A serum-free culture method for inducing differentiation of muscle satellite cells into myotubes

Abstract

The application discloses a serum-free culture method for inducing differentiation of muscle satellite cells into myotubes. The inventors have found that the muscle satellite cells can be efficiently induced to differentiate into myotubes with a differentiation rate of up to 46.2% by sequentially adding dexamethasone and insulin into a serum-free basic culture medium. The serum-free culture method has the advantages that no serum or other components with unknown compositions need to be added into the culture medium, the chemical composition of the culture medium is clear, the stability of the induced culture is good, the cost is controllable, and the method is safer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology, and particularly relates to a serum-free culture method for inducing differentiation of muscle satellite cells into myotubes. BACKGROUND

[0002] Meat products account for a significant proportion of human diet, and meat not only has unique texture and flavor, but also provides a large amount of nutrients, including minerals and essential amino acids, for humans. At present, people mainly obtain meat by relying on traditional farming, however, with the increasing concern about environmental degradation and personal health, the inherent drawbacks of traditional farming have gradually emerged, such as large greenhouse gas emissions, large consumption of water resources, large occupation of land area, overuse of antibiotics, and spread of various pathogens.

[0003] According to the results of the United Nations global population estimation, the global population will reach nearly 10 billion by 2050. At that time, the demand for meat products cannot be met by relying on traditional animal husbandry. Therefore, meat production needs to be carried out in a more efficient and sustainable way.

[0004] Cell culture meat is obtained by culturing cells in vitro, using synthetic biology, regenerative medicine and tissue engineering and other related technologies to differentiate cells into myotubes to form muscle-like tissue, and then directly producing meat products through product processing. Compared with traditional animal husbandry, cell culture meat can reduce the use of 96% of water resources and 99% of land area, and at the same time, cell culture meat is produced in a relatively controllable environment, which can effectively avoid the pollution of pathogenic microorganisms and the use of antibiotics, and the in vitro production cycle is shorter and the carbon emission content is lower. Developing cell culture meat technology can efficiently and sustainably provide sufficient and healthy meat products for humans.

[0005] Although cell culture meat has many advantages, the industry is still in its early stages of development, and most companies are still in the laboratory research stage, and the industry itself still faces many challenges. In terms of cost, the high cost of culture medium is the biggest challenge for the commercialization of cell culture meat. In terms of technology, the low efficiency of cell differentiation into myotubes makes cell culture meat lack the texture and taste of natural meat products. The muscle cells in pork, beef and chicken meat that people eat daily will fuse to form long strip-shaped myotubes with multiple nuclei, and the myotubes are connected to each other to develop into mature muscle fibers. These muscle fibers make natural meat products have rich tissue structure and texture, and can provide unique taste.

[0006] Mouse myoblast cells (C2C12) are muscle precursor cells, which can be differentiated into muscle cells under specific induction conditions, and then the muscle cells further fuse to form myotubes containing multiple nuclei. Based on this characteristic, C2C12 is often used as a tool cell to explore the mechanism behind muscle cell formation and muscle damage repair in basic research and the biomedicine industry. Myotubes are also crucial in the composition of meat products. To achieve the taste and flavor of natural meat, in vitro cultured cells need to be differentiated into myotubes for cell cultured meat.

[0007] The currently reported invention patents for inducing C2C12 differentiation into myotubes include CN103881966A using 2%-5% horse serum for induction, CN107937337A and CN106754666A using 2% horse serum, 1% double antibody, 8-10 µg / L daidzin, 20-30 mg / L pomegranate extract and 10-15 mg / L gypsophila extract for induction, CN111363718A using 2% horse serum and 10 µM retinol for induction; The published literature (Han et al; 2017; 14(5): 434-443.doi: 10.7150 / ijms.18427) uses 10% fetal bovine serum, 10 µM dexamethasone and 100 nM insulin for induction. However, these methods are not the best solution for application in the field of cell cultured meat. First of all, the use of animal serum does not conform to the concept of animal protection in the cell cultured meat industry. Secondly, the composition of serum is complex, and the stability of the quality of different batches of serum is difficult to control, which will further affect the stability of the process. Finally, the use of serum will make the cost of cell cultured meat production high, increasing the difficulty of commercial development. Therefore, it is of great significance in the cell cultured meat industry to develop serum-free differentiation medium to induce the formation of myotubes. SUMMARY

[0008] The purpose of the present application is to overcome at least one of the deficiencies of the prior art and provide two serum-free culture methods for inducing muscle satellite cells to differentiate into myotubes.

