CN117625518A - Method for preparing cell culture snowflake meat - Google Patents

Abstract

The invention discloses a method for manufacturing cell-cultured snowflake meat and belongs to the technical fields of stem cell culture and cell-cultured meat. The invention discloses a culture medium that realizes simultaneous myogenesis and adipogenesis of muscle stem cells, which can respectively realize rapid myogenesis and adipogenesis differentiation of muscle stem cells and long-term maintenance of myogenesis and adipogenesis differentiation state of muscle stem cells. The invention discloses a method for making cell-cultured meat that utilizes muscle stem cells to simultaneously produce muscle fibers and fat, and also discloses a method of controlling the proportion of fat and muscle fibers in cell-cultured meat by regulating the proportion of muscle stem cells with different myogenic differentiation abilities. . The present invention realizes the myogenic and adipogenic differentiation of muscle stem cells in two-dimensional and three-dimensional culture conditions under the same induction condition. It does not need to cultivate multiple types of cells and can achieve the simultaneous acquisition of muscle fibers and fat in cultured meat. Reducing the workload of cell culture provides new ideas and methods for the industrialization and customized production of cell cultured meat.

Description

A method for manufacturing cell-cultured snowflake meat

Technical field

The present invention relates to a method for producing cell-cultured snowflake meat. Specifically, it relates to a method for producing cell-cultured snowflake meat that can utilize muscle stem cells to simultaneously produce muscle fiber and fat. It belongs to the technical fields of stem cell culture and cell-cultured meat.

Background technique

Cell cultured meat (Cultured meat), as an important part of future food, is based on the growth and development and damage repair mechanism of muscle tissue, using animal cells cultured in vitro to obtain muscle fibers, fats and other cells that make up muscle tissue, and then collect and A new type of meat food that is shaped and processed into food. Compared with traditional meat production, cell cultured meat has good resource conversion rate and sustainable development potential. Its production process does not involve the feeding and slaughtering of livestock, which greatly shortens the meat production cycle and can provide real animals. Protein will definitely become an important part of the future meat product market.

Commercially available meat contains tissue such as muscle fiber and fat, which provides rich protein and fatty acids, while also ensuring the high-quality taste of meat products. In the process of making cell-cultured meat, muscle stem cells (Muscle StemCell, also known as muscle satellite cells, Satellite Cell) are usually used as seed cells. They are expanded and differentiated in vitro to produce myoblasts, which are further differentiated and fused to produce a large number of muscle fibers to form muscle tissue. . However, currently, the use of muscle stem cells as seed cells is mainly used for the production of cell-cultured muscle fibers. If you want to produce cell-cultured adipose tissue, you need to use other seed cells, such as mesenchymal stem cells (Mesenchymal stem cells), resulting in a complicated production process. Cost increase. If only muscle stem cells are used as seed cells, their adipogenic differentiation ability can be explored, and the adipogenic differentiation strategy of muscle stem cells can be developed, which will provide a new method for producing cell-cultured snowflake meat containing both muscle fibers and fat.

Contents of the invention

The invention provides a method for making cell-cultured meat that utilizes muscle stem cells to simultaneously produce muscle fibers and fat. At the same time, the ratio of muscle fibers and fat in the cell-cultured meat can be regulated by adjusting the ratio of muscle stem cells with different myogenic differentiation capabilities.

The first object of the present invention is to provide a culture medium that rapidly induces muscle stem cells to differentiate into myocytes and adipocytes simultaneously. The cells can form obvious myotubes and lipid droplets after 4 days of induction. The composition of the culture medium is ( a) or (b):

(a) Basic culture medium, fetal calf serum, insulin, rosiglitazone, and oleic acid;

(b) Basic medium, horse serum, rosiglitazone, oleic acid;

In one embodiment, the basal culture includes DMEM or F12.

In one embodiment, in (a), the concentration of fetal bovine serum is 5% (v/v), the concentration of insulin is 1-50 mg/L, the concentration of rosiglitazone is 1-50 mg/L, and the oil concentration is 5% (v/v). The acid concentration used is 10 to 300 μM.

