Medium for myogenic differentiation and pluripotent differentiation of fish precursor fat cells, method for inducing myogenic differentiation and pluripotent differentiation and application

Abstract

The invention provides a fish precursor fat cell myogenic differentiation and pluripotent differentiation culture medium, a method for inducing myogenic differentiation and pluripotent differentiation and application, and particularly belongs to the technical field of cell biology. The differentiation culture medium for inducing the fish precursor fat cells to differentiate into the muscle cells comprises a myogenic differentiation culture medium and a differentiation maintenance culture medium. According to the myogenic differentiation culture medium, the large yellow croaker precursor fat cells can be induced to be differentiated towards muscle cells through the regulation and control effect of growth factors, and the differentiation maintaining culture medium can maintain myogenic differentiation and enhance the quality of myotubes; by combining the adipogenic differentiation culture medium, the directional pluripotent differentiation of the precursor adipocytes to different types of cells can be finally realized, and myotubes and adipocytes are efficiently formed. The preparation of the cell culture meat provides a new strategy and method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cell biology technology, and particularly relates to fish precursor adipocyte myogenic differentiation, pluripotent differentiation culture medium and a method and application for inducing myogenic differentiation and pluripotent differentiation. BACKGROUND

[0002] Stem cells are a kind of cells with unlimited self-renewal ability and can produce at least one type of highly differentiated progeny cells. Functionally, stem cells are cells with multi-directional differentiation potential and self-renewal ability, and can differentiate to produce a certain specific type of cells in vivo. Once differentiated into progenitor cells, stem cells lose the ability of self-renewal, and the number of progenitor cells is only supplemented by the proliferation and differentiation of stem cells, and the progenitor cells maintain a high proliferation ability. According to different differentiation potential, stem cells can be divided into totipotent stem cells, pluripotent stem cells and unipotent stem cells. Among them, pluripotent stem cells have the ability to produce multiple types of cells. For example, bone marrow mesenchymal stem cells can differentiate into bone, cartilage, muscle, fat, etc. Stem cells have extremely broad application prospects in the fields of cell repair, developmental biology, pharmacology, etc. At present, with the development of synthetic biology, the application of stem cells in this field has attracted widespread attention. Precursor adipocytes are important stem cells in adipose tissue, and their differentiation is particularly important. However, current research on precursor adipocytes is mostly focused on differentiation into adipocytes. At present, there is no method that can realize the pluripotent differentiation of precursor adipocytes. SUMMARY

[0003] The present application provides a fish precursor adipocyte myogenic differentiation, pluripotent differentiation culture medium and a method and application for inducing myogenic differentiation and pluripotent differentiation. The differentiation medium for inducing fish precursor adipocyte to differentiate into muscle cells can induce precursor adipocytes to differentiate into muscle cells, and ultimately the present application can realize the directional pluripotent differentiation of precursor adipocytes into different types of cells, specifically efficiently forming muscle tubes and adipocytes.

[0004] The present application provides a set of differentiation medium for inducing fish precursor adipocyte to differentiate into muscle cells, including myogenic differentiation medium and differentiation maintenance medium; the myogenic differentiation medium takes F12 medium as the basic medium, and includes 1-10% fetal bovine serum by volume percentage, 1-10 μM RepSox by molar concentration, 1-15 nM LY411575 by molar concentration, 10-100 nM vitamin C by molar concentration, and 1% penicillin and streptomycin mixed solution by volume percentage; the differentiation maintenance medium takes DMEM medium as the basic medium, and includes 1-10% fetal bovine serum by volume percentage, 1-5 ng / mL hydrocortisone by mass concentration, and 1% penicillin and streptomycin mixed solution by volume percentage.

[0005] The present invention also provides the application of the differentiation culture medium described in the above technical solution for inducing fish precursor adipocytes to differentiate into myocytes in the induction of fish precursor adipocytes into myoblastic differentiation.