[0009] The technical solution adopted by the present application is:

[0010] The first aspect of the present application provides:

[0011] A serum-free culture method for inducing muscle satellite cells to differentiate into myotubes, comprising the following steps:

[0012] Healthy muscle satellite cells are added to the No. 1 differentiation medium and cultured for 12 h-72 h;

[0013] The No. 1 differentiation medium is removed, the No. 2 differentiation medium is added, and further cultured to differentiate into myotubes; wherein:

[0014] The first differentiation medium is a serum-free basal medium to which is added dexamethasone at a final concentration of 1-50 μM;

[0015] The second differentiation medium is a serum-free basal medium to which is added insulin at a final concentration of 50-250 nM.

[0016] Another serum-free culture method for inducing differentiation of muscle satellite cells into myotubes comprises directly culturing healthy muscle satellite cells in a third differentiation medium to differentiate the muscle satellite cells into myotubes, wherein the third differentiation medium is a serum-free basal medium to which is added insulin at a final concentration of 50-250 nM and transferrin at a final concentration of 1-20 μg / mL.

[0017] In some examples of the serum-free culture method, the first differentiation medium is a serum-free basal medium to which is added dexamethasone at a final concentration of 8-12 μM; and / or the second differentiation medium is a serum-free basal medium to which is added insulin at a final concentration of 80-130 nM; and / or the third differentiation medium is a serum-free basal medium to which is added insulin at a final concentration of 50-150 nM and transferrin at a final concentration of 1-20 μg / mL.

[0018] In some examples of the serum-free culture method, the serum-free basal medium is selected from one of DMEM, RPMI 1640, MEM, DMEM / F12, F10, CD293, Medium 231, Medium 106, and a basal medium modified based thereon.

[0019] In some examples of the serum-free culture method, the culturing time of the muscle satellite cells in the first differentiation medium is 24-72 h.

[0020] In some examples of the serum-free culture method, the culturing time after the addition of the second differentiation medium is 5-7 d.

[0021] In some examples of the serum-free culture method, the culturing time of the muscle satellite cells in the third differentiation medium is 5-10 d.

[0022] In some examples of the serum-free culture method, the muscle satellite cells are selected from one of mouse myoblast cells, bovine muscle satellite cells, and porcine muscle satellite cells.

[0023] In some examples of the serum-free culture method, the muscle satellite cells are inoculated at a density of 0.1-5*10 5

[0024] In some examples of the serum-free culture method, the culturing condition is 37°C, 5% CO2.​

[0025] A second aspect of the present invention provides:

[0026] A serum-free culture medium combination that can induce myosatellite cell differentiation into myotubes includes differentiation medium 1 and differentiation medium 2, wherein:

[0027] The No. 1 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 1-50 μM dexamethasone;

[0028] The No. 2 differentiation medium is a serum-free basal medium supplemented only with insulin at a final concentration of 50-250 nM.

[0029] In some examples of serum-free culture medium combinations, the No. 1 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 8-12 μM dexamethasone; the No. 2 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 80-130 nM insulin.

[0030] In some examples of serum-free culture medium combinations, the serum-free basal medium is selected from one of DMEM, RPMI1640, MEM, DMEM / F12, F10, CD293, Medium 231, Medium 106, and basal media modified based thereon, preferably DMEM medium.

[0031] A third aspect of the present invention provides:

[0032] Serum-free culture medium that can induce myosatellite cells to differentiate into myotubes is a serum-free basal medium supplemented only with a final concentration of 50-250 nM insulin and 1-20 μg / mL transferrin.

[0033] In some examples of serum-free culture media, the No. 3 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 50-150 nM insulin and 1-20 μg / mL transferrin.

[0034] In some examples of serum-free culture media, the No. 1 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 8-12 μM dexamethasone; the No. 2 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 80-130 nM insulin, preferably DMEM medium.

