A serum-free culture medium for bovine adipose mesenchymal stem cells suitable for cell culture meat and a preparation method thereof

Abstract

The invention discloses a kind of bovine adipose mesenchymal stem cell serum-free culture medium suitable for cell culture meat, belongs to the field of animal cell culture technology, including DMEM basic culture, L-alanyl-L-glutamine, recombinant transferrin, recombinant bovine insulin, sodium selenite, zinc sulfate heptahydrate, cortisol, recombinant human albumin, recombinant basic fibroblast growth factor, recombinant transforming growth factor β1, recombinant insulin-like growth factor, ethanolamine, mercaptoethanol and 2-phosphoric acid-L-ascorbic acid sodium salt. The inventor of the present application has obtained a new bovine adipose-derived stem cell (Adipose-derived stem cell, ADSC) serum-free culture medium through repeated experimental verification, which can effectively culture bovine adipose mesenchymal stem cells.

Description

Technical Field

[0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a serum-free culture medium for bovine adipose mesenchymal stem cells suitable for cell culture meat and a preparation method thereof. Background Art

[0002] Compared with plant meat and fermented meat made from microorganisms, cell-cultured meat is a new type of meat that is closest to traditional meat. Cell-cultured meat has the advantages of no microbial contamination, high content of high-quality protein and good eating experience. The carbon footprint and water resource requirements of the production process are much lower than those of traditional livestock breeding. It is one of the most promising development directions for farmed meat substitutes in the future. At the same time, the technical barriers of the cell-cultured meat industry are also higher and more challenging. Among them, the development of low-cost, animal-free culture media suitable for cell-cultured meat is an important foundation for cell-cultured meat to meet food safety standards and eventually enter large-scale production.

[0003] Mesenchymal stem cells (MSCs) are derived from the early mesoderm of embryonic development. They are adult stem cells with self-renewal and multidirectional differentiation potentials and can maintain their biological characteristics after in vitro large-scale expansion. They can proliferate and differentiate into a variety of tissues such as bone, cartilage, muscle, ligament, tendon, fat and matrix cells. Therefore, mesenchymal stem cells are one of the important cell sources for cultured meat.

[0004] At present, there are mainly the following types of culture media for culturing MSCs: one is to add 5-20% animal serum to the basic culture medium (such as α-MEM culture medium, DMEM culture medium, etc.). However, animal serum is expensive and has limited production, so it is not suitable for large-scale production of cell-cultured meat. In addition, animal serum may be contaminated with mycoplasma, prions, etc., and there are risks such as human xenoprotein contamination or allergies, which makes it difficult to meet the standards for consumption; the second type is to add serum substitutes (such as human platelet lysate, etc.) to the basic culture medium. However, this type of culture medium also has animal-derived components and unknown proteins, and has the disadvantages of large batch differences and unstable sources, and is not suitable for large-scale production of cell-cultured meat; the third type is to supplement the basic culture medium with serum substitutes that are free of animal origin and have clear chemical components, which can not only meet the nutritional requirements of cells, but also avoid the natural defects of the first two types of culture media. Therefore, a cell culture medium with a clear chemical formula and low cost suitable for the growth of MSCs cells is an important prerequisite for the large-scale production of cell-cultured meat. Summary of the invention

[0005] The purpose of the present invention is to solve the existing problems. The inventors have obtained a new serum-free culture medium for bovine adipose-derived stem cells (ADSC) through repeated experimental verification, which can effectively culture bovine adipose-derived stem cells.

[0006] The present invention is achieved through the following technical solutions:

[0007] A serum-free medium for bovine adipose mesenchymal stem cells suitable for cell culture meat, the medium comprising DMEM basal medium, 75 mg / L L-alanyl-L-glutamine, 0-5.5 mg / L recombinant transferrin, 0-10 mg / L recombinant bovine insulin, 0.001-0.01 mg / L sodium selenite, 0.1-2 mg / L zinc sulfate heptahydrate, 10-50 ng / mL cortisol, 0.1-1.5 mg / mL recombinant human albumin, 1-10 ng / mL recombinant basic fibroblast growth factor, 0-2 ng / mL recombinant transforming growth factor β1, 5-20 ng / mL recombinant insulin-like growth factor, 2-100 mg / mL ethanolamine, 0.1-3 mg / L mercaptoethanol and 10-50 mg / L 2-phospho-L-ascorbic acid sodium salt, wherein the DMEM basal medium comprises: 4500 mg / L D-glucose, 3700 mg / L sodium bicarbonate, 584 mg / L L-glutamine and 110 mg / L sodium pyruvate.

