A method for producing cell cultured marbled meat

Abstract

The present invention discloses a method for manufacturing cell-cultured marbled meat, and belongs to the technical field of stem cell culture and cell-cultured meat. The present invention discloses a culture medium for realizing simultaneous myogenic and adipogenic differentiation of muscle stem cells, which can respectively realize rapid myogenic and adipogenic differentiation of muscle stem cells and maintain the myogenic and adipogenic differentiation states of muscle stem cells for a long time. The present invention discloses a method for manufacturing cell-cultured meat that utilizes muscle stem cells to simultaneously produce muscle fibers and fat, and discloses a method for controlling the ratio of fat and muscle fibers in cell-cultured meat by regulating the ratio of muscle stem cells with different myogenic differentiation abilities. The present invention realizes simultaneous myogenic and adipogenic differentiation of muscle stem cells in two-dimensional and three-dimensional culture conditions under the same induction conditions, and can realize simultaneous acquisition of muscle fibers and fat in cultured meat without culturing multiple types of cells, thereby reducing the workload of cell culture and providing new ideas and methods for the industrialized and customized production of cell-cultured meat.

Description

Technical Field

[0001] The invention relates to a method for producing cell-cultured marbled meat, in particular to a method for producing cell-cultured marbled meat which can utilize muscle stem cells to simultaneously produce muscle fibers and fat, and belongs to the technical field of stem cell culture and cell-cultured meat. Background Art

[0002] Cultured meat is an important part of future food. It is a new type of meat food that is made by culturing animal cells in vitro based on the growth and development of muscle tissue and the mechanism of damage repair. It obtains muscle fibers, fat and other cells that constitute muscle tissue, and then collects, shapes and processes them into food. Compared with traditional meat production, cultured meat has a good resource conversion rate and sustainable development potential. Its production process does not involve the feeding and slaughtering of livestock, which greatly shortens the production cycle of meat. At the same time, it can provide real animal protein, and will definitely become an important part of the future meat product market.

[0003] Commercially available meat contains tissues such as muscle fibers and fat, which provide rich proteins and fatty acids, while also ensuring the high-quality taste of meat products. In the process of producing cell-cultured meat, muscle stem cells (Muscle Stem Cell, also known as muscle satellite cells) are usually used as seed cells, which are differentiated into myoblasts through in vitro amplification, and then further differentiated and fused to produce a large number of muscle fibers to form muscle tissue. However, at present, muscle stem cells are mainly used as seed cells for the production of cell-cultured muscle fibers. If cell-cultured adipose tissue is to be manufactured, other seed cells, such as mesenchymal stem cells, are required, which complicates the production process and increases costs. If only muscle stem cells are used as seed cells, their adipogenic differentiation ability is explored, and a muscle stem cell adipogenic differentiation strategy is developed, a new method is provided for the manufacture of cell-cultured marbled meat containing both muscle fibers and fat. Summary of the invention

[0004] The present invention provides a method for producing cell-cultured meat by using 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 abilities.

[0005] The first object of the present invention is to provide a culture medium for rapidly inducing simultaneous myogenic and adipogenic differentiation of muscle stem cells, wherein the cells can form obvious myotubes and lipid droplets after 4 days of induction, wherein the composition of the culture medium is (a) or (b):

[0006] (a) Basic culture medium, fetal bovine serum, insulin, rosiglitazone, and oleic acid;

[0007] (b) basal medium, horse serum, rosiglitazone, and oleic acid;

[0008] In one embodiment, the basal medium comprises DMEM or F12.

[0009] 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 concentration of oleic acid is 10-300 μM.

[0010] In one embodiment, insulin is used at a concentration of 10 mg / L, rosiglitazone is used at a concentration of 10 mg / L, and oleic acid is used at a concentration of 100 μM.

[0011] In one embodiment, in (b), the horse serum is used at a concentration of 2% (v / v), rosiglitazone is used at a concentration of 1-50 μM, and oleic acid is used at a concentration of 10-300 μM.

[0012] In one embodiment, rosiglitazone is used at a concentration of 10 μM and oleic acid is used at a concentration of 100 μM.

[0013] In one embodiment, the muscle stem cells include but are not limited to muscle stem cells of mammals or oviparous animals.

[0014] In one embodiment, the muscle stem cells include muscle stem cells from pigs, chickens, ducks, cows or sheep.

