Fetal bovine serum-free culture medium for promoting survival and proliferation of muscle stem cells in vitro and its application

Abstract

The present invention discloses a fetal bovine serum-free culture medium for promoting the survival and proliferation of muscle stem cells in vitro and its application, and belongs to the technical field of stem cell culture and culture medium. The present invention provides a fetal bovine serum-free culture medium SFBM that can ensure the growth and survival of muscle stem cells, and develops a fetal bovine serum-free culture medium SFPM that can quickly promote the proliferation of muscle stem cells based on SFBM, in which muscle stem cells can grow normally without affecting their differentiation potential. It effectively avoids the problems that may be caused by fetal bovine serum, utilizes waste (pig blood) when separating muscle stem cells, greatly reduces the cost of culture medium, provides sufficient cell sources for the research of seed cells and muscle stem cells in the cell culture meat industry, and is conducive to the large-scale production and commercial use of cells.

Description

Technical Field

[0001] The invention relates to a fetal bovine serum-free culture medium for promoting the survival and proliferation of muscle stem cells in vitro and an application thereof, belonging to the technical field of stem cell culture and culture medium. Background Art

[0002] Muscle stem cells (Muscle Stem Cell, also known as muscle satellite cells, Satellite Cell) are multipotent stem cells in muscle tissue and can be isolated from fresh muscle. They have been widely used in tissue engineering, medicine, cell culture meat and other fields because of their rich sources, easy separation, in vitro proliferation ability and multidirectional differentiation potential such as myogenic differentiation, adipogenic differentiation and osteogenic differentiation.

[0003] In the process of cell culture, culture medium is the most important component to maintain cell survival and proliferation. Most of the commonly used cell culture media now add serum substances such as fetal bovine serum / calf serum / horse serum. Serum is a complex mixture obtained by removing fibrin from plasma. It contains proteins, peptides, vitamins and growth factors necessary for various cells, which are very important for cell proliferation and differentiation. Among them, fetal bovine serum (FBS) is taken from fetal cows that have not been exposed to the external environment. Its serum contains the least antibodies and other components that are harmful to cells. It is rich in nutrients and cytokines necessary for cell growth. It can promote efficient cell proliferation and has the best quality. Therefore, it is the most commonly used in cell culture. However, the output of fetal bovine serum is low, the cost is high, and there are differences in quality between batches. It also brings some animal ethical issues and is not suitable for large-scale industrialization and commercial use of cells.

[0004] Therefore, it is urgent to find a culture medium that does not use fetal bovine serum and can ensure the efficient proliferation of muscle stem cells. This can greatly reduce the cost of muscle stem cell culture and facilitate large-scale production and commercial use of cells. Summary of the invention

[0005] The purpose of the present invention is to provide two high-efficiency muscle stem cell proliferation culture media without using fetal bovine serum and their application, which can ensure the rapid proliferation of muscle stem cells without using fetal bovine serum and ensure that their differentiation potential remains unchanged.

[0006] The first object of the present invention is to provide a serum-free basal medium SFBM (Serum-free basal medium) that can ensure the growth and proliferation of muscle stem cells. The serum-free medium includes a basal medium and additives.

[0007] The basic culture medium includes DMEM and / or F12.

[0008] The basic culture medium is a culture medium obtained by mixing DMEM and F12 in equal proportions.

[0009] The additive comprises component A, component B and component C; component A comprises bovine serum albumin (BSA), insulin, transferrin, selenite, ethanolamine, L-glutamine and dexamethasone, or comprises bovine serum albumin (BSA), L-glutamine, glucocorticoid and 1× insulin-transferrin-selenium-aminoethanol (ITS-X); component B comprises platelet-derived factor, basic growth factor, epidermal growth factor and insulin-like growth factor; component C comprises inorganic salt, α-ketoglutaric acid, HPPES solution and MEM amino acid solution.

[0010] In one embodiment, the component C may further contain ascorbic acid (VC) or its derivatives and / or amino acids.

[0011] In one embodiment, the glucocorticoid is selected from one or more of dexamethasone or a salt thereof, a dexamethasone solvate, hydrocortisone or a salt thereof, and a hydrocortisone solvate.

[0012] In one embodiment, the ascorbic acid (VC) or its derivatives are selected from one or more of vitamin C (ascorbic acid), ascorbyl glucoside, ethyl ascorbic acid, magnesium ascorbic acid phosphate, sodium ascorbic acid phosphate, and other solvates of ascorbic acid.