[0008] Preferably, the culture medium consists of DMEM basal medium, L-alanyl-L-glutamine 75 mg / L, recombinant transferrin 4 mg / L, recombinant bovine insulin 8 mg / L, sodium selenite 0.005 mg / L, zinc sulfate heptahydrate 1 mg / L, cortisol 25 ng / mL, recombinant human albumin 1.25 mg / mL, recombinant basic fibroblast growth factor 5 ng / mL, recombinant transforming growth factor β1 0.5 ng / mL, recombinant insulin-like growth factor 10 ng / mL, ethanolamine 50 mg / mL, mercaptoethanol 2.2 mg / L and 2-phospho-L-ascorbic acid sodium salt 30 mg / L, wherein the DMEM basal medium contains: 4500 mg / L D-glucose, 3700 mg / L sodium bicarbonate, 584 mg / L L-glutamine and 110 mg / L sodium pyruvate.

[0009] A method for preparing a serum-free culture medium for bovine adipose mesenchymal stem cells suitable for cell culture meat comprises the following steps:

[0010] (1) Prepare reagents of corresponding concentrations for use: L-alanyl-L-glutamine 43.5 mg / L, recombinant transferrin 1 mg / L, recombinant bovine insulin 5 mg / L, sodium selenite 0.4 mg / L, zinc sulfate heptahydrate 0.01 g / L, cortisol 0.5 mg / mL, recombinant human albumin 0.1 g / mL, recombinant basic fibroblast growth factor 10 μg / mL, recombinant transforming growth factor β 10.5 ng / mL, recombinant insulin-like growth factor 100 μg / mL, ethanolamine 50 mg / mL, mercaptoethanol 10 mg / L, and 2-phospho-L-ascorbic acid sodium salt 0.1 g / L;

[0011] (2) Filter with a 0.2 μm low protein adsorption filter membrane;

[0012] (3) Add the filtered L-alanyl-L-glutamine, recombinant transferrin, recombinant bovine insulin, sodium selenite, zinc sulfate heptahydrate, cortisol, recombinant human albumin, recombinant basic fibroblast growth factor, recombinant transforming growth factor-β1, recombinant insulin-like growth factor, ethanolamine, mercaptoethanol and 2-phospho-L-ascorbic acid sodium salt solution to the DMEM basal medium and mix well to obtain a serum-free medium for bovine adipose mesenchymal stem cells.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] 1. The culture medium of the present invention does not contain animal-derived components, the infection risk is controllable, and it meets food production standards;

[0015] 2. The components contained in the culture medium of the present invention are all derived from recombinant proteins or chemically synthesized, and the composition is clear;

[0016] 3. When the culture medium prepared by the method of the present invention is used, it is not necessary to coat the culture dish or culture bottle in advance;

[0017] 4. When the culture medium prepared by the method of the present invention is used, the cells have good wall adhesion and the cell morphology has no obvious difference from that of the serum cultured cells;

[0018] 5. The culture medium prepared by the method of the present invention has a high cell proliferation rate when used, is relatively cheap, and is suitable for large-scale cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Screening Plackett-Burman test results for the first batch of culture medium components;

[0020] Figure 2 Screening Plackett-Burman test results for the second batch of culture medium ingredients;

[0021] Figure 3This is a comparison diagram of P7 generation bovine adipose mesenchymal stem cells in the present invention, serum-free culture medium of other brands, and 10% serum culture medium. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0023] The components in the present invention can be conventional commercially available products (Table 1).

[0024] Table 1. Serum-free medium composition information

[0025]

[0026]

[0027] Example 1 Screening of serum-free culture medium components for bovine adipose-derived mesenchymal stem cells

[0028] Screening method: Two batches of Plackett-Burman experimental design were performed on various components in serum-free culture medium (Tables 2 and 3). Each batch of Plackett-Burman experiment had 12 treatment groups, and each treatment group had 3 replicates. In the first batch of experiments, two concentration levels of ITS (%), fibronectin (FN, μg / mL), recombinant human serum albumin (ReHA, mg / mL), recombinant basic fibroblast growth factor (FGF2, ng / mL), recombinant insulin-like growth factor (IGF1, ng / mL), recombinant epidermal growth factor (EGF, ng / mL), recombinant transforming growth factor β1 (TGFβ1, ng / mL) and recombinant vascular endothelial growth factor (VEGF, ng / mL) were screened. The concentration levels were determined by references and previous experiments.