[0015] When the muscle stem cells grow to 80-90% density, replace the ROI medium or RO medium and incubate at 37°C and 5% CO 2 Under the same conditions, the culture medium was replaced every 24 hours, and differentiation was induced for about 4 days, during which obvious myotubes and lipid droplets were formed at the same time.

[0016] 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, wherein the culture medium consists of two parts: culture medium A+culture medium B; culture medium A consists of: basal culture medium, fetal bovine serum, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), indomethacin (IDM), insulin, and rosiglitazone; culture medium B consists of: basal culture medium, fetal bovine serum, and insulin.

[0017] In one embodiment, in culture medium A, fetal bovine serum is used at a concentration of 10% (v / v), dexamethasone is used at a concentration of 0.1-5 μM, IBMX is used at a concentration of 1-100 μM, IDM is used at a concentration of 0.1-5 μM, insulin is used at a concentration of 10-300 nM, and rosiglitazone is used at a concentration of 1-30 μM.

[0018] 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.

[0019] 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.

[0020] 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, wherein the method comprises using the culture medium for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells to carry out differentiation culture of muscle stem cells.

[0021] In one embodiment, the method is to place muscle stem cells in a common culture medium and culture them until the cell density is 80-90%, replace culture medium A, replace the culture medium every 24 hours, and after inducing differentiation for 3 days, replace culture medium B, culture for 2 days, and cycle the culture for 3 times.

[0022] In one embodiment, the common culture medium is basal medium and 10% (v / v) fetal bovine serum.

[0023] In one embodiment, the basal medium comprises DMEM or F12.

[0024] In one embodiment, the culture conditions of the method are 37°C, 5% CO 2 .

[0025] The fourth object of the present invention is to provide a method for regulating the ratio of fat and muscle fibers in cell-cultured meat, wherein muscle stem cells of different generations are mixed in a certain proportion, and cultured for myogenic and adipogenic differentiation in the above-mentioned culture medium that rapidly induces simultaneous myogenic and adipogenic differentiation of muscle stem cells or in the above-mentioned culture medium that maintains long-term myogenic and adipogenic differentiation of muscle stem cells.

[0026] The present invention also provides the use of the above-mentioned culture medium for rapidly inducing simultaneous myogenic and adipogenic differentiation of muscle stem cells or the above-mentioned culture medium for maintaining long-term myogenic and adipogenic differentiation of muscle stem cells in the field of cell cultured meat.

[0027] Beneficial effects:

[0028] Intramuscular preadipocytes and muscle stem cells (also called muscle satellite cells) are two different cells. Intramuscular preadipocytes mainly differentiate into adipose tissue, while muscle stem cells are pluripotent and have the potential to differentiate into muscle fibers, fat, bones, etc. The production of cell-cultured meat requires tissue components such as muscle fibers and fat. Usually, two or more types of cells need to be cultured separately and induced or co-cultured separately to form a culture medium containing both muscle fibers and fat. However, because muscle stem cells have the potential for multidirectional differentiation, the present invention is optimized based on the culture medium for inducing adipocyte adipogenesis and applied to muscle stem cells, so that they can simultaneously differentiate into myofascination and adipogenesis, thereby achieving the effect of simultaneously producing muscle fibers and fat with only one type of cell.

[0029] The present invention identifies three culture media that can achieve simultaneous myogenic and adipogenic differentiation of muscle stem cells, which can respectively achieve rapid myogenic and adipogenic differentiation of muscle stem cells and long-term maintenance of myogenic and adipogenic differentiation of muscle stem cells. It was also found that the myogenic and adipogenic differentiation abilities of muscle stem cells cultured in vitro for different times were different. Cells with shorter in vitro culture times had stronger myogenic differentiation abilities, and cells with longer culture times could obtain more fat. Therefore, the proportion of fat and muscle fibers in cell-cultured meat can be controlled by regulating the proportion of muscle stem cells with different myogenic differentiation abilities in the cultured meat. Myogenic and adipogenic differentiation of muscle stem cells can be achieved simultaneously under the same induction conditions. Muscle fibers and fat can be obtained simultaneously in cultured meat without culturing multiple types of cells, which greatly reduces the workload of culturing cells and provides a new idea and method for the industrialized and customized production of cell-cultured meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The cell morphology of the P4 cell myogenic differentiation group, ROI group and RO group at different culture times (4× field of view);

[0031] Figure 2 The results of Oil Red O staining of P4 cells in myogenic differentiation group, ROI group and RO group after 4 days of culture;

[0032] Figure 3 The proportion of cells producing lipid droplets and muscle fibers in the P4 cell myogenic differentiation group, ROI group, and RO group after 4 days of culture;

