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CN115537388A - Bovine adipose-derived mesenchymal stem cell serum-free medium suitable for cell culture of meat and preparation method thereof - Google Patents

Bovine adipose-derived mesenchymal stem cell serum-free medium suitable for cell culture of meat and preparation method thereof Download PDF

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CN115537388A
CN115537388A CN202211249948.1A CN202211249948A CN115537388A CN 115537388 A CN115537388 A CN 115537388A CN 202211249948 A CN202211249948 A CN 202211249948A CN 115537388 A CN115537388 A CN 115537388A
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growth factor
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高寒
梁逸
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Hangzhou Ji Mi Biotechnology Co ltd
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Abstract

The invention discloses a serum-free culture medium for bovine adipose-derived mesenchymal stem cells, which is suitable for cell culture meat, belongs to the technical field of animal cell culture, and comprises DMEM basic culture, L-alanyl-L-glutamine, recombinant transferrin, recombinant bovine insulin, sodium selenite, zinc sulfate heptahydrate, cortisol, recombinant human serum albumin, recombinant basic fibroblast growth factor, recombinant transforming growth factor beta 1, recombinant insulin-like growth factor, ethanolamine, mercaptoethanol and 2-phosphoric acid-L-ascorbic acid sodium salt. Repeated experiments prove that the inventor obtains a novel bovine Adipose-derived mesenchymal stem cell (ADSC) serum-free culture medium, and can effectively culture the bovine Adipose mesenchymal stem cells.