[0035] The beneficial effects of this invention are:

[0036] The culture methods of some examples of the present invention have simple culture medium components and can achieve differentiation rates as high as 31.9% and 46.2% without the use of serum, effectively inducing myosatellite cells to differentiate into myotubes.

[0037] The culture methods of some examples of the present invention do not require the addition of serum or other components of unknown composition to the culture medium, which has a clear chemical composition, good stability of induction culture, more controllable cost, and is also safer.

[0038] The differentiation culture medium components in some examples of this invention are clearly defined, making it easier to control the technology and ensuring the stability of the process. Attached Figure Description

[0039] Figure 1 These are staining images of myotube characteristic protein MHC 7 days after C2C12 cell differentiation was induced using serum starvation (Fig. A) and 2% horse serum (Fig. B).

[0040] Figure 2 These are MHC staining images of C2C12 cells 7 days after induction of differentiation in Examples 1 and 2.

[0041] Figure 3 This is an MHC staining photograph taken 4 days after bovine muscle satellite cell differentiation was induced in Example 3.

[0042] Figure 4 This is an MHC staining photograph of C2C12 cells 7 days after induction of differentiation in Example 4.

[0043] Figure 5 It is the result of manual cell counting.

[0044] Figure 6 It is the result of automatic cell counting. Detailed Implementation

[0045] The abbreviations used in this invention have the following meanings, unless otherwise specified, they all refer to the meanings commonly used in the art.

[0046] The meanings of the abbreviations are as follows:

[0047] PBS: Phosphate Buffer

[0048] PFA: Paraformaldehyde

[0049] FBS: Fetal bovine serum

[0050] BSA: Bovine serum albumin

[0051] DAPI: 4',6-Diamidinyl-2-phenylindole

[0052] MHC: Myosin Heavy Chain

[0053] A serum-free culture method for inducing myosatellite cell differentiation into myotubes includes the following steps:

[0054] Healthy muscle satellite cells were transferred into differentiation medium 1 and cultured for 12 h-72 h.

[0055] Remove differentiation medium No. 1, add differentiation medium No. 2, and culture further until differentiation into myotubes; wherein:

[0056] The No. 1 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 1-50 μM dexamethasone;

[0057] The No. 2 differentiation medium is a serum-free basal medium supplemented only with insulin at a final concentration of 50-250 nM.

[0058] Another serum-free culture method for inducing myotube differentiation of muscle satellite cells includes directly culturing healthy muscle satellite cells in differentiation medium No. 3 until the muscle satellite cells differentiate into myotubes. The differentiation medium No. 3 is a serum-free basal medium supplemented only with a final concentration of 50-250 nM insulin and 1-20 μg / mL transferrin.

[0059] In some examples of serum-free culture methods, differentiation medium No. 1 is a serum-free basal medium supplemented only with a final concentration of 8-12 μM dexamethasone; differentiation medium No. 2 is a serum-free basal medium supplemented only with a final concentration of 80-130 nM insulin. Studies have shown that this final concentration results in higher induction efficiency.

[0060] In some examples of serum-free culture methods, the No. 3 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 50-150 nM insulin and 1-10 μg / mL transferrin. Studies have shown that this final concentration results in higher induction efficiency.

[0061] In this invention, serum-free basal medium refers to a synthetic culture medium with a well-defined chemical composition that does not contain any added natural components (such as serum) of unknown origin. These media are well-known to those skilled in the art; they can be purchased as commercial products or prepared according to their composition. In some examples of serum-free culture methods, the serum-free basal medium is selected from one of DMEM, RPMI 1640, MEM, DMEM / F12, F10, CD293, Medium 231, Medium 106, and basal media modified from these. Experimental data show that DMEM medium can better promote cell differentiation and is a better choice.

[0062] In some serum-free culture methods, the muscle satellite cells are cultured in differentiation medium 1 for 24-72 hours. Cultured in differentiation medium 1 for a period of time, the muscle satellite cells can be initially induced, which facilitates further differentiation induction.

[0063] The differentiation induction culture time of cells in differentiation medium No. 2 can be adjusted according to the differentiation status of the cells. In some serum-free culture methods, the culture time after adding differentiation medium No. 2 is 5-7 days.