[0013] In one embodiment, the amino acid is selected from one or more of glycine, alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and glutamine.

[0014] In one embodiment, the inorganic salt is selected from one or more of calcium chloride, potassium chloride, copper sulfate, sodium bicarbonate, ferric nitrate, sodium chloride, ferrous sulfate, disodium hydrogen phosphate, magnesium chloride, sodium hydrogen phosphate, magnesium sulfate, and zinc sulfate.

[0015] In one embodiment, the concentrations of the components in the additive in the serum-free medium are as follows: 1-8 mg / mL bovine serum albumin (BSA), 0.5-5 μg / mL insulin, 10-200 μg / mL transferrin, 1-20 ng / mL selenite, 0.5-5 μg / mL ethanolamine, 1-5 mM L-glutamine, 10 -7 ~10 -6M dexamethasone, 1-50 ng / mL platelet-derived factor, 1-50 ng / mL basic fibroblast growth factor, 1-50 ng / mL epidermal growth factor, 10-100 ng / mL insulin-like growth factor, 2-10 mM inorganic salt, 10-100 μg / mL ascorbic acid (VC) or its derivatives, 5-100 μM amino acids, 0.5-5 mM α-ketoglutaric acid, 1×HPPES solution, 1×MEM amino acid solution.

[0016] Muscle stem cells can adhere to and grow in SFBM culture medium, ensuring the normal growth of muscle stem cells in the absence of FBS.

[0017] In one embodiment, bovine serum albumin is used at a concentration of 5 mg / mL, insulin is used at a concentration of 2.5 μg / mL, transferrin is used at a concentration of 100 μg / mL, selenite is used at a concentration of 6.7 ng / mL, ethanolamine is used at a concentration of 2 μg / mL, L-glutamine is used at a concentration of 2 mM, dexamethasone is used at a concentration of 10 -7 M, platelet-derived factor was used at a concentration of 10 ng / mL, basic fibroblast growth factor was used at a concentration of 10 ng / mL, epidermal growth factor was used at a concentration of 10 ng / mL, insulin-like growth factor was used at a concentration of 50 ng / mL, and NaHCO 3 The concentration of inorganic salts was 6.5 mM, the concentration of ascorbic acid (VC) or its derivatives was 50 μg / mL, the concentration of amino acids was 50 μM, and the concentration of α-ketoglutaric acid was 2 mM.

[0018] In one embodiment, the muscle stem cells include but are not limited to muscle stem cells of mammals or oviparous animals.

[0019] The second object of the present invention is to provide a fetal bovine serum-free culture medium SFPM for promoting rapid proliferation of muscle stem cells. The SFPM culture medium is prepared by adding 1-10% (v / v) pig blood cryopreservation solution to the serum-free basal culture medium.

[0020] In one embodiment, the porcine blood cryothaw solution is used at a concentration of 2% (v / v).

[0021] In one embodiment, the method for preparing the pig blood freeze-thaw solution is to remove red blood cells from the pig blood, freeze-thaw repeatedly for multiple times, and take the supernatant and filter it.

[0022] In one embodiment, the porcine blood is piglet blood.

[0023] In one embodiment, the repeated freezing and thawing conditions are freezing at -80°C and thawing at 37°C.

[0024] The third object of the present invention is to provide a method for culturing muscle stem cells, wherein the method comprises culturing muscle stem cells using the culture medium SFBM and the culture medium SFPM.

[0025] The fourth object of the present invention is to provide the use of the culture medium SFBM and the culture medium SFPM in cell culture meat production. In these two culture media, muscle stem cells can still have differentiation potential without the addition of fetal bovine serum, and can fuse to form multinucleated myotubes and muscle fibers.

[0026] Beneficial effects:

[0027] The present invention obtains substances with clear components that can ensure the normal growth and rapid proliferation of muscle stem cells by screening from multiple formulas, invents a serum-free culture medium SFBM that can ensure the growth and survival of muscle stem cells, and develops a fetal bovine serum-free culture medium SFPM that can quickly promote the proliferation of muscle stem cells based on SFBM, in which muscle stem cells can grow normally without affecting their differentiation potential. It effectively avoids the problems that may be caused by fetal bovine serum, greatly reduces the cost of the culture medium, provides sufficient cell sources for the research of seed cells and muscle stem cells in the cell culture meat industry, and is conducive to the large-scale production and commercial use of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The cell morphology changes of muscle stem cells in different basic culture media;

[0029] Figure 2 MTT assay for the proliferation activity of muscle stem cells in different basal culture media;