[0006] The present invention also provides a method for inducing myogenic differentiation of fish precursor adipocytes based on the differentiation culture medium for inducing differentiation of fish precursor adipocytes into myocytes as described in the above technical solution, comprising the following steps:

[0007] Proliferate and culture fish precursor fat cells;

[0008] Fish progenitor fat cells, after proliferation culture, are inoculated into the myogenic differentiation medium for myogenic differentiation culture, and then replaced with the differentiation maintenance medium for differentiation maintenance culture to obtain myocytes.

[0009] The present invention also provides a culture medium for inducing pluripotent differentiation of fish precursor adipocytes, comprising the differentiation culture medium for inducing fish precursor adipocytes to differentiate into myocytes and the adipogenic differentiation culture medium described in the above technical solution.

[0010] Preferably, the adipogenic differentiation medium is based on DMEM medium, which includes 1% to 20% horse serum by volume and a mixed solution of penicillin and streptomycin by volume of 1%.

[0011] The present invention also provides the application of the differentiation culture medium for inducing fish precursor adipocytes to differentiate into myocytes as described in the above-mentioned technical solution, or the culture medium for inducing fish precursor adipocytes to differentiate into multiple cells as described in the above-mentioned technical solution, in inducing fish precursor adipocytes to differentiate into multiple cells.

[0012] Preferably, the pluripotent differentiation includes myoblastic differentiation and adipogenic differentiation.

[0013] The present invention also provides a method for inducing pluripotent differentiation of fish precursor adipocytes based on the culture medium for inducing pluripotent differentiation of fish precursor adipocytes as described in the above-described technical solution, comprising the following steps:

[0014] Proliferate and culture fish precursor fat cells;

[0015] Fish progenitor fat cells, after proliferation culture, were inoculated into the myoblast differentiation medium for myoblast differentiation culture, and then replaced with the differentiation maintenance medium for differentiation maintenance culture to obtain myoblasts.

[0016] Fish progenitor adipocytes, after proliferation and culture, were inoculated into the adipogenic differentiation medium for adipogenic differentiation culture to obtain adipocytes.

[0017] Preferably, the myoblast differentiation culture time is 24-72 h; the differentiation maintenance culture time is 48-240 h; and the adipogenic differentiation culture time is 24-120 h.

[0018] Preferably, during the proliferation culture, the inoculation density of fish precursor fat cells is 10-1. 4 ~10 7 per mL.

[0019] This invention provides a differentiation culture medium for inducing the differentiation of fish preadipocytes into myocytes. The myoblast differentiation medium in this invention can induce fish preadipocytes to differentiate into myocytes, while the differentiation maintenance medium can maintain and enhance differentiation capacity, specifically by enhancing the quality of myotubes, making them longer, thicker, and more adherent. Experimental results show that, using large yellow croaker preadipocytes as experimental material, and utilizing the myoblast differentiation medium and differentiation maintenance medium of this invention, the differentiation of preadipocytes into myotubes can be achieved. In the field of synthetic biology, such as cell cultured meat, compared to unipotent stem cells like myoblasts, the myoblast differentiation of large yellow croaker preadipocytes can achieve pluripotent differentiation, which is beneficial for advancing the field of synthetic biology.

[0020] This invention also provides a culture medium and method for inducing pluripotent differentiation of fish preadipocytes. Utilizing differentiation media for inducing myocyte differentiation and adipogenic differentiation media, fish preadipocytes are induced to differentiate into myotubes and adipocytes. The method of this invention has the advantages of simple operation and high differentiation efficiency. The proposed scheme of this invention can achieve pluripotent differentiation of fish preadipocytes. The results of the examples show that large yellow croaker preadipocytes can form a large number of myotubes under the culture of myogenic differentiation medium and differentiation maintenance medium, and can also efficiently differentiate into adipocytes under the induction of adipogenic differentiation medium. Therefore, the method provided by this invention offers a new approach for the preparation of cell-cultured meat. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The images provided by this invention show the isolation and identification of primary preadipocytes from large yellow croaker; where A is the cell isolation result and B is the cell identification result.

[0023] Figure 2The images show the myoblastic differentiation results of the large yellow croaker precursor adipocytes provided by this invention; where A is a phase-contrast microscopy observation image; B is an immunofluorescence staining image of desmin in myotubes; and C is a fusion index result image.

