Culture medium and method for producing cell cultured meat by co-culturing myoblasts from different fish

Abstract

The present invention provides a culture medium and method for co-culturing myoblasts of different fish to produce cell-cultured meat, which specifically belongs to the field of cell engineering technology. The culture medium for co-culturing myoblasts of different fish to produce cell-cultured meat described in the present invention includes a proliferation culture medium and a differentiation culture medium; the proliferation culture medium is based on DMEM / F12 culture medium and also contains fetal bovine serum; the differentiation culture medium is based on DMEM culture medium and also contains horse serum. In a specific embodiment, the culture medium described in the present invention can make large yellow croaker myoblasts differentiate towards fat cells and eel myoblasts differentiate towards muscle cells, and finally obtain artificial fish meat similar to the composition of real fish meat cells.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering, and in particular relates to a culture medium and a method for co-culturing different fish myoblasts to produce cell cultured meat. Background Art

[0002] Cultured meat is made by extracting animal muscle stem cells and adipose stem cells for in vitro culture, which then differentiate into muscle fibers and adipocytes. Cultured meat involves the separation and purification of stem cells, the proliferation and differentiation of stem cells, and the co-culture of different types of cells. During the production process, only culture medium, a certain temperature and humidity, and carbon dioxide are needed to provide the environment required for nutrition and growth. It will not cause environmental pollution, does not consume the feed and water required in traditional meat production, and occupies a small area, saving space costs. It is the main research and development direction of artificial meat in the future.

[0003] Fish meat is tender, delicious and nutritious, and is a good source of some vitamins and minerals. Therefore, developing fish cell culture technology to produce cell-cultured meat to meet human needs is a means to solve the decline of fish resources. Real fish meat is composed of different types of cells, the main components of which are muscle cells and fat cells. How to mimic the in vivo environment so that different types of stem cells differentiate towards muscle cells or fat cells under in vitro co-culture conditions is an important part of the factory production of cell-cultured meat. Therefore, inventing a co-culture medium or method to produce cell-cultured meat is a technical problem that technicians in this field need to solve. Summary of the invention

[0004] The purpose of the present invention is to provide a culture medium and method for co-culturing different fish myoblasts to produce cell cultured meat. The use of the culture medium of the present invention for co-culturing different fish myoblasts can simulate the in vivo environment so that different fish myoblasts can be differentiated into muscle cells or fat cells under the conditions of in vitro co-culture, and can provide technical support for the subsequent large-scale production of cell cultured meat.

[0005] The invention provides a culture medium for co-culturing myoblasts of different fishes to produce cell cultured meat, the culture medium comprising a proliferation culture medium and a differentiation culture medium; the proliferation culture medium uses DMEM / F12 culture medium as a basic culture medium and also contains fetal bovine serum; the differentiation culture medium uses DMEM culture medium as a basic culture medium and also contains horse serum.

[0006] Preferably, in the proliferation medium, the volume concentration of fetal bovine serum is 50-200 μL / mL.

[0007] Preferably, the proliferation medium further contains basic fibroblast growth factor (bGFG).

[0008] Preferably, in the proliferation medium, the mass concentration of basic fibroblast growth factor is 0-50 ng / mL.

[0009] Preferably, the volume concentration of horse serum in the differentiation medium is 10-100 μL / mL.

[0010] Preferably, the fish include large yellow croaker and eel.

[0011] The present invention also provides a method for producing cell-cultured meat by co-culturing different fish myoblasts in the culture medium based on the above technical scheme, comprising the following steps: mixing different fish myoblasts and inoculating them into a proliferation culture medium for proliferation co-culture, and then performing differentiation co-culture in a differentiation culture medium to obtain cell-cultured meat.

[0012] Preferably, the fish include large yellow croaker and eel.

[0013] Preferably, when the fish are large yellow croaker and eel, the total amount of inoculation of large yellow croaker and eel is 10 4 ~10 6 / mL; the inoculation ratio of large yellow croaker myoblasts to eel myoblasts is 1:9~9:1.

[0014] Preferably, the culture temperature is 25-30° C.; the proliferation co-culture time is 24-120 h; and the differentiation co-culture time is 24-240 h.