[0030] Figure 3 The cell morphology changes of muscle stem cells in different screening groups;

[0031] Figure 4 MTT assay analysis of cell proliferation activity of muscle stem cells in different screening groups;

[0032] Figure 5 Cell morphological changes of muscle stem cells in 5% FBS, SFBM, and SFPM groups;

[0033] Figure 6 MTT assay analysis of cell proliferation activity of muscle stem cells in 5% FBS, SFBM, and SFPM groups;

[0034] Figure 7 Cell morphology and MyHC immunofluorescence results of muscle stem cells during differentiation in 5% FBS, SFBM, and SFPM groups. DETAILED DESCRIPTION

[0035] Example 1 Development of Fetal Bovine Serum-Free Culture Medium for Muscle Stem Cells

[0036] To develop a culture medium without fetal bovine serum for muscle stem cells, we first determined the basic proteins, supplements, cytokines and other components that can ensure cell survival, and investigated the effects of the basic culture medium DMEM and DMEM / F12 in a culture medium without fetal bovine serum. The basic components and concentrations used are as follows:

[0037] Table 1 Components and concentrations of supplements in the fetal bovine serum-free basal medium for muscle stem cells

[0038]

[0039] Primary muscle stem cells were seeded into 96-well plates at 7000 cells / well and incubated at 37°C and 5% CO 2 Under the conditions of 10% fetal bovine serum (FBS), muscle stem cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) to adhere to the wall. After adhesion, the supernatant was removed and the culture medium was replaced with DMEM+A+B, DMEM+2% FBS+A+B, DMEM / F12+A+B, and DMEM / F12+2% FBS+A+B. The amount of culture medium added to each group was the same, and 3 parallels were set for each group. The cell morphology was observed every day, and the amount of live cells was detected by MTT method after 3 days of culture. The specific operation was as follows:

[0040] 1) Aspirate the culture medium, dilute the MTT working stock solution (phenol red-free DMEM) at a ratio of 1:4, add 100 μL to each well, and culture at 37°C for 4 h;

[0041] 2) Remove the MTT working solution, add 150 μL DMSO (dimethyl sulfoxide) to each well, and incubate at 37°C on a shaker for 15 min;

[0042] 3) Use an ELISA reader to detect the absorbance wave (490 nm) and analyze the data.

[0043] Cell morphology changes of muscle stem cells in different culture media Figure 1 The results of cell proliferation activity detection analysis (MTT absorbance detection) are as follows Figure 2 .Depend on Figure 1 and Figure 2 The results showed that muscle stem cells grew better when DMEM / F12 was used as the basal culture medium, and that the culture medium with serum was superior to the culture medium without serum. Therefore, it was determined that the culture medium to be developed subsequently would be based on DMEM / F12.

[0044] In order to further ensure the growth and proliferation ability of muscle stem cells in fetal bovine serum-free medium, insulin-transferrin-selenium-aminoethanol (ITS-X), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), NaHCO 3 , MEM Amino Acids Solution, ascorbic acid (VC), tyrosine, α-ketoglutaric acid and B-27 additive (B27(-VA)), the group settings are shown in Table 2, and the component concentrations used are shown in Table 3.

[0045] Primary muscle stem cells were cultured at 10 4 Cells were seeded into 96-well plates and incubated at 37°C and 5% CO 2 Under the conditions of culture, muscle stem cells were allowed to adhere to the wall in DMEM medium containing 10% FBS. After adhesion, the culture medium of 16 groups was replaced respectively, and 3 parallels were set up for each group. The culture medium containing serum was used as the control group (1, 2, 3). The cell morphology was observed every day, and the amount of live cells was detected by MTT method after 3 days of culture.

[0046] Table 2 Screening group settings for fetal bovine serum-free culture medium for muscle stem cells

[0047]

[0048] Table 3 Concentrations of components used in screening culture medium without fetal bovine serum for muscle stem cells

[0049]

[0050]