[0024] Figure 3 The diagram shows the myoblastic differentiation results of large yellow croaker myoblasts provided by this invention; wherein, A is an immunofluorescence staining image of desmin in myotubes; and B is a fusion index result diagram.

[0025] Figure 4 The image shows the adipogenic differentiation results of the large yellow croaker precursor adipocytes provided by this invention; where A is a phase contrast microscope observation image; and B is a Nile red fluorescence staining image of the adipocytes. Detailed Implementation

[0026] This invention provides a set of differentiation media for inducing the differentiation of fish preadipocytes into myocytes, including a myogenic differentiation medium and a differentiation maintenance medium. The myogenic differentiation medium uses F12 medium as the basal medium and includes 1%–10% fetal bovine serum, 1–10 μM RepSox, 1–15 nM LY411575, 10–100 nM vitamin C, and a 1% (v / v) mixed solution of penicillin and streptomycin. The differentiation maintenance medium uses DMEM medium as the basal medium and includes 1%–10% (v / v) fetal bovine serum, 1–5 ng / mL hydrocortisone, and a 1% (v / v) mixed solution of penicillin and streptomycin.

[0027] This invention does not have any special restrictions on the source of the raw materials used in the culture medium; conventional commercially available products can be used.

[0028] The myogenic differentiation medium described in this invention uses F12 medium as the basal medium. F12 medium has a complex composition, is rich in various trace elements, and is suitable for culturing animal cells with low serum content.

[0029] The myogenic differentiation culture medium of this invention comprises 1% to 10% by volume of fetal bovine serum, specifically 2%, 4%, or 5%. The role of fetal bovine serum in inducing the differentiation of fish preadipocytes into myocytes is to provide nutrition and necessary trace elements.

[0030] The myogenic differentiation culture medium of this invention includes RepSox at a molar concentration of 1–10 μM, specifically 4 μM, 5 μM, or 6 μM. RepSox plays a role in promoting myogenic differentiation during the induction of fish preadipocytes into myocytes.

[0031] The myogenic differentiation culture medium of this invention includes LY411575 at a molar concentration of 1–15 nM, specifically 8 nM, 10 nM, or 12 nM. LY411575 plays a role in promoting myogenic differentiation during the induction of fish preadipocyte differentiation into myocytes.

[0032] The myogenic differentiation culture medium of this invention includes vitamin C at a molar concentration of 10–100 nM, specifically 40 nM, 50 nM, or 60 nM. Vitamin C plays a role in reducing cell death during the induction of fish preadipocyte differentiation into myocytes.

[0033] The myogenic differentiation culture medium of this invention comprises a 1% (v / v) mixed solution of penicillin and streptomycin. The penicillin and streptomycin mixed solution is available from Beijing Solarbio Science & Technology Co., Ltd., catalog number P1400.

[0034] The myogenic differentiation culture medium described in this invention can induce fish precursor adipocytes to differentiate into myocytes. In this invention, the fish precursor adipocytes include those of the large yellow croaker. Specifically, the large yellow croaker precursor adipocytes are derived from the adipose tissue of the large yellow croaker peritoneum.

[0035] The differentiation maintenance medium described in this invention is based on DMEM medium.

[0036] The differentiation maintenance culture medium of this invention comprises fetal bovine serum at a volume percentage of 1% to 10%, specifically 6%, 8%, or 10%. The role of fetal bovine serum in the differentiation maintenance culture stage is to provide nutrients and necessary trace elements.

[0037] The differentiation maintenance culture medium of this invention includes hydrocortisone at a concentration of 1–5 ng / mL, specifically 1 ng / mL, 3 ng / mL, or 5 ng / mL. Hydrocortisone plays a role in enhancing the quality of myotubes during the differentiation maintenance culture stage.

[0038] The differentiation maintenance medium of this invention enhances the quality of myotubes, making them longer, thicker, and with stronger adhesion. Combined with the myogenic differentiation medium, it can maintain cell myogenic differentiation and enhance differentiation capacity.