[0033] Figure 4 The MyHC immunofluorescence staining results of the P4 cell myogenic differentiation group, ROI group and RO group after 4-day culture;

[0034] Figure 5 The cell morphology of the P4 cell myogenic differentiation group and myogenic adipogenic differentiation group at different culture times (4× field of view);

[0035] Figure 6The results of Oil Red O staining of P4 cell myogenic differentiation group and myogenic adipogenic differentiation group on the 4th and 10th days;

[0036] Figure 7 The MyHC immunofluorescence staining results of the P4 cell myogenic differentiation group and myogenic adipogenic differentiation group on the 4th and 10th days;

[0037] Figure 8 The Oil Red O staining results of cells of different generations cultured under different culture conditions for 4 days (20× field of view);

[0038] Fig. 9 The MyHC immunofluorescence staining results of cells of different generations cultured under different culture conditions for 4 days (10× field of view);

[0039] Fig.10 The percentage of cells at different generations that produce lipid droplets after culturing for 4 days under different culture conditions;

[0040] Fig.11 The percentage of cells that formed myofibers after 4 days of culture at different generations under different culture conditions;

[0041] Fig.12 The cell ratios of fat and muscle fibers formed by cells of different generations under different culture conditions. DETAILED DESCRIPTION

[0042] The culture medium involved in the following examples is:

[0043] Basic culture medium (by volume percentage): 90% DMEM culture medium, 10% fetal bovine serum (FBS).

[0044] Differentiation medium (by volume percentage): 98% DMEM medium, 2% horse serum.

[0045] ROI culture medium (by volume percentage): 95% DMEM culture medium, 5% fetal bovine serum, 10 mg / L insulin, 10 mg / L rosiglitazone, 100 μM oleic acid.

[0046] RO culture medium (by volume percentage): 98% DMEM culture medium, 2% horse serum, 10 μM rosiglitazone, 100 μM oleic acid.

[0047] LMF medium (composed of A and B): Medium A (by volume percentage): 90% DMEM, 10% fetal bovine 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.

[0048] The experimental methods involved in the following embodiments are:

[0049] 1. Oil Red O staining: Oil Red O is a strong lipid solvent and lipid dye, which is easy to combine with triglycerides to form small lipid droplets. Because its solubility in intracellular lipids is greater than that in the original solvent, it can be used for fat staining. The specific operation is as follows:

[0050] (1) Before staining, remove the culture medium from the culture dish and wash it 2-3 times with PBS;

[0051] (2) Fix with 4% paraformaldehyde at room temperature in the dark for 10 min;

[0052] (3) Remove the waste liquid, add fresh 4% paraformaldehyde and fix in the dark for 1 h, then wash with PBS 2-3 times;

[0053] (4) Mix the stock solution and the diluent in a ratio of 5:2, filter to remove impurities, add an appropriate amount of dye solution to just cover the cell layer, stain at 37°C for 10-15 minutes, and wash with PBS for 5-20 seconds;

[0054] (5) Stain with counterstain for 3-5 min and wash with PBS for 30-60 s;

[0055] (6) When observing cells under a microscope, fat appears red and cell nuclei appear dark blue.

[0056] The Oil Red O staining kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0057] 2. Immunofluorescence identification:

[0058] (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;

[0059] (2) Fix with 150 μL 4% paraformaldehyde (precooled at 4°C), permeabilize at room temperature for 15 min, remove the paraformaldehyde, and carefully wash with PBS buffer three times;

[0060] (3) After adding 150 μL of PBS containing 0.5% TritonX-100 for 15 min, the solution was removed and the cells were carefully washed three times with PBS buffer;

[0061] (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;

[0062] (5) Add 100 μL of MyHC antibody diluted in PBS containing 1% bovine serum albumin (BSA), wrap with tin foil, incubate at room temperature for 1 h, and then incubate at 4°C overnight;

[0063] (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 PBS solution containing 1% BSA, wrap with tin foil, and incubate at room temperature for 1-1.5 hours.

[0064] (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.

[0065] Example 1 Rapid induction of simultaneous myogenic and adipogenic differentiation of muscle stem cells

[0066] Primary muscle stem cells with differentiation potential (P4 in vitro) were cultured at 10 4 Cells / well were inoculated into 96-well plates, and myogenic differentiation group, ROI group and RO group were set up respectively, with 3 parallels for each group. 2 The muscle stem cells were cultured under the conditions to allow them to adhere to the wall, and when the cell density reached 80-90%, the differentiation medium, ROI medium and RO medium were replaced respectively.