[0051] Changes in cell morphology of muscle stem cells in different groups Figure 3 The results of cell proliferation activity detection analysis (MTT absorbance detection) are as follows Figure 4 .Depend on Figure 3 and Figure 4 The results showed that the cells grew better in groups 6, 7, 10, and 11, and were able to maintain the growth and proliferation of muscle stem cells. Therefore, a serum-free culture medium (SFBM) for muscle stem cell proliferation was obtained: bovine serum albumin (BSA) 5mg / mL, insulin μg / mL, transferrin 100μg / mL, sodium selenite 6.7ng / mL, ethanolamine 2μg / mL, L-glutamine 2mM, dexamethasone 10 -7 M, PDGF-BB 10ng / mL, bFGF 10ng / mL, EGF 10ng / mL, LR 3 -IGF1 50 ng / mL, NaHCO 36.5mM, ascorbic acid (VC) 50μg / mL, tyrosine 50μM, α-ketoglutaric acid 2mM, 1X HPPES solution, 1X MEM amino acid solution; among them, 2.5μg / mL insulin, 100μg / mL transferrin, 6.7ng / mL sodium selenite, and 2μg / mL ethanolamine can be replaced with 1×ITS-X.

[0052] The concentration of bovine serum albumin (BSA) in SFBM medium was adjusted to 1-8 mg / mL, the concentration of insulin was adjusted to 0.5-5 μg / mL, the concentration of transferrin was adjusted to 10-200 μg / mL, the concentration of sodium selenite was adjusted to 1-20 ng / mL, the concentration of ethanolamine was adjusted to 0.5-5 μg / mL, the concentration of L-glutamine was adjusted to 1-5 mM, and the concentration of dexamethasone was adjusted to 10 -7 ~10 -6 M, PDGF-BB concentration adjusted to 1-50 ng / mL, bFGF concentration adjusted to 1-50 ng / mL, EGF concentration adjusted to 1-50 ng / mL, LR 3 -IGF1 concentration adjusted to 10-100 ng / mL, NaHCO 3 The concentration is adjusted to 2-10mM, the ascorbic acid (VC) concentration is adjusted to 10-100μg / mL, the tyrosine concentration is adjusted to 5-100μM, and the α-ketoglutaric acid concentration is adjusted to 0.5-5mM. Muscle stem cells can be cultured in the same way to ensure their normal growth and proliferation.

[0053] Example 2 Development of a culture medium without fetal bovine serum for efficient proliferation of muscle stem cells

[0054] In order to further increase the proliferation rate of muscle stem cells in SFBM medium, the blood of young pigs was collected when muscle stem cells were separated, and after removing red blood cells, it was frozen at -80°C and then quickly thawed at 37°C. After repeated freezing and thawing 3-4 times, the supernatant was taken and filtered through a 0.22μm filter to obtain pig blood freeze-thaw solution. 2% (v / v) pig blood freeze-thaw solution was added to the SFBM medium to obtain a fetal bovine serum-free medium SFPM that can quickly promote the proliferation of muscle stem cells.

[0055] Primary muscle stem cells were seeded into 96-well plates at 7000 cells / well and incubated at 37°C and 5% CO 2 Under the conditions of culture, muscle stem cells were allowed to adhere to the wall in DMEM medium containing 10% FBS. After adhesion, DMEM medium containing 5% FBS, SFBM medium, and SFPM medium were replaced respectively. Three parallels were set up for each group. Cell morphology was observed every day, and the amount of viable cells was detected by MTT method after 2 days of culture.

[0056] The cell morphology of muscle stem cells in the three groups is as follows Figure 5 The results of cell proliferation activity detection analysis (MTT absorbance detection) are as follows Figure 6 .Depend on Figure 5 and Figure 6 The results showed that cells were able to achieve rapid proliferation in the culture medium SFPM, and the effect was much better than the 5% FBS group. Therefore, a fetal bovine serum-free culture medium SFPM that can rapidly promote the proliferation of muscle stem cells was obtained.

[0057] Adjusting the concentration of pig blood cryopreservation solution in SFPM culture medium to 1-10% (v / v), and culturing muscle stem cells in the same manner can still ensure their efficient proliferation. Selecting different batches of pig blood cryopreservation solution, and culturing muscle stem cells in the same manner can still ensure their efficient proliferation.

[0058] Example 3 Application of two fetal bovine serum-free muscle stem cell culture media in the production of cell cultured meat

[0059] The present invention provides two fetal bovine serum-free muscle stem cell culture media (SFBM, SFPM), in which muscle stem cells can grow and proliferate. As seed cells for cell cultured meat, they must be guaranteed to have efficient differentiation ability, so the differentiation potential of muscle stem cells in the two culture media was examined.