[0029] Table 2. Plackett-Burman experimental design for the first batch of media component screening

[0030] Treatment Group ITS FN ReHA FGF2 IGF1 EGF TGFβ1 VEGF 1 0 0 0.8 10 5 0 0 0 2 5 2 0.8 0 0 0 0 5 3 5 2 0 0 5 0 0.5 0 4 0 2 0.8 0 5 10 0 5 5 0 2 0.8 10 0 10 0.5 0 6 5 0 0.8 0 5 10 0.5 0 7 5 0 0 10 5 10 0 5 8 0 0 0 0 0 10 0.5 5 9 0 0 0 0 0 0 0 0 10 5 2 0 10 0 10 0 0 11 5 0 0.8 10 0 0 0.5 5 12 0 2 0 10 5 0 0.5 5

[0031] After culturing cells with 12 experimental formulations for 72 hours, the cells were counted and the results of the 12 treatment groups were analyzed ( Figure 1 ). The results showed that the addition of ITS, ReHA, FGF2, IGF and TGFβ1 promoted cell proliferation and were therefore retained in the culture medium formula.

[0032] In the second batch of experiments, two concentration levels of sodium selenite (SS, 0 and 0.01%), ferric citrate (FC, μg / mL), zinc sulfate heptahydrate (ZnSO4, mg / L), hydrocortisone (HC, ng / mL), ethanolamine (Ethan., mg / L), mercaptoethanol (EGF, mg / L), HEPES buffer (mg / L) and 2-phospho-L-ascorbic acid sodium salt (P_LAA, μg / mL) were screened.

[0033] Table 3. Plackett-Burman experimental design for screening of the second batch of culture medium components

[0034] Treatment Group SS FC ZnSO4 HC Ethan. ME HEPES P_LAA 1 0 0 10 25 50 0 0 0 2 0.01 2 1 0 0 0 0 20 3 0.01 2 0 0 50 0 3000 0 4 0 2 1 0 50 2.2 0 20 5 0 2 1 25 0 2.2 3000 0 6 0.01 0 1 0 50 2.2 3000 0 7 0.01 0 0 25 50 2.2 0 20 8 0 0 0 0 0 2.2 3000 20 9 0 0 0 0 0 0 0 0 10 0.01 2 0 25 0 2.2 0 0 11 0.01 0 1 25 0 0 3000 20 12 0 2 0 25 50 0 3000 20

[0035] After culturing cells with 12 experimental formulations for 72 hours, the cells were counted and the results of the 12 treatment groups were analyzed ( Figure 2 ). The results showed that the addition of sodium selenite, zinc sulfate heptahydrate, hydrocortisone, ethanolamine, mercaptoethanol and 2-phospho-L-ascorbic acid sodium salt promoted cell proliferation and were therefore retained in the culture medium formulation. However, ferric citrate and HEPES buffer had no promoting effect in the current formulation and were therefore not retained in the culture medium.

[0036] Example 2 Determination of the ratio of main components of serum-free medium

[0037] After determining the components of the culture medium, two orthogonal test designs (Tables 4 and 5) were used to optimize the ratio of the two batches of screened culture medium components. Each orthogonal test had 12 treatment groups, each with 3 replicates, and each component was designed with two concentration levels, high and low.

[0038] Table 4. The first orthogonal experimental design

[0039]

[0040]

[0041] Table 5. Second orthogonal experimental design

[0042] Treatment Group SS ZnSO4 HC Ethan. ME P_LAA 1 0.005 1 25 50 0.1 5 2 0.01 1 50 2 0.1 30 3 0.01 2 50 50 0.1 5 4 0.005 1 50 50 2.2 30 5 0.005 1 25 2 2.2 5 6 0.01 1 50 50 2.2 5 7 0.01 2 25 50 2.2 30 8 0.005 2 50 2 2.2 30 9 0.005 2 50 2 0.1 5 10 0.01 2 25 2 2.2 5 11 0.01 1 25 2 0.1 30 12 0.005 2 25 50 0.1 30

[0043] The results showed that the bovine adipose-derived mesenchymal stem cells cultured in the following ratio had the best morphology, higher proliferation efficiency, and lower overall culture medium cost: the culture medium included DMEM high-glucose basal medium, recombinant transferrin 4mg / L, recombinant bovine insulin 8mg / L, sodium selenite 0.005mg / L, zinc sulfate heptahydrate 1mg / L, cortisol 25ng / mL, recombinant human albumin 1.25mg / mL, recombinant basic fibroblast growth factor 5ng / mL, recombinant transforming growth factor β1 0.5ng / mL, recombinant insulin-like growth factor 10ng / mL, ethanolamine 50mg / mL, mercaptoethanol 2.2mg / L, and 2-phospho-L-ascorbic acid sodium salt 30mg / L. In addition, 75mg / L L-alanyl-L-glutamine was added to the culture medium as a supplementary nitrogen source.