[0064] The differentiation induction culture time of cells in differentiation medium No. 3 can be adjusted according to the differentiation status of the cells. In some serum-free culture methods, the culture time of the muscle satellite cells in differentiation medium No. 3 is 5-10 days.

[0065] Muscle satellite cells can be common mammalian muscle satellite cells. In some serum-free culture methods, the muscle satellite cells are selected from mouse myoblasts, bovine muscle satellites, or porcine muscle satellite cells. These cells are more common and are also common cell cultures for meat.

[0066] The seeding density of muscle satellite cells can be adjusted according to the cell growth status. In some serum-free culture methods, the seeding density of muscle satellite cells is 0.1-5 x 10^6 cells / year. 5 per mL.

[0067] In some examples of serum-free culture methods, the culture conditions are 37°C and 5% CO2.

[0068] A second aspect of the present invention provides:

[0069] A serum-free culture medium combination that can induce myosatellite cell differentiation into myotubes includes differentiation medium 1 and differentiation medium 2, wherein:

[0070] The No. 1 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 1-50 μM dexamethasone;

[0071] The No. 2 differentiation medium is a serum-free basal medium supplemented only with insulin at a final concentration of 50-250 nM.

[0072] In some examples of serum-free culture medium combinations, the No. 1 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 8-12 μM dexamethasone; the No. 2 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 80-130 nM insulin.

[0073] In some examples of serum-free culture medium combinations, the serum-free basal medium is selected from one of DMEM, RPMI1640, MEM, DMEM / F12, F10, CD293, Medium 231, Medium 106, and basal media modified based thereon.

[0074] A third aspect of the present invention provides:

[0075] Serum-free culture medium that can induce myosatellite cells to differentiate into myotubes is a serum-free basal medium supplemented only with a final concentration of 50-250 nM insulin and 1-20 μg / mL transferrin.

[0076] In some examples of serum-free culture media, the No. 3 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 50-150 nM insulin and 1-10 μg / mL transferrin.

[0077] In some examples of serum-free culture media, the serum-free basal medium is selected from one of DMEM, RPMI 1640, MEM, DMEM / F12, F10, CD293, Medium 231, Medium 106, and basal media modified based thereon.

[0078] The technical solution of the present invention will be further explained below with reference to experiments.

[0079] Reagent preparation

[0080] 1) Preparation of transferrin: Add the specified amount of transferrin to ddH2O.

[0081] 2) Preparation of dexamethasone: Add the specified amount of dexamethasone to anhydrous ethanol.

[0082] 3) Insulin preparation: Add the specified amount of insulin to Tris-HCl (pH=6.0).

[0083] 4) Differentiation medium No. 1, DMEM serum-free basal medium supplemented with 1-50 μM dexamethasone.

[0084] 5) Differentiation medium No. 2, DMEM serum-free basal medium supplemented with 50-250 nM insulin.

[0085] 6) Differentiation medium No. 3, DMEM serum-free basal medium supplemented with 50-250 nM insulin and 1-20 μg / mL transferrin.

[0086] 7) Preparation of 5% Triton X-100: Dissolve 500 µL of Triton X-100 in 10 mL of PBS.

[0087] 8) Preparation of 5% BSA: Dissolve 500 µg BSA in 10 mL PBS.

[0088] Cell sample preparation

[0089] 1) Before the experiment, remove the complete culture medium and trypsin from the refrigerator and preheat them to 37°C;

[0090] 2) Wipe the outside of the culture medium bottles and PBS bottles with alcohol swabs and place them in the operating table;

[0091] 3) Remove the culture dish from the incubator and observe the cell growth under an inverted microscope. When the cells are in the logarithmic growth phase, passage them.