[0060] Primary muscle stem cells with differentiation potential (5th generation in vitro, P5) were cultured at 10 4 Cells / well were inoculated into 96-well plates, and 5% FBS group (DMEM medium containing 5% FBS), SFBM group and SFPM group were set up respectively, and 3 parallels were set up for each group. Muscle stem cells were allowed to adhere to the plate in DMEM medium containing 10% FBS and incubated at 37°C and 5% CO 2 The cells were cultured under the same conditions, and when the cell density reached 80-90%, the supernatant was removed, and the culture medium was replaced according to the groups. After 5 days of differentiation induction, myosin heavy chain (MyHC) immunofluorescence staining was performed.

[0061] Differentiation medium (by volume percentage): 98% DMEM medium, 2% horse serum.

[0062] Immunofluorescence identification:

[0063] (1) Aspirate the culture medium from the wells to be treated and carefully wash twice with PBS buffer to remove most of the non-adherent cells;

[0064] (2) Fix with 150 μL of 4% paraformaldehyde (precooled at 4°C), permeabilize at room temperature for 15 min, remove the paraformaldehyde, and carefully wash with PBS buffer three times;

[0065] (3) Add 150 μL of 0.5% TritonX-100 in PBS for 15 min, remove the solution, and carefully wash with PBS buffer three times;

[0066] (4) Add 150 μL of blocking solution (1% BSA, PBST containing a final concentration of 22.52 mg / mL glycine; PBST is PBS containing 0.1% Tween20), incubate at room temperature for 30 min; remove the solution and carefully wash with PBS buffer three times;

[0067] (5) Add 100 μL of antibody diluted in 1% bovine serum albumin (BSA) in PBS, wrap with tin foil, incubate at room temperature for 1 h, and then incubate at 4°C overnight;

[0068] (6) After overnight treatment, place at room temperature for 1 hour, then wash with PBS three times, 5 minutes each time. Add 100 μL of fluorescently labeled secondary antibody diluted 1:200 in 1% BSA in PBS solution, wrap with tin foil, and incubate at room temperature for 1-1.5 hours.

[0069] (7) Wash three times with PBS, add 100 μL of 20 μM DAPI, incubate at room temperature in the dark for 10 min, wash three times with PBS, 5 min each time, add 100 μL PBS, and observe and photograph under a fluorescence microscope.

[0070] The cell morphology and MyHC immunofluorescence of the three groups are shown in Figure 7 . The results show that under the three groups of conditions, muscle stem cells can proliferate normally. The cells in the SFPM group can adhere to the wall in 3 to 4 hours, and the cells in the other two groups can adhere to the wall for more than 8 hours. In the SFPM culture medium, the cell morphology and adhesion are significantly improved, and the cells can adhere to the wall faster and show normal cell morphology. In the 5% FBS group, SFBM group and SFPM group, they can spontaneously fuse to form multinucleated myotubes, which does not affect their myogenic differentiation ability.

[0071] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A fetal bovine serum-free culture medium SFPM for promoting rapid proliferation of muscle stem cells, characterized in that: The SFPM culture medium is prepared by adding 1-10% pig blood freeze-thaw solution to the serum-free basal culture medium SFBM; the method for preparing the pig blood freeze-thaw solution is to remove red blood cells from pig blood, freeze-thaw repeatedly for multiple times, and take the supernatant and filter it; The serum-free basal medium SFBM comprises a basal medium and additives; The basic culture medium includes DMEM and F12; The concentrations of the components in the additive in the serum-free medium are as follows: 1-8 mg / mL bovine serum albumin, 0.5-5 μg / mL insulin, 10-200 μg / mL transferrin, 1-20 ng / mL selenite, 0.5-5 μg / mL ethanolamine, 1-5 mM L-glutamine, 10 -7 ~10 -6 M dexamethasone, 1~50 ng / mL platelet-derived growth factor PDGF-BB, 1~50ng / mL basic fibroblast growth factor bFGF, 1~50 ng / mL epidermal growth factor EGF, 10~100 ng / mL insulin-like growth factor LR 3 -IGF1, 2~10 mM sodium bicarbonate, 10~100 μg / mL ascorbic acid, 5~100 μM tyrosine, 0.5~5 mM α-ketoglutaric acid, 1×HPPES solution, 1×MEM amino acid solution.

2. The fetal bovine serum-free culture medium SFPM according to claim 1, characterized in that The conditions for repeated freezing and thawing are freezing at -80°C and thawing at 37°C.

3. A method for culturing muscle stem cells, characterized in that: The method comprises culturing muscle stem cells using the fetal bovine serum-free culture medium SFPM as described in claim 1 or 2.

4. Use of the fetal bovine serum-free culture medium SFPM according to claim 1 or 2 in the production of cell cultured meat.

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