In one embodiment, the concentration of insulin is 10 mg/L, the concentration of rosiglitazone is 10 mg/L, and the concentration of oleic acid is 100 μM.

In one embodiment, in (b), the horse serum is used at a concentration of 2% (v/v), the rosiglitazone is used at a concentration of 1 to 50 μM, and the oleic acid is used at a concentration of 10 to 300 μM.

In one embodiment, rosiglitazone is used at a concentration of 10 μM, and oleic acid is used at a concentration of 100 μM.

In one embodiment, the muscle stem cells include, but are not limited to, mammalian or oviparous muscle stem cells.

In one embodiment, the muscle stem cells comprise porcine, chicken, duck, bovine or ovine muscle stem cells.

When the muscle stem cells grow to 80-90% density, replace the ROI medium or RO medium, culture at 37°C and 5% _CO2 , replace the medium every 24 hours, induce differentiation for about 4 days, and form obvious muscle cells at the same time. tubes and lipid droplets.

The second object of the present invention is to provide a culture medium for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells. The culture medium consists of two parts: culture medium A + culture medium B; the composition of culture medium A is: basic culture medium , fetal bovine serum, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), indomethacin (IDM), insulin, rosiglitazone; medium B: basal medium, fetal bovine Serum, insulin.

In one embodiment, in culture medium A, the concentration of fetal bovine serum is 10% (v/v), the concentration of dexamethasone is 0.1-5 μM, the concentration of IBMX is 1-100 μM, and the concentration of IDM is 0.1-5 μM. 5 μM, insulin is used at a concentration of 10 to 300 nM, and rosiglitazone is used at a concentration of 1 to 30 μM.

In one embodiment, dexamethasone is used at a concentration of 1 μM, IBMX is used at a concentration of 1 μM, IDM is used at a concentration of 1 μM, insulin is used at a concentration of 100 nM, and rosiglitazone is used at a concentration of 10 μM.

In one embodiment, in culture medium B, the concentration of fetal bovine serum is 10% (v/v), and the concentration of insulin is 10-300 nM.

The third object of the present invention is to provide a culture method for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells. The method is to use the above-mentioned culture medium for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells to perform differentiation and culture of muscle stem cells. .

In one embodiment, the method is to culture muscle stem cells in a common medium until the cell density is 80-90%, replace medium A every 24 hours, induce differentiation for 3 days, and then replace the culture medium. Base B, culture for 2 days, culture cycle 3 times.

In one embodiment, the common culture medium is basal culture medium and 10% (v/v) fetal calf serum.

In one embodiment, the basal culture includes DMEM or F12.

In one embodiment, the culture conditions of the method are at 37°C, 5% _CO2 .

The fourth object of the present invention is to provide a method for regulating the proportion of fat and muscle fiber in cell cultured meat. Muscle stem cells of different generations are mixed according to a certain ratio, and the muscle stem cells are rapidly induced to differentiate into myogenesis and adipogenesis simultaneously in the above culture medium. The myogenic and adipogenic differentiation culture is performed in the medium or the above-mentioned medium that maintains long-term myogenic and adipogenic differentiation of muscle stem cells.

The present invention also provides the application of the above-mentioned medium for rapidly inducing simultaneous myogenic and adipogenic differentiation of muscle stem cells or the above-mentioned medium for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells in the field of cell cultured meat.

Beneficial effects:

Intramuscular preadipocytes and muscle stem cells (also called muscle satellite cells) are two different types of cells. Intramuscular preadipocytes mainly differentiate into adipose tissue, while muscle stem cells are pluripotent and have the ability to differentiate into muscle fibers and fat. , bone and other potential; making cell-cultured meat requires muscle fiber, fat and other tissue components. Usually, two or more types of cells need to be cultured separately and induced or co-cultured separately to form a cell that contains both muscle fiber and fat. However, because muscle stem cells have the ability to differentiate in multiple directions Potential, so the present invention optimizes and applies it to muscle stem cells based on the culture medium for inducing adipocyte adipogenesis, so that it can form muscle and adipocytes at the same time, achieving the effect of using only one type of cells to produce muscle fibers and fat at the same time.