[0039] The present invention also provides the application of the differentiation culture medium described in the above technical solution for inducing fish precursor adipocytes to differentiate into myocytes in the induction of fish precursor adipocytes into myoblastic differentiation.

[0040] The present invention also provides a method for inducing myogenic differentiation of fish precursor adipocytes based on the differentiation culture medium for inducing differentiation of fish precursor adipocytes into myocytes as described in the above technical solution, comprising the following steps:

[0041] Proliferate and culture fish precursor fat cells;

[0042] Fish progenitor fat cells, after proliferation culture, are inoculated into the myogenic differentiation medium for myogenic differentiation culture, and then replaced with the differentiation maintenance medium for differentiation maintenance culture to obtain myocytes.

[0043] This invention relates to the proliferation and culture of fish preadipocytes. The proliferation culture is performed in a cell culture dish. In a specific embodiment, the seeding density of the fish preadipocytes during the proliferation culture can be 10-1. 4 ~10 7 cells / mL, which can be 10 5 ~10 6 Specifically, it can be 10 cells / mL. 5 cells / mL or 10 6 Cells / mL. In a specific embodiment, the cell culture medium used for proliferation culture is DMEM / F12 as the basal medium, comprising 9%–11% fetal bovine serum and 1% penicillin and streptomycin mixed solution by volume, specifically comprising 10% fetal bovine serum and 1% penicillin and streptomycin mixed solution by volume. In this invention, the temperature of the proliferation culture is 25–30°C, which can be 26–28°C, specifically 26°C, 27°C, or 28°C. In a specific embodiment, the proliferation culture is carried out in a low-temperature incubator containing 5% CO2. In a specific embodiment, the proliferation culture time is 20–96 hours, and differentiation culture is carried out when the cell confluence reaches 75%–85%.

[0044] After proliferation culture, the fish precursor adipocytes proliferated in this invention are inoculated into the myoblast differentiation medium for myoblast differentiation culture, and then replaced with the differentiation maintenance medium for differentiation maintenance culture to obtain myocytes. In a specific embodiment, the myoblast differentiation culture time is 24–72 h, specifically 36 h, 48 h, or 60 h. The differentiation maintenance culture time is 18–240 h, specifically 144 h, 168 h, or 180 h. In this invention, the differentiation maintenance culture temperature is 25–30 °C, specifically 26–28 °C, specifically 26 °C, 27 °C, or 28 °C. In a specific embodiment, the differentiation maintenance culture is carried out in a low-temperature incubator containing 5% CO2.

[0045] This invention also provides a culture medium for inducing pluripotent differentiation of fish preadipocytes, comprising the differentiation culture medium for inducing fish preadipocytes to differentiate into myocytes as described in the above-mentioned technical solution, and an adipogenic differentiation culture medium. The myoblastic differentiation culture medium of this invention can induce preadipocytes to differentiate into myocytes; the differentiation maintenance culture medium can maintain and enhance the differentiation effect; the adipogenic differentiation culture medium can induce preadipocytes to differentiate into adipocytes, and the culture medium for inducing pluripotent differentiation of fish preadipocytes of this invention can achieve pluripotent differentiation of fish preadipocytes. In a specific embodiment, the adipogenic differentiation culture medium is based on DMEM culture medium, comprising 1%–20% (v / v) horse serum and 1% (v / v) a mixed solution of penicillin and streptomycin. The adipogenic differentiation culture medium of this invention comprises 1%–20% (v / v) horse serum, specifically 10%.

[0046] This invention also provides the application of the differentiation culture medium for inducing fish precursor adipocytes to differentiate into myocytes, as described in the above-mentioned technical solutions, or the culture medium for inducing pluripotent differentiation of fish precursor adipocytes, as described in the above-mentioned technical solutions, in inducing pluripotent differentiation of fish precursor adipocytes. In this invention, the pluripotent differentiation includes myoblastic differentiation and adipogenic differentiation. The culture medium for inducing pluripotent differentiation of fish precursor adipocytes of this invention can be used to culture fish precursor adipocytes and induce them to differentiate into myocytes or adipocytes. The results of the examples show that, under the induction of the myoblastic differentiation culture medium, large yellow croaker precursor adipocytes can efficiently differentiate into a large number of myotubes, with a fusion index approximately three times that of myoblasts. Under the induction of the adipogenic differentiation culture medium, 100% adipocytes can be formed.