[0015] The present invention provides a culture medium for co-culturing myoblasts of different fish to produce cell-cultured meat. The use of the culture medium of the present invention for co-culturing myoblasts of different fish can simulate the in vivo environment so that myoblasts of different fish can differentiate into muscle cells or fat cells respectively under the conditions of in vitro co-culture, and can provide technical support for the subsequent large-scale production of cell-cultured meat. The experimental results show that when large yellow croaker myoblasts and eel myoblasts are co-cultured using the culture medium of the present invention, large yellow croaker myoblasts differentiate into fat cells, and eel myoblasts differentiate into muscle cells, and finally artificial fish meat similar to the composition of real fish meat cells is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1A growth diagram of co-culture of large yellow croaker myoblasts and eel myoblasts at different cell inoculation ratios provided by the present invention; wherein A is a typical electron microscope image of co-culture of large yellow croaker myoblasts and eel myoblasts at different cell inoculation ratios; B is a cell viability result diagram of co-culture of large yellow croaker myoblasts and eel myoblasts at different cell inoculation ratios;

[0018] Figure 2 The in vitro differentiation result diagram of the large yellow croaker myoblast and the eel myoblast provided by the present invention; wherein A is a typical electron microscope picture of the large yellow croaker myoblast and the eel myoblast after differentiation; B is an Oil Red O staining and Desmin immunofluorescence staining picture of the large yellow croaker myoblast and the eel myoblast after differentiation;

[0019] Figure 3 The in vitro differentiation result diagram of the co-culture of large yellow croaker myoblasts and eel myoblasts at different cell seeding ratios provided by the present invention; wherein A is a desmin immunofluorescence staining picture after differentiation under the co-culture of large yellow croaker myoblasts and eel myoblasts at different cell seeding ratios; B is an Oil Red O staining picture after differentiation under the co-culture of large yellow croaker myoblasts and eel myoblasts at different cell seeding ratios; C is a fusion index result diagram after differentiation under the co-culture of large yellow croaker myoblasts and eel myoblasts at different cell seeding ratios. DETAILED DESCRIPTION

[0020] The present invention provides a culture medium for co-culturing different fish myoblasts to produce cell cultured meat, the culture medium comprising a proliferation culture medium and a differentiation culture medium; the proliferation culture medium is based on DMEM / F12 culture medium and also contains fetal bovine serum; the differentiation culture medium is based on DMEM culture medium and also contains horse serum. The present invention does not specifically limit the sources of the various raw materials of the culture medium, and conventional commercially available DMEM / F12 culture medium, DMEM culture medium, fetal bovine serum and horse bovine serum can be used. In a specific embodiment, in the proliferation culture medium, the volume concentration of fetal bovine serum is 50~200 μL / mL. In a specific embodiment, the volume concentration of fetal bovine serum can be 50 μL / mL, 100 μL / mL or 200 μL / mL. In the present invention, the proliferation culture medium also contains basic fibroblast growth factor. The present invention does not specifically limit the source of basic fibroblast growth factor, and conventional commercially available basic fibroblast growth factor can be used. In a specific embodiment, in the proliferation culture medium, the mass concentration of basic fibroblast growth factor is 0~50 ng / mL. In a specific embodiment, in the proliferation medium, the mass concentration of basic fibroblast growth factor can be 0 ng / mL, 10 ng / mL or 50 ng / mL. In a specific embodiment, in the differentiation medium, the volume concentration of horse serum is 10~100 μL / mL. In a specific embodiment, in the differentiation medium, the volume concentration of horse serum can be 20 μL / mL, 80 μL / mL or 100 μL / mL. In the present invention, the fish includes large yellow croaker and eel. In a specific embodiment, the fish are large yellow croaker and eel. Large yellow croaker and eel myoblasts are co-cultured using the culture medium of the present invention, and the large yellow croaker myoblasts differentiate into adipocytes, and the eel myoblasts differentiate into muscle cells.