The present invention identifies three culture media that can achieve simultaneous myogenesis and adipogenesis of muscle stem cells, which can respectively achieve rapid myogenesis and adipogenesis differentiation of muscle stem cells and long-term maintenance of the myogenesis and adipogenesis differentiation state of muscle stem cells. At the same time, it was found that muscle stem cells have different myogenic and adipogenic differentiation abilities at different times cultured in vitro. Cells cultured for a shorter time in vitro have stronger myogenic differentiation ability, and cells cultured for a longer time can obtain more fat. Therefore, they can be regulated through regulation. The proportion of muscle stem cells with different myogenic differentiation abilities in cultured meat is used to control the proportion of fat and muscle fibers in cell cultured meat. Myogenic and adipogenic differentiation of muscle stem cells can be achieved simultaneously under the same induction conditions. Without culturing multiple types of cells, muscle fibers and fat in cultured meat can be obtained at the same time, which greatly reduces the workload of culturing cells and provides better conditions for cell culture. The industrialized and customized production of meat provides a new idea and method.

Description of drawings

Figure 1 shows the cell morphology of P4 generation cells in myogenic differentiation group, ROI group and RO group at different culture times (4× visual field);

Figure 2 shows the Oil Red O staining of P4 generation cells in myogenic differentiation group, ROI group and RO group after 4 days of culture;

Figure 3 shows the proportion of cells producing lipid droplets and muscle fibers in the P4 generation cell myogenic differentiation group, ROI group and RO group after culture for 4 days;

Figure 4 shows the MyHC immunofluorescence staining of P4 generation cells in myogenic differentiation group, ROI group and RO group after 4 days of culture;

Figure 5 shows the cell morphology of the P4 generation cells in the myogenic differentiation group and myoadipogenic differentiation group at different culture times (4×field of view);

Figure 6 shows the Oil Red O staining on days 4 and 10 of the myogenic differentiation group and myoadipogenic differentiation group of P4 generation cells;

Figure 7 shows the MyHC immunofluorescence staining on days 4 and 10 in the myogenic differentiation group and myoadipogenic differentiation group of P4 generation cells;

Figure 8 shows the Oil Red O staining of cells of different generations under different culture conditions for 4 days (20× visual field);

Figure 9 shows the MyHC immunofluorescence staining of cells of different passages cultured under different culture conditions for 4 days (10× field of view);

Figure 10 shows the proportion of cells producing lipid droplets after cells of different passages were cultured under different culture conditions for 4 days;

Figure 11 shows the proportion of cells that formed muscle fibers after cells of different passages were cultured under different culture conditions for 4 days;

Figure 12 shows the proportion of cells forming fat and muscle fibers in different passages of cells under different culture conditions.

Detailed ways

Culture media involved in the following examples:

Basal medium (by volume percentage): 90% DMEM medium, 10% fetal bovine serum (FBS).

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

ROI medium (by volume percentage): 95% DMEM medium, 5% fetal calf serum, 10 mg/L insulin, 10 mg/L rosiglitazone, 100 μM oleic acid.

RO medium (by volume percentage): 98% DMEM medium, 2% horse serum, 10 μM rosiglitazone, 100 μM oleic acid.

LMF medium (composed of A and B): Medium A (by volume percentage): 90% DMEM, 10% fetal calf serum, 1 μM dexamethasone, 1 μM 3-isobutyl-1-methylxanthine (IBMX ), 1 μM indomethacin (IDM), 100 nM insulin, 10 μM rosiglitazone; medium B (by volume percentage): 90% DMEM, 10% fetal bovine serum, 100 nM insulin.