[0047] The present invention also provides a method for inducing pluripotent differentiation of fish precursor adipocytes based on the culture medium for inducing pluripotent differentiation of fish precursor adipocytes as described in the above-described technical solution, comprising the following steps:

[0048] Proliferate and culture fish precursor fat cells;

[0049] Fish progenitor adipocytes, after proliferation culture, are inoculated into the myoblast differentiation medium for myoblast differentiation culture, and then replaced with the differentiation maintenance medium for differentiation maintenance culture to obtain myocytes; fish progenitor adipocytes, after proliferation culture, are inoculated into the adipogenic differentiation medium for adipogenic differentiation culture to obtain adipocytes.

[0050] The method for inducing pluripotent differentiation of fish precursor adipocytes according to the present invention specifically refers to a method for inducing directed pluripotent differentiation of large yellow croaker precursor adipocytes into both myocyte and adipocyte directions. In a specific embodiment, during the proliferation culture, the seeding density of fish precursor adipocytes is 10-1. 4 ~10 7 cells / mL, which can be 105 ~10 6 Specifically, it can be 10 cells / mL. 5 cells / mL or 10 6 The culture time for myoblast differentiation is 24–72 h, specifically 36 h, 48 h, or 60 h; the culture time for differentiation maintenance is 48–240 h, specifically 144 h, 168 h, or 180 h; and the culture time for adipogenic differentiation is 24–120 h, specifically 60 h, 72 h, or 84 h. In this invention, the temperatures for the proliferation culture, myoblast differentiation culture, differentiation maintenance culture, and adipogenic differentiation culture are 25–30 °C, specifically 26–28 °C, specifically 26 °C, 27 °C, or 28 °C. In a specific embodiment, the above cultures are carried out in a low-temperature incubator containing 5% CO2.

[0051] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes the culture medium for myoblastic and pluripotent differentiation of fish preadipocytes provided by the present invention, as well as the methods and applications for inducing myoblastic and pluripotent differentiation, but these should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] Myogenic differentiation medium: F12 medium was used as the basal medium, containing 2% fetal bovine serum, 5 μM RepSox, 10 nM LY411575, 50 nM vitamin C and 1% penicillin and streptomycin mixed solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number P1400).

[0054] Differentiation maintenance medium: DMEM medium was used as the basal medium, containing 8% fetal bovine serum, 2 ng / mL hydrocortisone, and a mixed solution of penicillin and streptomycin at 1% volume.

[0055] Adipogenic differentiation medium: DMEM medium as the basal medium, containing 10% horse serum and a mixed solution of 1% penicillin and streptomycin.

[0056] Cell culture medium for proliferation: DMEM / F12 medium as the base medium, containing 10% fetal bovine serum and a mixed solution of penicillin and streptomycin at 1% by volume.

[0057] Example 2

[0058] Myogenic differentiation medium: Based on F12 medium, containing 4% fetal bovine serum, 3 μM RepSox, 8 nM LY411575, 60 nM vitamin C, and a 1% penicillin and streptomycin mixed solution.

[0059] Differentiation maintenance medium: DMEM medium was used as the basal medium, containing 5% fetal bovine serum, 4 ng / mL hydrocortisone, and a mixed solution of penicillin and streptomycin at 1% volume.

[0060] Adipogenic differentiation medium: DMEM medium as the basal medium, containing 12% horse serum and a mixed solution of 1% penicillin and streptomycin;

[0061] Cell culture medium for proliferation: DMEM / F12 medium as the base medium, containing 9% fetal bovine serum and 1% penicillin and streptomycin mixed solution by volume.

[0062] Example 3

[0063] Myogenic differentiation medium: Based on F12 medium, containing 3% fetal bovine serum, 4 μM RepSox, 12 nM LY411575, 40 nM vitamin C, and a 1% penicillin and streptomycin mixed solution.