[0044] Example 3 Comparison of the medium formula with serum medium and commercially available serum-free medium

[0045] According to the culture medium formula determined by the present invention, a serum-free culture medium is prepared. At the same time, a serum-containing culture medium for conventional use is prepared: fetal bovine serum is added to the DMEM high-glucose culture medium at a ratio of 10%, and then a recombinant basic fibroblast growth factor at a concentration of 5 ng / mL is added. The above two culture media are compared with a serum-free mesenchymal stem cell culture medium of a certain brand on the market. First, the adherent bovine adipose mesenchymal stem cells are digested with 0.25% trypsin, and then added to the two culture media at a concentration of 30,000 cells per milliliter, and placed in a 12-well plate with a volume of 1 mL per well. The 12-well culture plate is placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. After 72 hours of culture, the cell state is observed and the concentration of live cells is counted. Figure 3 This is a comparison of P7 bovine adipose mesenchymal stem cells cultured in three different culture media. As can be seen from the figure, the cells cultured in the three culture media are all spindle-shaped and fibrous, indicating that the serum-free culture medium formula can maintain a good cell morphology. The results of cell counting show that the serum-free culture medium of the present invention can achieve the same level of cell proliferation as the commercially available serum-free culture medium, and the proliferation promotion efficiency is higher than that of the 10% serum culture medium.

Claims

1. A serum-free culture medium for bovine adipose mesenchymal stem cells suitable for cell culture meat, characterized in that: The culture medium is composed of DMEM basal culture medium, L-alanyl-L-glutamine 75 mg / L, recombinant transferrin 0-5.5 mg / L, recombinant bovine insulin 0-10 mg / L, sodium selenite 0.001-0.01 mg / L, zinc sulfate heptahydrate 0.1-2 mg / L, cortisol 10-50 ng / mL, recombinant human albumin 0.1-1.5 mg / mL, recombinant basic fibroblast growth factor 1-10 ng / mL, recombinant transforming growth factor β1 0-2 ng / mL, recombinant insulin-like growth factor 5-20 ng / mL, ethanolamine 2-100 mg / mL, mercaptoethanol 0.1-3 mg / L and 2-phospho-L-ascorbic acid sodium salt 10-50 mg / L, wherein the DMEM basal culture medium contains: 4500 mg / L D-glucose, 3700 mg / L sodium bicarbonate, 584 mg / L L-glutamine and 110 mg / L sodium pyruvate.

2. The serum-free medium for bovine adipose mesenchymal stem cells suitable for cell culture of meat according to claim 1, characterized in that: The culture medium consists of DMEM basal culture medium, 75 mg / L L-alanyl-L-glutamine, 4 mg / L recombinant transferrin, 8 mg / L recombinant bovine insulin, 0.005 mg / L sodium selenite, 1 mg / L zinc sulfate heptahydrate, 25 ng / mL cortisol, 1.25 mg / mL recombinant human albumin, 5 ng / mL recombinant basic fibroblast growth factor, 10.5 ng / mL recombinant transforming growth factor β, 10 ng / mL recombinant insulin-like growth factor, 50 mg / mL ethanolamine, 2.2 mg / L mercaptoethanol and 30 mg / L 2-phospho-L-ascorbic acid sodium salt, wherein the DMEM basal culture medium contains: 4500 mg / L D-glucose, 3700 mg / L sodium bicarbonate, 584 mg / L L-glutamine and 110 mg / L sodium pyruvate.

3. A method for preparing a serum-free medium for bovine adipose mesenchymal stem cells suitable for cell culture meat according to claim 1, characterized in that: The steps include: (1) Prepare reagents of corresponding concentrations for use: L-alanyl-L-glutamine 43.5 mg / L, recombinant transferrin 1 mg / L, recombinant bovine insulin 5 mg / L, sodium selenite 0.4 mg / L, zinc sulfate heptahydrate 0.01 g / L, cortisol 0.5 mg / mL, recombinant human albumin 0.1 g / mL, recombinant basic fibroblast growth factor 10 μg / mL, recombinant transforming growth factor β1 0.5 ng / mL, recombinant insulin-like growth factor 100 μg / mL, ethanolamine 50 mg / mL, mercaptoethanol 10 mg / L, and 2-phospho-L-ascorbic acid sodium salt 0.1 g / L; (2) Filter with a 0.2 μm low protein adsorption filter membrane; (3) Add the filtered L-alanyl-L-glutamine, recombinant transferrin, recombinant bovine insulin, sodium selenite, zinc sulfate heptahydrate, cortisol, recombinant human albumin, recombinant basic fibroblast growth factor, recombinant transforming growth factor-β1, recombinant insulin-like growth factor, ethanolamine, mercaptoethanol and 2-phospho-L-ascorbic acid sodium salt solution to the DMEM basal medium and mix well to obtain a serum-free medium for bovine adipose mesenchymal stem cells.

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