[0021] The present invention also provides a method for producing cell-cultured meat by co-culturing different fish myoblasts in the culture medium based on the above technical solution, comprising the following steps: mixing different fish myoblasts and inoculating them into a proliferation culture medium for proliferation co-culture, and then performing differentiation co-culture in a differentiation culture medium to obtain cell-cultured meat. In the present invention, the proliferation co-culture and differentiation co-culture in the culture process refer to different fish myoblasts sharing a set of proliferation culture medium during the co-culture process, and different fish myoblasts sharing a set of differentiation culture medium during the co-culture process after proliferation. The consistency of the culture medium during the co-culture process can make the culture simpler and more efficient, and better mimic the living environment of cells in the body.

[0022] In the present invention, the fish include large yellow croaker and eel. In a specific embodiment, the fish are large yellow croaker and eel. In the present invention, when the fish are large yellow croaker and eel, the total amount of inoculation of large yellow croaker and eel is 10 4 ~106 / mL; the inoculation ratio of large yellow croaker myoblasts to eel myoblasts is 1:9~9:1. In a specific embodiment, the inoculation ratio of large yellow croaker myoblasts to eel myoblasts is 1:8 or 2:7. The above inoculation ratio can make the two types of myoblasts better differentiate into muscle cells and adipocytes. In a specific embodiment, the total inoculation amount of large yellow croaker and eel can be 1×10 5 ~2×10 5 In another specific embodiment, the total inoculation amount of large yellow croaker and eel is 1×10 5 / mL. In a specific embodiment, the culture temperature is 25-30°C; the time of the proliferation co-culture is 24-120 h; the time of the differentiation co-culture is 24-240 h. In a specific embodiment, the culture temperature can be 25°C, 27°C or 30°C. In a specific embodiment, the time of the proliferation co-culture can be 24-72h; or it can be 24 h, 48 h or 72 h. In a specific embodiment, the time of the differentiation co-culture can be 72-240h, or it can be 72 h, 168 h or 240 h. In the co-culture process of the present invention, there is no need to replace the proliferation culture medium, and the differentiation culture medium is replaced every 3 days, and half is replaced each time. The co-culture method of the present invention can effectively simulate the in vivo environment so that different fish stem cells share a set of proliferation and differentiation culture media under the conditions of in vitro co-culture, and finally differentiate towards muscle cells or fat cells respectively. This method is simple to operate and has excellent results, and can provide technical support for the subsequent large-scale production of cell cultured meat.

[0023] To further illustrate the present invention, the culture medium and method for co-culturing different fish myoblasts to produce cell-cultured meat provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0025] DMEM / F12 medium (Procell ® , PM150312); DMEM culture medium (gibco ® , 8123447); fetal bovine serum (WISENT ® , 086-150); horse serum (biosharp ® , BL209A); ​​bFGF (Beyotime ® , P6443).

[0026] Example 1

[0027] Evaluation of the growth of co-cultured large yellow croaker myoblasts and eel myoblasts at different cell seeding ratios.

[0028] About 5000 muscle satellite cells were seeded in each well of a 96-well plate, and 100 μL proliferation medium (DMEM / F12+100 μL / mL fetal bovine serum+10 ng / mL bFGF) was added to each well. The plates were cultured continuously in a 27°C incubator for 5 days. The cell viability was determined using the CCK-8 kit, and the growth of large yellow croaker myoblasts and eel myoblasts in different cell seeding ratios in in vitro co-culture was evaluated.

[0029] The results are as follows Figure 1 As shown in B. The results showed that when the inoculation ratio of large yellow croaker myoblasts and eel myoblasts was 1:8, the cell proliferation effect became better and better with the increase of culture time, and was significantly better than the inoculation ratio of 2:7, and also better than eel myoblasts and large yellow croaker myoblasts cultured alone. Figure 1 The electron microscope image of A in the figure shows that the cell morphology of eel myoblasts is more slender than that of large yellow croaker myoblasts. In co-culture, the proportion of slender cells is also high due to the high inoculation ratio of eels. This result shows that co-culture does not affect the growth of the two types of myoblasts and is more conducive to their growth than single culture. This also shows the possibility of co-culturing the two types of cells. Compared with the traditional co-culture method of culturing different types of cells separately or using different growth culture media, the two types of myoblasts in the present invention use the same proliferation and differentiation culture medium, which provides a basis for the subsequent large-scale production of cell cultured meat.