[0064] Differentiation maintenance medium: DMEM medium was used as the basal medium, containing 6% fetal bovine serum, 3 ng / mL hydrocortisone, and a mixed solution of penicillin and streptomycin at 1% volume.

[0065] Adipogenic differentiation medium: DMEM medium as the basal medium, containing 8% horse serum and a mixed solution of 1% penicillin and streptomycin by volume;

[0066] Cell culture medium for proliferation: DMEM / F12 medium as the base medium, containing 11% fetal bovine serum and 1% penicillin and streptomycin mixed solution by volume.

[0067] Example 4

[0068] Isolation and identification of preadipocytes from large yellow croaker.

[0069] Primary preadipocytes of large yellow croaker were isolated by digesting the peritoneal adipose tissue of adult large yellow croaker with trypsin at room temperature (25°C) for 30 min. Figure 1As shown in Figure A. Then, the isolated large yellow croaker precursor adipocytes were immunofluorescently stained with the precursor adipocyte-specific marker hoxc9. The results are shown in Figure A. Figure 1 B in the figure shows that approximately 99% of the cells are hoxc9+. These data indicate that the isolated cells possess the characteristics of preadipocytes, meaning that the primary cells obtained were preadipocytes.

[0070] Example 5

[0071] The large yellow croaker precursor adipocytes obtained in Example 4 were subjected to myoblast differentiation using the proliferation culture medium, myoblast differentiation medium, and differentiation maintenance medium described in Example 1.

[0072] Large yellow croaker precursor adipocytes were seeded into 48-well plates containing cell proliferation culture medium for proliferation culture, and the cell density was 1×10⁶ cells / well. 5 Cells were cultured at a density of 75%–85% at a rate of [number] cells / mL until confluence reached 75%–85%. Myogenic differentiation was initially performed on myogenic differentiation medium for 48 hours, followed by maintenance differentiation medium for 168 hours to obtain differentiated myotubes. The culture temperature was 27°C in a low-temperature incubator containing 5% CO2. Desmin staining was performed on the differentiated myotubes, and they were photographed using a laser confocal microscope. Results are as follows: Figure 2 China A and Figure 2 As shown in Figure B, preadipocytes differentiated into numerous myotubes under the induction of myoblastic differentiation medium. Their fusion index was calculated, and the results are as follows: Figure 2 As shown in Figure C, the fusion index reached approximately 32%.

[0073] Example 6

[0074] The proliferation culture medium, myogenic differentiation medium and differentiation maintenance medium of large yellow croaker myoblasts were used to induce myogenic differentiation.

[0075] Large yellow croaker myoblasts were seeded into 48-well plates containing cell proliferation culture medium for proliferation culture, and the cell density was 1×10⁶ cells / well. 5 Cells were cultured at a density of 75%–85% at a rate of [number] cells / mL until confluence reached 75%–85%. Differentiation was then initiated using myoblastic differentiation medium for 48 hours, followed by maintenance differentiation medium for 168 hours to obtain differentiated myotubes. The culture temperature was 27°C in a low-temperature incubator containing 5% CO2. The differentiated myotubes were stained with desmin and photographed using a laser confocal microscope. Results are as follows: Figure 3 As shown in Figure A, myoblasts differentiated into partial myotubes under the induction of myoblast differentiation medium. Their fusion index was calculated, and the results are as follows: Figure 3 As shown in Figure B, the fusion index reached approximately 11%.

[0076] Example 7

[0077] The large yellow croaker precursor adipocytes obtained in Example 4 were subjected to adipogenic differentiation using the cell culture medium for proliferation culture and the adipogenic differentiation medium in Example 1.

[0078] Large yellow croaker precursor adipocytes were seeded into 48-well plates containing cell proliferation culture medium for proliferation culture, and the cell density was 1×10⁶ cells / well. 5 Cells were cultured at a density of 10 cells / mL until they reached 75%–85% confluence, at which point they were cultured for differentiation. Adipogenic differentiation was performed on adipogenic differentiation medium for 72 hours. Differentiated adipocytes were stained with Nile Red and photographed using a laser confocal microscope. Results are as follows: Figure 4 As shown, almost all of the precursor adipocytes differentiated into adipocytes under the induction of adipogenic differentiation medium.