[0067] The culture medium was replaced every 24 hours, and observation and photography were performed under a microscope every day. On the 4th day of differentiation culture, Oil Red O staining and myosin heavy chain (MyHC) immunofluorescence staining were performed to examine the generation of lipid droplets and MyHC in each group.

[0068] Cell morphology of the myogenic differentiation group, ROI group, and RO group at different culture times Figure 1 As shown, the Oil Red O staining on the 4th day of differentiation culture was as follows Figure 2 As shown, MyHC immunofluorescence staining is as follows Figure 3 As shown. It can be seen that the growth of the three groups of cells was similar on the first day of differentiation culture ( Figure 1 ), with the increase of induction time, muscle stem cells in the three culture conditions were able to differentiate into multinucleated myotubes, and there were obvious clusters of cells in the ROI and RO groups ( Figure 1 ). The results of Oil Red O and the proportion of cells producing lipid droplets on the 4th day showed that there were only a few red oil droplets near the myotubes in the myogenic differentiation group, while significant lipid droplets appeared in the ROI and RO groups, existing side by side with the myotubes, and the lipid droplets in the ROI group were more obvious. 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 myofibroblasts produced showed that under the three culture conditions, muscle stem cells could form multinucleated myotubes and express MyHC protein. Therefore, it was determined that muscle stem cells in the ROI and RO groups could quickly and simultaneously produce myofibers and fat, which could be formed in about 4 days of induction ( Figure 3 , Figure 4 ).

[0069] The insulin concentration in the ROI culture medium was adjusted to 1-50 mg / L, the rosiglitazone concentration was adjusted to 1-50 mg / L, and the oleic acid concentration was adjusted to 10-300 μM; the rosiglitazone concentration in the RO culture medium was adjusted to 1-50 μM, and the oleic acid concentration was adjusted to 10-300 μM. The same method was used to induce myogenic and adipogenic differentiation of muscle stem cells, and the simultaneous formation of muscle fibers and fat could be achieved.

[0070] Example 2 Long-term stable myogenic and adipogenic differentiation of muscle stem cells

[0071] Primary muscle stem cells with differentiation potential (P4 in vitro) were cultured at 10 4 Cells / well were inoculated into 96-well plates, and myogenic differentiation group and myogenic adipogenic differentiation group were set up respectively, with 3 parallels for each group. The cells were first incubated in basal medium at 37°C and 5% CO 2 The muscle stem cells were cultured under the conditions to allow them to adhere to the wall, and when the cell density reached 80-90%, the differentiation medium and LMF medium (composed of A and B) were replaced respectively.

[0072] When the muscle stem cells of the myogenic and adipogenic differentiation groups grew to 80-90% density, medium A was replaced and the cells were incubated at 37°C and 5% CO. 2 After 3 days of differentiation induction, the medium B was replaced and the cells were cultured at 37°C and 5% CO 2 The cells were cultured under the same conditions for 2 days and cultured 3 times in total, which could maintain the morphology of myotubes and lipid droplets for a long time. The lipid droplets gradually increased with the increase of culture time.

[0073] The culture medium of the two groups of cells was replaced every 24 hours, and the cells 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 to examine the generation of lipid droplets and MyHC in each group.

[0074] Cell morphology of myogenic differentiation group and myogenic adipogenic differentiation group at different culture times Figure 5 As shown, the Oil Red O staining on the 4th and 10th days is as follows Figure 6 As shown, MyHC immunofluorescence staining is as follows Figure 7As shown. It can be seen that the growth of cells in the two groups was similar on the first day of culture. On 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 shedding. As the culture time increased, the cell shedding became more serious. However, in the myogenic differentiation group, the cells did not show obvious shedding until the tenth day of culture, and they could survive and adhere well. Figure 5 ). The results of Oil Red O on the 4th and 10th days showed that there were only a small amount of red oil droplets near the myotubes in the myogenic differentiation group, and the lipid droplet content did not increase significantly with the increase of culture time, and was accompanied by cell shedding; the cells in the myogenic and adipogenic differentiation group had good adhesion ability on the 4th and 10th days, and no obvious cell shedding was observed. At the same time, the lipid droplet content produced was significantly more than that in the myogenic differentiation group. With the increase of culture time, the fat content gradually increased ( Figure 6 ). MyHC immunofluorescence results showed that under both culture conditions, muscle stem cells could form multinucleated myotubes and express MyHC protein, but on the 10th day, the myotubes produced in the myogenic differentiation group showed obvious detachment, while the myotubes in the myogenic adipogenic differentiation group could still adhere well ( Figure 7 ). Therefore, it was determined that LMF culture medium can greatly prolong the survival time of muscle stem cells under differentiation culture conditions, and simultaneously produce muscle fibers and fat, and is suitable for two-dimensional and three-dimensional culture of muscle stem cells.