Experimental methods involved in the following examples:

1. Oil Red O staining: Oil Red O is a strong lipid solvent and lipid dye. It is easier to combine with triglycerides to form small lipid droplets because its solubility in intracellular lipids is higher than in the original solvent. Large for fat staining. The specific operations are as follows:

(1) Before staining, aspirate the culture medium in the culture dish and wash it 2-3 times with PBS;

(2) Use 4% paraformaldehyde to fix for 10 minutes at room temperature in the dark;

(3) Remove the waste liquid, add new 4% paraformaldehyde and fix in the dark for 1 hour, then wash 2-3 times with PBS;

(4) Mix the stock solution and diluent in a ratio of 5:2, and filter to remove impurities. Add an appropriate amount of dye solution to cover the cell layer, stain at 37°C for 10-15 minutes, and wash with PBS for 5-20 seconds;

(5) Stain with counterstain solution for 3-5 minutes and wash with PBS for 30-60 seconds;

(6) Observe cells under a microscope. Fat is red and cell nuclei are dark blue.

Oil Red O stain kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

2. Immunofluorescence identification:

(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;

(2) Fix with 150 μL 4% paraformaldehyde (pre-cooled at 4°C), permeate for 15 minutes at room temperature, remove the paraformaldehyde, and wash carefully three times with PBS buffer;

(3) Add 150 μL of PBS containing 0.5% TritonX-100 and treat for 15 minutes, then remove the solution and wash carefully 3 times with PBS buffer;

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

(5) Add 100 μL of MyHC antibody diluted in PBS containing 1% bovine serum albumin (BSA), cover with tin foil, incubate at room temperature for 1 hour and then overnight at 4°C;

(6) After overnight treatment, let it come to room temperature for 1 hour, then wash with PBS 3 times, 5 minutes each time. Add 100 μL of fluorescently labeled secondary antibody diluted 1:200 in PBS solution containing 1% BSA, wrap it with tin foil, and incubate at room temperature for 1-1.5 h.

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

Example 1 Rapid induction of simultaneous myogenic and adipogenic differentiation of muscle stem cells

Primary muscle stem cells with differentiation potential (4th passage cultured in vitro, P4) were seeded into 96-well plates at 10 ⁴ cells/well, and myogenic differentiation groups, ROI groups and RO groups were set up respectively. Each group was set to 3 A parallel. First, culture the muscle stem cells in the basal medium at 37°C and 5% _CO2 to adhere to the wall. When the cell density reaches 80-90%, the differentiation medium, ROI medium and RO medium are replaced respectively.

The culture medium was changed every 24 hours, and observations and photos were taken under a microscope every day. When the differentiation culture reached the 4th day, Oil Red O staining and myosin heavy chain (MyHC) immunofluorescence staining were performed to examine the lipid droplets and lipid droplets of each group. The generation of MyHC.

The cell morphology of the myogenic differentiation group, ROI group and RO group at different culture times is shown in Figure 1, the Oil Red O staining on the 4th day of differentiation and culture is shown in Figure 2, and the MyHC immunofluorescence staining is shown in Figure 3. It can be seen that the growth conditions of the three groups of cells were similar on the first day of differentiation and culture (Figure 1). As the induction time increased, the muscle stem cells in the three groups of culture conditions were able to significantly differentiate to form multinucleated myotubes. In the ROI group and RO group, There are also obvious aggregated cells (Figure 1). The Oil Red O results and the proportion of cells producing lipid droplets on the 4th day showed that there were only a small amount of red oil droplets near the myotubes in the cells of the myogenic differentiation group. Significant lipid droplets appeared in the ROI group and RO group, existing side by side with the myotubes, and in the ROI group Lipid droplets were more obvious, and the proportion of cells producing lipid droplets in the ROI group was about 30% higher than that in the myogenic differentiation group (Figure 2, Figure 3). The results of MyHC immunofluorescence and the proportion of myofiber-producing cells show that under the three groups of culture conditions, muscle stem cells can form multinucleated myotubes and express MyHC protein. Therefore, it is determined that muscle stem cells can quickly and simultaneously produce muscle fibers and fat in the ROI group and RO group. It can be formed in about 4 days of induction (Figure 3, Figure 4).

Adjust the concentration of insulin in the ROI medium to 1 to 50 mg/L, the concentration of rosiglitazone to 1 to 50 mg/L, and the concentration of oleic acid to 10 to 300 μM; adjust the concentration of rosiglitazone in the RO medium to 1 to 50 μM, adjust the oleic acid concentration to 10 to 300 μM, and induce myogenic and adipogenic differentiation of muscle stem cells according to the same method, which can achieve the simultaneous formation of muscle fibers and fat.