Description

Bovine adipose-derived mesenchymal stem cell serum-free medium suitable for cell culture of meat and preparation method thereof
Technical Field
The invention belongs to the technical field of animal cell culture, and particularly relates to a bovine adipose-derived mesenchymal stem cell serum-free medium suitable for cell culture of meat and a preparation method thereof.
Background
Compared with plant meat and fermented meat made of microorganisms, the cell culture meat is novel meat which is closest to traditional meat. The cell culture meat has the advantages of no microbial pollution, high-quality protein content, good eating experience and the like, and the carbon footprint and water resource requirements in the production process are much lower than those of the traditional livestock breeding, so that the cell culture meat is one of the most promising development directions in the future culture of meat substitutes. Meanwhile, the technical barrier of the cell culture meat industry is also higher and more challenging. Among them, the development of a low-cost animal-derived component-free medium suitable for cell culture meat is an important basis for cell culture meat to meet food safety standards and eventually to be able to enter mass production.
Mesenchymal Stem Cells (MSCs) are derived from the mesoderm in the early embryonic development stage, are adult Stem Cells which have the potential of self-renewal and multidirectional differentiation and can still maintain the biological characteristics thereof after in vitro large-scale amplification, and can proliferate and differentiate to various tissues such as bone, cartilage, muscle, ligament, tendon, fat and stromal Cells. Therefore, mesenchymal stem cells are one of the important cell sources for culturing meat.
At present, the media used to culture MSCs are mainly of the following types: one is a basal medium (such as alpha-MEM medium, DMEM medium, etc.) supplemented with 5-20% animal serum. However, animal serum is expensive and has a limited yield, and is not suitable for large-scale production of cell culture meat. In addition, animal serum may have mycoplasma, prion and other pollution, and human xenoprotein pollution or allergy and other risks, and is difficult to meet edible standards; the second category is the addition of serum replacement (e.g., human platelet lysate, etc.) to basal media. However, the culture medium has animal-derived components, has unknown protein, has the defects of large batch difference, unstable source and the like, and is not suitable for producing cell culture meat on a large scale; the third type is to supplement serum substitute components which are not derived from animals and have definite chemical components in a basal culture medium, so that the nutrient requirement of cells can be met, and the natural defects of the first two types of culture media can be avoided. Therefore, the cell culture medium which is suitable for the growth of the MSCs and has definite chemical formula and low cost is an important prerequisite for the development of large-scale production of cell culture meat.
Disclosure of Invention
Aiming at the existing problems, the inventor obtains a novel serum-free culture medium of bovine Adipose-derived mesenchymal stem cells (ADSCs) through repeated experimental verification, and can effectively culture the bovine Adipose mesenchymal stem cells.
The invention is realized by the following technical scheme:
a bovine adipose-derived mesenchymal stem cell serum-free culture medium suitable for cell culture meat comprises a DMEM basic culture medium, and further comprises the following components and concentrations thereof: 75mg/L of L-alanyl-L-glutamine, 0-5.5mg/L of recombinant transferrin, 0-10mg/L of recombinant bovine insulin, 0.001-0.01mg/L of sodium selenite, 0.1-2mg/L of zinc sulfate heptahydrate, 10-50ng/mL of cortisol, 0.1-1.5mg/mL of recombinant human albumin, 1-10ng/mL of recombinant basic fibroblast growth factor, 1-2 ng/mL of recombinant transforming growth factor beta, 5-20ng/mL of recombinant insulin-like growth factor, 2-100mg/mL of ethanolamine, 0.1-3mg/L of mercaptoethanol and 10-50mg/L of 2-phosphate-L-ascorbic acid sodium salt, wherein the DMEM basal medium comprises: 4500mg/L D-glucose, 3700mg/L sodium bicarbonate, 584mg/L L-glutamine and 110mg/L sodium pyruvate.
Preferably, the culture medium comprises a DMEM basic culture medium, and further comprises the following components and concentrations thereof: 75mg/L of L-propylamino-L-glutamine, 4mg/L of recombinant transferrin, 8mg/L of recombinant bovine insulin, 0.005mg/L of sodium selenite, 1mg/L of zinc sulfate heptahydrate, 25ng/mL of cortisol, 1.25mg/mL of recombinant human serum albumin, 5ng/mL of recombinant basic fibroblast growth factor, 1.5 ng/mL of recombinant transforming growth factor beta, 10ng/mL of recombinant insulin-like growth factor, 50mg/mL of ethanolamine, 2.2mg/L of mercaptoethanol and 30mg/L of 2-phosphoric acid-L-ascorbic acid sodium salt, wherein the DMEM basal medium comprises: 4500mg/L D-glucose, 3700mg/L sodium bicarbonate, 584mg/L L-glutamine and 110mg/L sodium pyruvate.
A preparation method of a bovine adipose-derived mesenchymal stem cell serum-free medium suitable for cell culture meat comprises the following steps:
(1) Preparing the corresponding concentrations for later use: 43.5mg/L of L-alanyl-L-glutamine, 1mg/L of recombinant transferrin, 5mg/L of recombinant bovine insulin, 0.4mg/L of sodium selenite, 0.01g/L of zinc sulfate heptahydrate, 0.5mg/mL of cortisol, 0.1g/mL of recombinant human serum albumin, 10 mug/mL of recombinant basic fibroblast growth factor, 100 mug/mL of recombinant insulin-like growth factor, 50mg/mL of ethanolamine, 10mg/L of mercaptoethanol and 0.1g/L of sodium 2-phosphate-L-ascorbate;
(2) Filtering with 0.2 μm low protein adsorption filter membrane;
(3) And adding the filtered L-propylamino-L-glutamine, recombinant transferrin, recombinant bovine insulin, sodium selenite, zinc sulfate heptahydrate, cortisol, recombinant human serum albumin, recombinant basic fibroblast growth factor, recombinant transforming growth factor-beta 1, recombinant insulin-like growth factor, ethanolamine, mercaptoethanol and 2-phosphoric acid-L-ascorbic acid sodium salt solution into a DMEM basic culture medium, and uniformly mixing to obtain the serum-free culture medium of the bovine adipose-derived mesenchymal stem cells.
Compared with the prior art, the invention has the following advantages:
1. the culture medium has no animal-derived components, has controllable infection risk and meets the food production standard;
2. the components contained in the culture medium are all derived from recombinant proteins or chemically synthesized, and the components are clear;