[0092] 4) Place the petri dish on the operating table and use a suction pump to remove the original culture medium;

[0093] 5) Slowly add 5 mL of PBS along the sidewall of the culture dish to wash twice;

[0094] 6) Add 2 mL of trypsin and spread it evenly on the bottom of the culture dish, then aspirate and incubate in an incubator (37℃) for 2 min;

[0095] 7) Add 2 mL of culture medium to stop digestion, gently pipette the cells from the bottom, and collect the cells into a 15 mL centrifuge tube;

[0096] 8) Place the centrifuge tubes in the centrifuge and centrifuge at 1000 rpm for 3 minutes;

[0097] 9) Resuspend the cells in 1 mL of culture medium, count the cells, and sort them at a ratio of 1*102. 5 Seed cells per well in a 24-well plate and add culture medium to a final volume of 1 mL / well. Incubate at 37°C with 5% CO2 for 2 days. When the cells reach over 90% confluence, differentiation induction can be performed.

[0098] Cell differentiation induction protocol 1

[0099] 1) Before the experiment, remove the differentiation medium and preheat it to 37°C;

[0100] 2) Wipe the outside of the culture medium bottles and PBS bottles with alcohol swabs and place them in the operating table;

[0101] 3) Remove the 24-well plate from the incubator and observe the cell growth under an inverted microscope to confirm that the cells are healthy and the cell density is above 90% before proceeding with differentiation.

[0102] 4) Place the 24-well plate in the operating table and use a suction pump to remove the original culture medium;

[0103] 5) Slowly add 1 mL of PBS along the sidewall of the plate to wash twice;

[0104] 6) Slowly add 1 mL of differentiation medium No. 1 along the side wall, and use DMEM + 10% FBS complete medium as a control experiment. Incubate at 37℃ in a 5% CO2 incubator.

[0105] 7) Remove the well plate from the incubator, place it on the operating table, and use a suction pump to remove the original culture medium;

[0106] 8) Slowly add 1 mL of PBS along the sidewall of the plate to wash twice;

[0107] 9) Slowly add 1 mL of differentiation medium No. 2 along the side wall, and use DMEM + 10% FBS complete medium as a control experiment. Incubate at 37℃ in a 5% CO2 incubator.

[0108] 10) When the cell morphology is observed to have changed from a single cell fusion into a myotube with multiple cell structures, subsequent immunofluorescence staining can be performed.

[0109] Table 1 shows the amount of inducer and culture time for different examples (Examples 1-3) of cell differentiation induction scheme.

[0110] Table 1

[0111]

[0112] Cell differentiation induction protocol two (Example 4)

[0113] 1) Before the experiment, remove the differentiation medium and preheat it to 37°C;

[0114] 2) Wipe the outside of the culture medium bottles and PBS bottles with alcohol swabs and place them in the operating table;

[0115] 3) Remove the 24-well plate from the incubator and observe the cell growth under an inverted microscope to confirm that the cells are healthy and the cell density is above 90% before proceeding with differentiation.

[0116] 4) Place the 24-well plate in the operating table and use a suction pump to remove the original culture medium;

[0117] 5) Slowly add 1 mL of PBS along the sidewall of the plate to wash twice;

[0118] 6) Slowly add 1 mL of differentiation medium No. 3 (insulin 100 nM + 5 μg / mL transferrin, DMEM) along the side wall, and use DMEM + 10% FBS complete medium as a control experiment. Incubate at 37℃ and 5% CO2 for 7 days.

[0119] Immunofluorescence staining experiment

[0120] 1) Place the well plate on the lab bench and use a suction pump to remove the original culture medium;

[0121] 2) Slowly add 1 mL of PBS along the sidewall of the plate to wash twice;

[0122] 3) Slowly add 200 µL of 4% PFA preheated at 37℃ along the side wall and fix at room temperature for 20 min, then aspirate and discard; add 1 mL of PBS to wash twice, each time on a shaker at 50 rpm for 5 min.

[0123] 4) Add 200 µL of 0.5% Triton X-100 to each well and permeate at room temperature for 10 min, then aspirate and discard; wash twice with 1 mL of PBS.

[0124] 5) Add 200 µL of 0.5% BSA to each well and block at room temperature for 30 min, then aspirate and discard; wash twice with 1 mL of PBS.

[0125] 6) Add 200 µL of primary antibody (dissolved 1:200 in blocking buffer) to each well and incubate overnight at 4°C (or 1.5 h at 37°C), then aspirate and discard; wash twice with 1 mL of PBS.