Example 2 Long-term stable myogenic and adipogenic differentiation of muscle stem cells

Primary muscle stem cells with differentiation potential (the 4th passage cultured in vitro, P4) were seeded into 96-well plates at 10 ⁴ cells/well, and myogenic differentiation groups and myoadipogenic differentiation groups were set up respectively. Each group was set 3 parallels were performed. First, culture the muscle stem cells in the basic medium at 37°C and 5% _CO2 to allow the muscle stem cells to adhere to the wall. When the cell density reaches 80-90%, the differentiation medium and LMF medium (composed of A and B composition).

When the muscle stem cells in the myogenic and adipogenic differentiation group grow to a density of 80-90%, medium A is replaced and cultured at 37°C and 5% _CO2 . After inducing differentiation for 3 days, medium B is replaced and cultured at 37°C and 5% CO2. Culturing for ₂ days under CO2 conditions, and culturing in this cycle for a total of 3 times, can maintain the morphology of myotubes and lipid droplets for a long time, and the number of lipid droplets gradually increases as the culture time increases.

The culture medium of the two groups of cells was changed every 24 hours, and they were observed and photographed under a microscope every day. On the 4th and 10th days of culture, Oil Red O staining and myosin heavy chain (MyHC) immunofluorescence staining were performed, respectively. The formation of lipid droplets and MyHC in each group was examined.

The cell morphology of the myogenic differentiation group and the myoadipogenic differentiation group at different culture times is shown in Figure 5, the Oil Red O staining on the 4th and 10th days is shown in Figure 6, and the MyHC immunofluorescence staining is shown in Figure 7 Show. It can be seen that the growth conditions of the two groups of cells were similar on the first day of culture. By the third day of culture, muscle stem cells could form obvious multinucleated myotubes under both culture conditions. However, after the sixth day, the cells in the myogenic differentiation group showed obvious As for the cell shedding, the cell shedding became more serious as the culture time increased. However, in the myogenic and adipogenic differentiation group, the cells did not fall off significantly until the 10th day of culture, and they could survive and adhere well (Figure 5). The Oil Red O results on days 4 and 10 showed that cells in the myogenic differentiation group only had a small amount of red oil droplets near the myotubes, and as the culture time increased, the content of lipid droplets did not increase significantly, and cells were shed. ; The adhesion ability of the cells in the myogenic and adipogenic differentiation group was very good on the 4th and 10th days, and no cells were obviously shed. At the same time, the content of lipid droplets produced was significantly more than that in the myogenic differentiation group. As the culture time increased, the fat content increased. gradually increased (Figure 6). MyHC immunofluorescence results showed that under both culture conditions, muscle stem cells could form multinucleated myotubes and express MyHC protein. However, on the 10th day, the myotubes produced in the myogenic differentiation group had obvious shedding, and the myotubes in the myoadipogenic differentiation group It still adheres well (Figure 7). Therefore, it was determined that LMF culture medium can greatly extend the survival time of muscle stem cells under differentiation culture conditions and produce muscle fibers and fat at the same time. It is suitable for two-dimensional and three-dimensional culture of muscle stem cells.

Adjust the concentration of dexamethasone in LMF A medium to 0.1-5 μM, the concentration of IBMX to 1-100 μM, the concentration of IDM to 0.1-5 μM, the concentration of insulin to 10-300 nM, and the concentration of rosiglitazone. Adjust the concentration of insulin in LMF B medium to 10 to 30 μM; adjust the concentration of insulin in LMF B medium to 10 to 300 nM, and induce muscle stem cell aging and adipogenic differentiation according to the same method, which can achieve the simultaneous formation of muscle fibers and fat.