[0079] The results of the above embodiments demonstrate that the two differentiation culture media provided by this invention, one for differentiation into myotubes and the other for differentiation into adipocytes, are simple to operate and have high differentiation efficiency. Large yellow croaker precursor adipocytes can form numerous myotubes under the culture medium of myoblast differentiation and the differentiation maintenance medium, with a fusion index approximately three times that of myoblasts. Simultaneously, they can also efficiently differentiate into adipocytes under the induction of adipogenic differentiation medium. Therefore, the method provided by this invention offers a new strategy and method for multi-cell co-culture and the preparation of cell-cultured meat.

[0080] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A group of differentiation culture media for inducing the differentiation of fish precursor adipocytes into myocytes, characterized in that, The medium includes a myogenic differentiation medium and a differentiation maintenance medium. The myogenic differentiation medium uses F12 medium as the basal medium and includes 1%–10% fetal bovine serum (FBS), 1–10 μM RepSox, 1–15 nM LY411575, 10–100 nM vitamin C, and a 1% (v / v) mixed solution of penicillin and streptomycin. The differentiation maintenance medium uses DMEM medium as the basal medium and includes 1%–10% FBS, 1–5 ng / mL hydrocortisone, and a 1% (v / v) mixed solution of penicillin and streptomycin.

2. The application of the differentiation medium for inducing fish preadipocytes to differentiate into myocytes as described in claim 1 in inducing myogenic differentiation of fish preadipocytes.

3. A method for inducing myoblastic differentiation of fish precursor adipocytes based on the differentiation culture medium for inducing differentiation of fish precursor adipocytes into myocytes as described in claim 1, comprising the following steps: Proliferate and culture fish precursor fat cells; Fish progenitor fat cells, after proliferation culture, are inoculated into the myogenic differentiation medium for myogenic differentiation culture, and then replaced with the differentiation maintenance medium for differentiation maintenance culture to obtain myocytes.

4. A culture medium for inducing pluripotent differentiation of fish precursor adipocytes, characterized in that, It includes the differentiation culture medium and adipogenic differentiation culture medium for inducing fish precursor adipocytes to differentiate into myocytes as described in claim 1.

5. The culture medium according to claim 4, characterized in that, The adipogenic differentiation medium is based on DMEM medium, which includes 1% to 20% horse serum by volume and a mixed solution of penicillin and streptomycin by volume of 1%.

6. The application of the differentiation medium for inducing fish precursor adipocytes to differentiate into myocytes as described in claim 1, or the medium for inducing fish precursor adipocytes to differentiate into multiple cells as described in claim 4 or 5, in inducing fish precursor adipocytes to differentiate into multiple cells.

7. The application according to claim 6, characterized in that, The pluripotent differentiation includes myoblastic differentiation and adipogenic differentiation.

8. A method for inducing pluripotent differentiation of fish precursor adipocytes based on the culture medium for inducing pluripotent differentiation of fish precursor adipocytes as described in claim 4 or 5, comprising the following steps: Proliferate and culture fish precursor fat cells; Fish progenitor fat cells, after proliferation culture, were inoculated into the myoblast differentiation medium for myoblast differentiation culture, and then replaced with the differentiation maintenance medium for differentiation maintenance culture to obtain myoblasts. Fish progenitor adipocytes, after proliferation and culture, were inoculated into the adipogenic differentiation medium for adipogenic differentiation culture to obtain adipocytes.

9. The method according to claim 8, characterized in that, The culture time for myoblast differentiation is 24–72 h; the culture time for differentiation maintenance is 48–240 h; and the culture time for adipogenic differentiation is 24–120 h.

10. The method according to claim 8, characterized in that, During the proliferation culture, the inoculation density of fish precursor fat cells was 10. 4 ~10 7 per mL.

Images