[0126] 7) Add 200 µL of secondary antibody (1:200 dissolved in blocking buffer) + DAPI (1:1000 dissolved in blocking buffer) to each well, and incubate at room temperature in the dark with shaking for 2 h; wash twice with 1 mL of PBS;

[0127] 8) Add 200 µL PBS to each well and observe the staining of cells and nuclei under fluorescence, and take pictures to record the results;

[0128] 9) Use ImageJ software to count the number of cell nuclei in the myotube and the total number of cell nuclei in the sample, and calculate the fusion index. The fusion index is the percentage of cell nuclei in the myotube relative to the total number of cell nuclei in the sample, and thus the differentiation efficiency is obtained.

[0129] result:

[0130] Figure 1 These are MHC staining images of C2C12 cells 7 days after differentiation was induced by serum starvation (Figure A) and 2% horse serum (Figure B). As can be seen from the images, the efficiency of C2C12 cells differentiating into myotubes is not high regardless of whether serum starvation or 2% horse serum is used. The number of myotubes is small and the length of myotubes is short.

[0131] Figure 2 These are MHC staining images of myotube characteristic proteins 7 days after C2C12 cell differentiation induced in Examples 1 and 2. Figure 2 A is the control experiment (DMEM + 10% FBS). Figure 2 B represents the result of Example 1. Figure 2 C represents the result of Example 2.

[0132] Figure 3This is an MHC staining photograph taken 4 days after bovine muscle satellite cell differentiation was induced in Example 3.

[0133] Figure 4 This is an MHC staining photograph of C2C12 cells 7 days after induction of differentiation in Example 4.

[0134] from Figures 2-4 As can be seen from Examples 1, 2, and 4, the myotubes induced by C2C12 contain more cell nuclei and have a larger diameter. Figure 3 The results show that the method is also applicable to the induction of myotube differentiation of bovine muscle satellite cells, which are difficult to culture and differentiate.

[0135] Figure 5 It is the result of manual cell counting.

[0136] Figure 6 This is the result of an automatic cell count.

[0137] Comparing manual and automatic cell counting using ImageJ software, Figure 5 This is the result of an artificial cell count; 43 cell nuclei were counted. Figure 6 The results of automatic counting show that 44 cell nuclei were counted, which demonstrates that automatic counting not only reduces manual labor and time, but also shows no significant difference from manual counting.

[0138] Statistical results of C2C12 differentiation efficiency showed that serum starvation resulted in a differentiation efficiency of 13.2%, 2% horse serum treatment resulted in a differentiation efficiency of 8.9%, Example 1 had a differentiation efficiency of 46.2%, Example 2 had a differentiation efficiency of 38.8%, and Example 4 had a differentiation efficiency of 31.9%. Meanwhile, the differentiation efficiency of myotubes induced by this invention in Example 3 was 24.6%. Statistical results indicate that the efficiency of myotube differentiation induced by this invention is significantly higher than that of traditional methods. The differentiation efficiency statistics for different examples are shown in Table 2.

[0139] Table 2

[0140]

[0141] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A serum-free culture method for inducing cell differentiation into myotubes, comprising the following steps: Add healthy cells to differentiation medium No. 1 and culture for 24-72 h; Remove differentiation medium No. 1, add differentiation medium No. 2, and culture for 5-7 days to further culture until differentiation into myotubes; wherein: The No. 1 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 8-12 μM dexamethasone; The No. 2 differentiation medium is a serum-free basal medium supplemented only with insulin at a final concentration of 80-130 nM; The serum-free basal medium is DMEM or a modified basal medium based on it; The cells are selected from mouse myoblasts, bovine muscle satellite cells, and porcine muscle satellite cells; the cell seeding density is 0.1-5*102. 5 per mL.

2. The serum-free culture method according to claim 1, characterized in that: The culture conditions were 37℃ and 5% CO2.

3. A serum-free culture medium combination capable of inducing cell differentiation into myotubes, consisting of differentiation medium No. 1 and differentiation medium No. 2, wherein: The No. 1 differentiation medium is a serum-free basal medium supplemented only with a final concentration of 8-12 μM dexamethasone; The No. 2 differentiation medium is a serum-free basal medium supplemented only with insulin at a final concentration of 80-130 nM; The serum-free basal medium is DMEM or a modified basal medium based on it.

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