[0030] Example 2

[0031] In vitro differentiation of large yellow croaker myoblasts and eel myoblasts.

[0032] After the confluence of large yellow croaker myoblasts and eel myoblasts reached more than 80%, differentiation medium was added respectively. The differentiation medium formula was DMEM + 20 μL / mL horse serum. The large yellow croaker myoblasts were cultured for 48 h and the eel myoblasts were cultured for 120 h in a 27°C incubator. After the differentiation was completed, the differentiated large yellow croaker myoblasts were stained with Oil Red O, and the differentiated eel myoblasts were stained with desmin immunofluorescence. The results are shown in Figure 2 As shown, almost all large yellow croaker myoblasts differentiated into adipocytes, while eel myoblasts formed more myotubes, indicating that using the same differentiation medium, large yellow croaker myoblasts can differentiate into adipocytes and eel myoblasts can differentiate into muscle cells.

[0033] Example 3

[0034] Evaluation of in vitro differentiation of large yellow croaker myoblasts and eel myoblasts co-cultured at different cell seeding ratios.

[0035] After the confluence of the myoblasts cultured in different proportions reached more than 80%, differentiation medium was added. The differentiation medium formula was DMEM + 20 μL / mL horse serum. Cultured in a 27°C incubator for 120 h. After differentiation was completed, desmin immunofluorescence staining was performed first, followed by Oil Red O staining. The results are shown in Figure 3 As shown, when the inoculation ratio of large yellow croaker myoblasts: eel myoblasts is 1:8, the effect of differentiation into myotubes is significantly better than 2:7, but the effect of differentiation into adipocytes is significantly worse than 2:7. This result shows that the co-culture mode of the present invention can make large yellow croaker myoblasts and eel myoblasts differentiate into muscle cells and adipocytes respectively when the proliferation medium and differentiation medium are consistent, and the ratio of myotube and adipocyte formation can be adjusted by adjusting the ratio of the two types of myoblasts, effectively simulating the living environment of different types of cells in the body. The fish meat of each type of fish has a different cell composition, resulting in a different taste, so the different product flavor requirements can be met by adjusting the ratio of the two types of myoblasts.

[0036] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium for co-culturing large yellow croaker muscle satellite cells and eel muscle satellite cells to produce cell cultured meat, characterized in that: The culture medium comprises a proliferation culture medium and a differentiation culture medium; the proliferation culture medium uses DMEM / F12 culture medium as a basic culture medium and further contains fetal bovine serum; the differentiation culture medium uses DMEM culture medium as a basic culture medium and further contains horse serum.

2. The culture medium according to claim 1, characterized in that In the proliferation medium, the volume concentration of fetal bovine serum is 50-200 μL / mL.

3. The culture medium according to claim 1, characterized in that The proliferation culture medium also contains basic fibroblast growth factor.

4. The culture medium according to claim 3, characterized in that In the proliferation medium, the mass concentration of basic fibroblast growth factor is 0-50 ng / mL.

5. The culture medium according to claim 1, characterized in that In the differentiation medium, the volume concentration of horse serum is 10-100 μL / mL.

6. A method for producing cell cultured meat by co-culturing large yellow croaker muscle satellite cells and eel muscle satellite cells based on the culture medium according to any one of claims 1 to 5, comprising the following steps: The large yellow croaker muscle satellite cells and the eel muscle satellite cells are mixed and inoculated into a proliferation medium for proliferation co-culture, and then differentiated co-cultured in a differentiation medium to obtain cell cultured meat.

7. The method according to claim 6, characterized in that The total inoculation amount of large yellow croaker muscle satellite cells and eel muscle satellite cells was 10 4 ~10 6 / mL; the inoculation ratio of large yellow croaker muscle satellite cells and eel muscle satellite cells is 1:9~9:

1.

8. The method according to claim 6, characterized in that The culture temperature is 25-30°C; the proliferation co-culture time is 24-120 h; the differentiation co-culture time is 24-240 h.

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