3. when the culture medium prepared by the method is applied, a culture dish or a culture bottle does not need to be coated in advance;
4. when the culture medium prepared by the method is applied, the adherence of cells is good, and the cell morphology has no obvious difference with that of serum cultured cells;
5. when the culture medium prepared by the method is applied, the cell proliferation rate is high, the price is relatively low, and the method is suitable for large-scale cell culture.
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FIG. 1 shows the results of a Plackett-Burman experiment for screening of first batch of medium components;
FIG. 2 shows the results of a Plackett-Burman experiment for the second batch of media composition screening;
FIG. 3 is a graph comparing P7 generation bovine adipose derived mesenchymal stem cells with other brands of serum-free medium and 10% serum medium.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
The components of the present invention may be selected from conventional commercially available products (Table 1).
TABLE 1 serum-free Medium composition information
Figure BDA0003887292890000031
Figure BDA0003887292890000041
Example 1 selection of serum-free Medium Components of bovine adipose-derived mesenchymal Stem cells
The screening method comprises the following steps: two batches of the Plackett-Burman experiment design (tables 2, 3) were run separately for various components in serum-free medium, with 12 treatment groups per batch of the Plackett-Burman experiment, and 3 replicates per treatment group. In the first experiment, two concentration levels of ITS (%), fibronectin (FN, μ g/mL), recombinant human serum albumin (ReHA, mg/mL), recombinant basic fibroblast growth factor (FGF 2, ng/mL), recombinant insulin-like growth factor (IGF 1, ng/mL), recombinant epidermal growth factor (EGF, ng/mL), recombinant transforming growth factor beta 1 (TGF beta 1, ng/mL) and recombinant vascular endothelial growth factor (VEGF, ng/mL) were screened, the concentration levels being determined by reference and earlier experiments.
TABLE 2 first batch Medium composition screening Plackett-Burman Experimental design
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
After 72h of cell culture with 12 experimental formulations, the cells were counted and the results of 12 treatment groups were analyzed (fig. 1). The results show that the addition of ITS, reHA, FGF2, IGF and TGF beta 1 promotes cell proliferation and is therefore retained in the medium formulation
Two concentration levels of sodium selenite (SS, 0 and 0.01%), ferric citrate (FC, μ g/mL), zinc sulfate heptahydrate (ZnSO 4, mg/L), hydrocortisone (h.c., ng/mL), ethanolamine (ethan., mg/L), mercaptoethanol (EGF, mg/L), HEPES buffer (mg/L) and sodium 2-phospho-L-ascorbate (P _ LAA, μ g/mL) were screened in the second experiment.
TABLE 3 second batch Medium composition screening Plackett-Burman Experimental design
Treatment group SS FC ZnSO4 H.C. 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
After 72h of cell culture with 12 experimental formulations, the cells were counted and the results of 12 treatment groups were analyzed (fig. 2). The results show that the addition of sodium selenite, zinc sulfate heptahydrate, hydrocortisone, ethanolamine, mercaptoethanol and sodium 2-phospho-L-ascorbate promotes cell proliferation and is therefore retained in the medium formulation. While ferric citrate and HEPES buffer did not promote in the current formulations and therefore were not retained in the culture medium.
EXAMPLE 2 determination of the proportion of the major constituents of serum-free Medium
After determining each component of the culture medium, the mixture ratio of the components of the culture medium screened in two batches is optimized through two times of orthogonal experimental design (tables 4 and 5). Each time, 12 treatment groups were tested orthogonally, with 3 replicates per treatment group, each component being designed for both high and low concentration levels.
TABLE 4 first orthogonal experimental design
Figure BDA0003887292890000051
Figure BDA0003887292890000061
TABLE 5 second orthogonal design of experiment
Treatment group SS ZnSO4 H.C. 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
As a result, it was found that: the bovine adipose-derived mesenchymal stem cells cultured by adopting the following mixture ratio have the advantages of optimal shape, higher proliferation efficiency and lower overall cost of the culture medium: the culture medium comprises a DMEM high-glycosyl basic culture medium, 4mg/L of recombinant transferrin, 8mg/L of recombinant bovine insulin, 0.005mg/L of sodium selenite, 1mg/L of zinc sulfate heptahydrate, 25ng/mL of cortisol, 1.25mg/mL of recombinant human serum albumin, 5ng/mL of recombinant basic fibroblast growth factor, 1.5 ng/mL of recombinant transforming growth factor beta, 10ng/mL of recombinant insulin-like growth factor, 50mg/mL of ethanolamine, 2.2mg/L of mercaptoethanol and 30mg/L of 2-phospho-L-ascorbic acid sodium salt. In addition, the medium was supplemented with 75mg/L L-propylamino-L-glutamine as a supplemental nitrogen source.
Example 3 comparison of the minimal Medium formulation with serum Medium and commercial serum-free Medium
According to the formula of the culture medium determined by the invention, a serum-free culture medium is prepared. Meanwhile, a conventionally used serum culture medium is prepared: adding fetal calf serum into DMEM high-sugar medium according to the proportion of 10%, and then adding recombinant basic fibroblast growth factor with the concentration of 5 ng/mL. The two culture mediums are compared with a serum-free mesenchymal stem cell culture medium of a certain brand on the market. First, bovine adipose-derived mesenchymal stem cells cultured adherently were digested with 0.25% trypsin, added to two media at a concentration of 3 ten thousand cells per mL, and placed in 12-well plates at a volume of 1mL per well. Placing 12-well culture plates at 37 deg.C, 5% CO 2 And culturing in a cell culture box with saturated humidity, observing the cell state after culturing for 72h, and counting the concentration of living cells. Fig. 3 is a graph comparing P7 generation bovine adipose-derived mesenchymal stem cells cultured in three different media. As can be seen from the figure, the cells cultured by the three culture media are all spindle-shaped and fiber-like, which indicates that the serum-free culture medium can maintain better cell morphology. The cell counting result shows that the serum-free culture medium can reach the same level of the commercial serum-free culture medium in terms of cell proliferation, and the proliferation promoting efficiency is higher than that of a 10% serum culture medium.