[0075] The concentrations of dexamethasone, IBMX, IDM, insulin, and rosiglitazone in LMF A culture medium were adjusted to 0.1-5 μM, 1-100 μM, 10-300 nM, and 1-30 μM, respectively; the concentration of insulin in LMF B culture medium was adjusted to 10-300 nM. The same method was used to induce muscle stem cell differentiation and adipogenesis, and the simultaneous formation of muscle fibers and fat could be achieved.

[0076] Example 3 Determination of the ability of muscle stem cells of different generations to differentiate into myoadipocytes

[0077] Different generations of muscle stem cells have different myogenic and adipogenic abilities. P4 muscle stem cells (the 4th generation of in vitro culture, P4) with differentiation potential and P8 muscle stem cells (the 8th generation of in vitro culture, P8) with later generations were selected to investigate the changes in their myogenic and adipogenic abilities. P4 and P8 cells were inoculated into 96-well plates, and myogenic differentiation group, myogenic adipogenic differentiation group, ROI group and RO group were set up respectively. Each group was set up with 3 parallels. Muscle stem cells were stretched in basal culture medium and incubated at 37°C and 5% CO 2After culturing for 2 to 3 days under the same conditions, the muscle stem cell differentiation medium, LMF medium, ROI medium and RO medium were replaced respectively. The differentiation medium, ROI medium and RO medium were replaced every 24 hours. The myogenic and adipogenic differentiation group was cultured in the same way as medium A in LMF medium for 3 days and then medium B for 2 days, and the culture cycle was repeated 3 times, with the culture replaced every 24 hours. Observation and photography were performed every day. When the cells were cultured to the 4th and 10th days, the 96-well plate cells were stained with Oil Red O and immunofluorescence staining of myosin heavy chain (MyHC) to examine the generation of lipid droplets and MyHC in each group, and the proportion of cells producing fat and the proportion of cells forming muscle fibers were calculated.

[0078] Oil red O staining of P4 and P8 cell myogenic differentiation groups, myogenic adipogenic differentiation groups, ROI groups and RO groups after 4 days of culture is shown in the figure. Figure 8 As shown, MyHC immunofluorescence staining is as follows Fig. 9 As shown, cells producing lipid droplets are more likely to be Fig.10 As shown, the cells that form myofibers are more numerous than e.g. Fig.11 As shown, the ratio of cells that form fat to muscle fibers is Fig.12 shown.

[0079] On the 4th day, the results of Oil Red O and the proportion of cells producing lipid droplets showed that there was no obvious clustered lipid droplets in the myogenic differentiation group, while the cells in the myogenic adipogenic differentiation group, ROI group and RO group all produced obvious lipid droplets ( Figure 8 ), and the proportion of cells producing lipid droplets in P8 cells was higher than that in P4 cells. The ROI group had the most lipid-producing cells ( Fig.10 ). The results of MyHC immunofluorescence and the analysis of the proportion of cells that formed myofibers showed that under the four culture conditions, P4 cells could differentiate well and form multinucleated myotubes. However, P8 cells had no obvious myotube formation and no obvious MyHC protein fluorescence under all the culture conditions. Fig. 9 ), the proportions were all below 10%, indicating that the myogenic differentiation ability of P8 cells was already very weak, and there was no significant difference among the groups ( Fig.11 ). From the results of the ratio of fat to muscle fibers formed by cells of different generations under different culture conditions, it can be seen that P4 cells produce more muscle fibers, and P8 cells have a higher fat / muscle fiber ratio. At 4 days of culture, the ROI group has the highest ratio ( Fig.12 ).

[0080] The results of this example show that there are significant differences in the myogenic differentiation and adipogenic differentiation abilities of cells of different generations. The differentiation ability of cells of the early generations is stronger and can produce more muscle fibers, while the myogenic differentiation of cells of the later generations is weakened. However, under the induction of adipogenic differentiation medium, they can still maintain good adipogenic differentiation ability and can produce fat.