Example 3 Determining the myogenic and adipogenic differentiation abilities of muscle stem cells at different generations

Muscle stem cells of different generations have different myogenic and adipogenic abilities. P4 generation muscle stem cells with differentiation potential (4th generation cultured in vitro, P4) and P8 generation muscle stem cells with lower generations (8th generation cultured in vitro, P8) were selected. To examine the changes in its muscle-forming and lipogenic abilities. P4 and P8 generation cells were seeded into 96-well plates respectively, and myogenic differentiation group, myoadipogenic differentiation group, ROI group and RO group were set up respectively. Each group was set up in 3 parallel groups. The muscles were cultured in basal medium. The stem cells were stretched and cultured at 37°C and 5% _CO2 for 2 to 3 days, and then the muscle stem cell differentiation medium, LMF medium, ROI medium and RO medium were replaced respectively. Differentiation medium, ROI medium and RO medium were replaced every 24 hours. In the myogenic and adipogenic differentiation group, culture medium A in LMF medium was cultured for 3 days and then culture medium B was replaced for 2 days. The culture was cycled 3 times and replaced every 24 hours. Observations and photos were taken every day. On the 4th and 10th days of culture, cells in 96-well plates were stained with Oil Red O and immunofluorescence staining with myosin heavy chain (MyHC) to examine the production of lipid droplets and MyHC in each group. situation, calculate the proportion of cells that produce fat and the proportion of cells that form muscle fibers.

The oil red O staining of the P4 and P8 generation cells in the myogenic differentiation group, myoadipogenic differentiation group, ROI group and RO group after culture for 4 days is shown in Figure 8, and the MyHC immunofluorescence staining is shown in Figure 9. Lipid production The proportion of cells in the drop is shown in Figure 10, the proportion of cells forming muscle fibers is shown in Figure 11, and the proportion of cells forming fat and muscle fibers is shown in Figure 12.

Analysis of the Oil Red O results and the proportion of cells producing lipid droplets on day 4 showed that no obvious aggregation of lipid droplets was produced in the myogenic differentiation group, while cells in the myogenic and adipogenic differentiation groups, ROI group and RO group all produced obvious lipid droplets (Figure 8) , and the proportion of cells producing lipid droplets in P8 generation cells was higher than that in P4 generation cells, and the ROI group had the most lipid-producing cells (Figure 10). MyHC immunofluorescence results and analysis of the proportion of cells forming myofibers showed that under four culture conditions, P4 generation cells could differentiate well under four conditions and could clearly form multinucleated myotubes, while P8 generation cells did not differentiate under each group of culture conditions. There was obvious myotube formation, but there was no obvious MyHC protein fluorescence (Figure 9), and the proportions were all below 10%, indicating that the myogenic differentiation ability of P8 generation cells was already very weak, and there was no significant difference between the groups (Figure 11). It can be seen from the results of the ratio of cells that form fat to muscle fibers in different generations of cells under different culture conditions that P4 generation cells produce more muscle fibers, and P8 generation cells have a higher fat/muscle fiber ratio. When cultured for 4 days, the ROI group has The highest ratio (Figure 12).

The results of this example show that there are significant differences in the myogenic differentiation and adipogenic differentiation capabilities of cells of different generations. The differentiation ability of cells in the early generations is stronger and can produce more muscle fibers. The myogenic differentiation of cells in later generations is weakened, but in the adipogenic differentiation Under the induction of culture medium, it can still maintain good adipogenic differentiation ability and can produce fat.

Example 4 Preparation of cell cultured snowflake meat with different fat/muscle fiber ratios

There are significant differences in the myogenic differentiation and adipogenic differentiation abilities of cells of different generations. The differentiation ability of cells of earlier generations is stronger and can produce more muscle fibers, while the myogenic differentiation of cells of later generations is weakened. However, under the induction of adipogenic differentiation medium, It can still maintain good adipogenic differentiation ability and can produce fat. Therefore, it is possible to control the proportion of cells with different differentiation capabilities and induce differentiation under myogenic and adipogenic medium conditions, thereby regulating the proportion of fat and muscle fibers, and providing new ideas for customized and personalized cell-cultured meat production.