Claims (3)

1. The serum-free culture medium for the bovine adipose-derived mesenchymal stem cells is suitable for cell culture of meat, and is characterized by comprising a DMEM basic culture medium and further comprising the following components in concentration: 75mg/L of L-alanyl-L-glutamine, 0-5.5mg/L of recombinant transferrin, 0-10mg/L of recombinant bovine insulin, 0.001-0.01mg/L of sodium selenite, 0.1-2mg/L of zinc sulfate heptahydrate, 10-50ng/mL of cortisol, 0.1-1.5mg/mL of recombinant human albumin, 1-10ng/mL of recombinant basic fibroblast growth factor, 1-2 ng/mL of recombinant transforming growth factor beta, 5-20ng/mL of recombinant insulin-like growth factor, 2-100mg/mL of ethanolamine, 0.1-3mg/L of mercaptoethanol and 10-50mg/L of 2-phosphate-L-ascorbic acid sodium salt, wherein the DMEM basal medium comprises: 4500mg/L D-glucose, 3700mg/L sodium bicarbonate, 584mg/L L-glutamine and 110mg/L sodium pyruvate.
2. The serum-free culture medium for the bovine adipose-derived mesenchymal stem cells, which is suitable for cell culture of meat, according to claim 1, comprises a DMEM (DMEM) basic culture medium, and further comprises the following components and concentrations thereof: 75mg/L of L-propylamino-L-glutamine, 4mg/L of recombinant transferrin, 8mg/L of recombinant bovine insulin, 0.005mg/L of sodium selenite, 1mg/L of zinc sulfate heptahydrate, 25ng/mL of cortisol, 1.25mg/mL of recombinant human serum albumin, 5ng/mL of recombinant basic fibroblast growth factor, 1.5 ng/mL of recombinant transforming growth factor beta, 10ng/mL of recombinant insulin-like growth factor, 50mg/mL of ethanolamine, 2.2mg/L of mercaptoethanol and 30mg/L of 2-phosphoric acid-L-ascorbic acid sodium salt, wherein the DMEM basal medium comprises: 4500 mg/LD-glucose, 3700mg/L sodium bicarbonate, 584mg/L L-glutamine and 110mg/L sodium pyruvate.
3. The method for preparing the serum-free medium for the bovine adipose-derived mesenchymal stem cells suitable for cell culture of meat according to claim 1, which comprises the following steps:
(1) Preparing the corresponding concentrations for later use: 43.5mg/L of L-alanyl-L-glutamine, 1mg/L of recombinant transferrin, 5mg/L of recombinant bovine insulin, 0.4mg/L of sodium selenite, 0.01g/L of zinc sulfate heptahydrate, 0.5mg/mL of cortisol, 0.1g/mL of recombinant human serum albumin, 10 ug/mL of recombinant basic fibroblast growth factor, 100 ug/mL of recombinant insulin-like growth factor, 50mg/mL of ethanolamine, 10mg/L of mercaptoethanol and 0.1g/L of sodium salt of 2-phospho-L-ascorbic acid;
(2) Filtering with 0.2 μm low protein adsorption filter membrane;
(3) And adding the filtered L-propylamino-L-glutamine, recombinant transferrin, recombinant bovine insulin, sodium selenite, zinc sulfate heptahydrate, cortisol, recombinant human serum albumin, recombinant basic fibroblast growth factor, recombinant transforming growth factor-beta 1, recombinant insulin-like growth factor, ethanolamine, mercaptoethanol and 2-phosphoric acid-L-ascorbic acid sodium salt solution into a DMEM basic culture medium, and uniformly mixing to obtain the serum-free culture medium of the bovine adipose-derived mesenchymal stem cells.
CN202211249948.1A 2022-10-12 2022-10-12 Bovine adipose-derived mesenchymal stem cell serum-free medium suitable for cell culture of meat and preparation method thereof Pending CN115537388A (en)

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