin immunofluorescence staining was performed, and the differentiation capacity of the cells was calculated by the fusion index.

[0090] The results are as follows Figure 5 As shown, the fusion index of the serum-free culture medium is approximately 9.6%, while that of the serum-containing culture medium is approximately 10.6%. The serum-free and serum-containing culture media have the same effect on maintaining cell stemness, indicating that the serum-free culture medium formulation obtained in this invention is feasible.

[0091] The serum-free culture medium provided by this invention is also suitable for other marine or freshwater fish.

[0092] Example 6

[0093] Application of serum-free culture medium in artificial meat

[0094] This embodiment creates a serum-free culture medium by adding exogenous components such as bovine serum albumin, linoleic acid, cholesterol, folic acid, dexamethasone, alpha-lipoic acid, TGFβ1, inositol, methylethanolamine, progesterone, L-carnitine, vitamin E, and HEPES to the basic DMEM / F12 medium. This medium allows for the normal proliferation of large yellow croaker muscle satellite cells. Cells cultured at 27°C for 72 hours showed the same results as those in serum-containing medium (DMEM / F12, 100 μL / mL FBS), exhibiting normal cell morphology and maintaining cell stemness, thus providing sufficient cells for the preparation of artificial meat.

[0095] In terms of cost, taking 500 mL of complete culture medium as an example, a general-purpose culture medium (DMEM / F12, 100 μL / mL FBS) costs approximately 313 yuan, while the serum-free culture medium of this invention costs approximately 210 yuan. Furthermore, compared to existing serum-free culture media, the serum-free exogenous additives provided by this invention are less expensive, contain fewer exogenous additives, and are mostly simple, readily available, and common compounds. Therefore, using the aforementioned serum-free culture medium can reduce culture costs and enable large-scale, low-cost culture of large yellow croaker muscle satellite cells, which is beneficial for industrial transformation.

[0096] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A serum-free culture medium, characterized in that, The culture medium was prepared from a basal medium and exogenous additives; the basal medium was DMEM / F12 medium; the exogenous additives were L-carnitine, linoleic acid, cholesterol, vitamin E, folic acid, dexamethasone, progesterone, lipoic acid, methylethanolamine, TGF β1, inositol, bovine serum albumin, and HEPES; the mass concentrations of the exogenous additives in the serum-free medium were as follows: L-carnitine 1.9 μg / mL, linoleic acid 0.5 μg / mL, cholesterol 3.3 μg / mL, vitamin E 100 μg / mL, folic acid 5 μg / mL, dexamethasone 2 ng / mL, progesterone 16.82 ng / mL, lipoic acid 70 ng / mL, methylethanolamine 0.2 μg / mL, TGF β1 1.5 ng / mL, inositol 5 μg / mL, bovine serum albumin 9 mg / mL, and HEPES 4.766 mg / mL.

2. The application of the serum-free culture medium of claim 1 in the in vitro culture of large yellow croaker muscle satellite cells.

3. The application of the serum-free culture medium according to claim 1 in the preparation of large yellow croaker cell-cultured meat.

4. The application according to claim 3, characterized in that, The large yellow croaker includes adult large yellow croaker or juvenile large yellow croaker.

5. A method for preparing large yellow croaker cell-cultured meat based on the serum-free culture medium of claim 1, comprising the following steps: Large yellow croaker muscle satellite cells were inoculated into the serum-free culture medium described in claim 1 and cultured.

6. The method according to claim 5, characterized in that, During the culture, the inoculum size of large yellow croaker muscle satellite cells was 1×10⁻⁶. 4 ~1×10 6 Serum-free culture medium (number per mL) 7. The method according to claim 5, characterized in that, The culture temperature is 25℃~27℃; the culture time is 24~72h.

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