The cultured cells are P3 generation muscle stem cells with strong myogenic differentiation ability (3rd generation cultured in vitro, P3) and P8 generation muscle stem cells with strong adipogenic differentiation ability (8th generation cultured in vitro, P8). Three groups are set up. , the P3/P8 ratios are 4:1, 1:1, 1:4 respectively. Mix the three groups of cells according to the proportion and mix them with fibrinogen hydrogel (10mg/mL) evenly, so that the initial cell concentration is 2 ×10 ⁶ cells/mL, inoculate into a 96-well plate, 40-60 μL per well. After the hydrogel is completely gelled and solidified, add 200 μL of DMEM medium containing 10% FBS to allow the cells to stretch. Cultivate in a cell culture incubator at 37°C and 5% _CO2 for 48 hours. Aspirate the medium and add 200 μL LMF medium for differentiation culture. Replace the LMF medium every 24 hours and incubate in a cell culture incubator at 37°C and 5% CO2. Culture for 10 days. Observations and photos were taken every day. When the differentiation culture reached the 4th and 10th days, Oil Red O staining and myosin heavy chain (MyHC) immunofluorescence staining were performed on the cells in the 96-well plate to examine the lipid droplets and MyHC in each group. To determine the production situation, take slices of the same thickness in the hydrogel, fix and stain them, and calculate the proportion of cells that produce fat and the proportion of cells that form muscle fibers. It can be seen that the proportions of fat and muscle fibers in the three groups are different, thus obtaining cell cultured snowflake meat products with different fat/muscle fiber ratios.

Although the present invention has been disclosed above in terms of preferred embodiments, they are 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 protection scope of the present invention should be defined by the claims.

Claims (10)

1. A culture medium for rapidly inducing muscle stem cells to differentiate into myocytes and adipocytes simultaneously, characterized in that the composition of the culture medium is (a) or (b): (a) Basic culture medium, fetal calf serum, insulin, rosiglitazone, and oleic acid; (b) Basal medium, horse serum, rosiglitazone, and oleic acid. 2. The culture medium according to claim 1, wherein the basic culture includes DMEM or F12. 3. The culture medium according to claim 1, characterized in that in (a), in terms of volume ratio, the concentration of fetal bovine serum is 5%, the concentration of insulin is 1-50 mg/L, and the concentration of rosiglitazone is 5%. The concentration is 1~50mg/L, and the usage concentration of oleic acid is 10~300μM. 4. The culture medium according to claim 1, wherein in (b), the horse serum concentration is 2%, the rosiglitazone concentration is 1 to 50 μM, and the oleic acid concentration is 2% by volume. 10～300μM. 5. A culture medium for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells, characterized in that the culture medium consists of two parts: culture medium A and culture medium B; The composition of culture medium A is: basal culture medium, fetal calf serum, dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, insulin, and rosiglitazone; culture medium B: basal culture base, fetal bovine serum, insulin. 6. The culture medium according to claim 5, characterized in that in culture medium A, the concentration of fetal bovine serum is 10%, the concentration of dexamethasone is 0.1-5 μM, the concentration of IBMX is 1-100 μM, and the concentration of IDM is 10%. The concentration is 0.1~5μM, the concentration of insulin is 10~300nM, and the concentration of rosiglitazone is 1~30μM; In medium B, fetal bovine serum was used at a concentration of 10%, and insulin was used at a concentration of 10 to 300 nM. 7. A culture method for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells. The method is to use the medium for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells according to claim 5 or 6 to perform differentiation and culture of muscle stem cells. 8. The method according to claim 7, characterized in that the method is to place muscle stem cells in a common medium and culture them until the cell density is 80-90%, and replace medium A every 24 hours. , after inducing differentiation for 3 days, replace medium B, culture for 2 days, and cycle culture 3 times. 9. A method for regulating the proportion of fat and muscle fiber in cell cultured meat, which is characterized by mixing muscle stem cells of different culture generations in proportion and adding them to the culture medium of any one of claims 1 to 4 or the medium of claim 5 or 6. Myogenic and adipogenic differentiation were cultured in the above-mentioned medium. 10. Application of the medium according to any one of claims 1 to 4 or the medium according to claims 5 or 6 in the field of cell cultured meat.