[0081] Example 4 Preparation of Marbled Meat with Cell Cultures of Different Fat / Muscle Fiber Ratios

[0082] There are significant differences in the myogenic and adipogenic differentiation abilities of cells of different generations. The differentiation ability of cells of the earlier generations is stronger and can produce more muscle fibers, while the myogenic differentiation of cells of the later generations is weakened. However, under the induction of adipogenic differentiation medium, they can still maintain good adipogenic differentiation ability and produce fat. Therefore, by controlling the ratio of cells with different differentiation abilities and inducing differentiation under the conditions of myogenic and adipogenic medium, the ratio of fat to muscle fibers can be regulated, providing new ideas for customized and personalized cell culture meat production.

[0083] The cultured cells were P3 muscle stem cells with strong myogenic differentiation ability (the third generation of in vitro culture, P3) and P8 muscle stem cells with strong adipogenic differentiation ability (the eighth generation of in vitro culture, P8). Three groups were set up, and the P3 / P8 ratios were 4:1, 1:1, and 1:4, respectively. The three groups of cells were mixed according to the proportion and then mixed evenly with fibrinogen hydrogel (10 mg / mL) to make the initial cell concentration 2×10 6 100 μL / mL, inoculated in 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 expand. 2 The cells were cultured in a cell culture incubator for 48 hours, the culture medium was removed, and 200 μL LMF culture medium was added for differentiation culture. The LMF culture medium was replaced every 24 hours, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 10 days. Observation and photography were performed every day. When the differentiation culture reached the 4th and 10th days, the cells in the 96-well plate were stained with Oil Red O and immunofluorescence staining of myosin heavy chain (MyHC) to examine the generation of lipid droplets and MyHC in each group. The same thickness slices were fixed and stained in the hydrogel to calculate the proportion of cells producing fat and the proportion of cells forming muscle fibers. It can be seen that the ratio of fat to muscle fibers in the three groups is different, thus obtaining cell cultured marbled meat products with different fat / muscle fiber ratios.

[0084] 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 method for maintaining myogenic and adipogenic differentiation of muscle stem cells, the method comprising: using a culture medium for maintaining myogenic and adipogenic differentiation of muscle stem cells to perform differentiation culture of muscle stem cells; The culture medium for maintaining myogenic and adipogenic differentiation of muscle stem cells consists of two parts: Medium A and Medium B; The composition of culture medium A is: basic culture medium, fetal bovine serum, dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, insulin, rosiglitazone; the composition of culture medium B is: basic culture medium, fetal bovine serum, insulin; In medium A, the concentration of fetal bovine serum is 10%, the concentration of dexamethasone is 0.1 to 5 μM, the concentration of IBMX is 1 to 100 μM, the concentration of IDM is 0.1 to 5 μM, the concentration of insulin is 10 to 300 nM, and the concentration of rosiglitazone is 1 to 30 μM; In medium B, the concentration of fetal bovine serum was 10%, and the concentration of insulin was 10-300 nM; The method comprises placing muscle stem cells in a basic culture medium and culturing them until the cell density reaches 80-90%, replacing the culture medium A, and replacing the culture medium every 24 hours. After inducing differentiation for 3 days, replacing the culture medium B, culturing for 2 days, and repeating the culture cycle for 3 times.

2. A method for regulating the ratio of fat and muscle fiber in cell cultured meat, characterized in that: Muscle stem cells of different culture generations are mixed in proportion, and cultured in a medium that maintains myogenic and adipogenic differentiation of muscle stem cells for myogenic and adipogenic differentiation; The culture medium for maintaining myogenic and adipogenic differentiation of muscle stem cells consists of two parts: culture medium A and culture medium B; The composition of culture medium A is: basic culture medium, fetal bovine serum, dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, insulin, rosiglitazone; the composition of culture medium B is: basic culture medium, fetal bovine serum, insulin; In medium A, the concentration of fetal bovine serum is 10%, the concentration of dexamethasone is 0.1 to 5 μM, the concentration of IBMX is 1 to 100 μM, the concentration of IDM is 0.1 to 5 μM, the concentration of insulin is 10 to 300 nM, and the concentration of rosiglitazone is 1 to 30 μM; In medium B, the concentration of fetal bovine serum was 10%, and the concentration of insulin was 10-300 nM; The method comprises placing mixed muscle stem cells of different culture generations in a basic culture medium and culturing them until the cell density reaches 80-90%, replacing the culture medium A, and replacing the culture medium every 24 hours. After inducing differentiation for 3 days, replacing the culture medium B, culturing for 2 days, and repeating the culture cycle for 3 times.

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