A serum-free method for inducing adipogenic transdifferentiation of fish muscle stem cells and its application

Abstract

The present invention belongs to the field of cell culture, and specifically relates to a method for serum-free induction of adipogenic transdifferentiation of fish muscle stem cells and its application. It includes a differentiation pretreatment medium, which includes DMEM / F12 basal medium, bovine blood extract, BMP‑4 and 5‑azacytidine; an adipogenic transdifferentiation medium, which includes DMEM / F12 medium, bovine blood extract, rosiglitazone, pristanic acid and lecithin. The present invention uses a special bovine blood extract and combines the pretreatment medium and the transdifferentiation medium to obtain a method for rapid adipogenic transdifferentiation of fish muscle stem cells under serum-free conditions, providing a feasible method for the subsequent large-scale production of cell cultured meat.

Description

Technical Field

[0001] The invention relates to the field of cell culture, and in particular to a serum-free method for inducing adipogenic transdifferentiation of fish muscle stem cells and an application thereof. Background Art

[0002] As an emerging technology, cell-cultured meat can produce meat by regulating cell proliferation and differentiation. It is a high-quality, safe and stable source of animal protein. Compared with traditional livestock and poultry farming, cell-cultured meat produces less greenhouse gases, occupies less arable land, and is less harmful to animals. Muscle stem cells are frequently used as seed cells in cell-cultured meat production. Due to their potential pluripotency, they can be transdifferentiated into fat cells. Fat cells can not only improve the flavor of meat, but the oil extracted from the cells also has great economic prospects.

[0003] Fish oil is rich in omega-3 polyunsaturated fatty acids, including DHA and EPA, which are of great value. DHA can promote brain development and improve memory; EPA can prevent cardiovascular and cerebrovascular diseases, such as coronary heart disease, hypertension and cerebral thrombosis, and also has anti-fatigue and anti-cancer effects. Therefore, fish oil is widely used in health care products, food, feed and clinical fields. In recent years, with the continuous improvement of residents' living standards, people's attention to nutritional health products has also increased, and fish oil health products are one of them.

[0004] Large yellow croaker ( Larimichthys crocea ) is rich in nutrition, tender in texture, and has high economic value. At present, our research team has achieved serum-free culture of muscle stem cells of large yellow croaker. The study of adipogenic transdifferentiation of muscle stem cells can not only further improve the preparation process of large yellow croaker cell cultured meat, but also lay a good foundation for the extraction and preparation of fish oil. Summary of the invention

[0005] The technical problem to be solved by the present invention is to develop a method for rapid adipogenic transdifferentiation of fish muscle stem cells under serum-free conditions, so as to make it a powerful means to solve the large-scale production of cell-cultured meat. However, there are currently no reports on serum-free induction of adipogenic transdifferentiation of fish muscle stem cells.

[0006] To solve the above problems, the present invention adopts a special bovine blood extract and combines the use of a pretreatment medium and a transdifferentiation medium to obtain a method for rapid adipogenic transdifferentiation of fish muscle stem cells under serum-free conditions, thereby providing a feasible method for the subsequent large-scale production of cell-cultured meat.

[0007] To achieve the above-mentioned purpose, the technical scheme of the present invention is: a serum-free method for inducing adipogenic transdifferentiation of fish muscle stem cells, which combines the use of a pretreatment medium and a transdifferentiation medium; wherein the pretreatment medium includes DMEM / F12 basal medium, bovine blood extract, BMP-4 and 5-azacytidine; and the transdifferentiation medium includes DMEM / F12 basal medium, bovine blood extract, rosiglitazone, pristanic acid and lecithin.

[0008] Other documents use bovine serum albumin, growth factors and other substances to maintain cell growth, but they are not suitable for the cells in the present invention. In order to reduce costs and maintain the normal physiological state of cells, the present invention uses a patent-pending bovine blood extract (application number 202311714192.8). BMP-4 can inhibit the myogenic differentiation of muscle stem cells and promote their adipogenic transdifferentiation. 5-azacytidine is a DNA methylation inhibitor. Studies have shown that 5-azacytidine can enable pluripotent stem cells to form new cell phenotypes and stabilize the expression of differentiation-related genes. In the present invention, there is no significant difference in the effects of BMP-4 and 5-azacytidine within the above concentration range. If it is lower than the above minimum dose, it cannot effectively induce cell differentiation, and if it exceeds the maximum dose, it will produce cytotoxicity.

[0009] After switching to bovine blood extract, conventional adipogenic differentiation medium will show cytotoxicity, so it is necessary to re-formulate an adipogenic differentiation medium suitable for bovine blood extract. Rosiglitazone is a PPARγ specific agonist, which promotes the accumulation of lipids in cells by inducing PPARγ to transform the cell morphology into fat-like. However, since the cells in the present invention are muscle stem cells, which are different from adipose stem cells, the efficiency of adipogenic differentiation is low. When adding rosiglitazone, we found that the cell differentiation cycle is long and the intracellular lipid accumulation is not obvious, so we choose to add lecithin, which is also called phosphatidylcholine. It is a naturally occurring diglyceride mixture of stearic acid, palmitic acid, linoleic acid and oleic acid. After adding lecithin, it can shorten the cell differentiation cycle and increase the lipid accumulation in cells. On this basis, we found that the speed of lipid accumulation in different cells is different, and this phenomenon of asynchronous differentiation leads to the loss of a large number of cells that have already completed differentiation. To solve this problem, we added pristanic acid to make the lipid accumulation of cells more uniform. The above method can quickly and effectively promote the adipogenic differentiation of muscle stem cells.

[0010] Furthermore, the pretreatment medium contains 5%-10% bovine blood extract by volume, 50ng / ml-100ng / ml of BMP-4, 50μg / ml-100μg / ml of 5-azacytidine, and the rest is DMEM / F12 basic medium. When the addition ratio of bovine blood extract is below 5%, the cells cannot adhere to the wall normally, and when it is higher than 10%, the cells will vacuolate; when BMP-4 is lower than 50 ng / ml, it has no obvious promoting effect on cell transdifferentiation, and when it is higher than 100 ng / ml, the cells will aggregate into clusters, affecting normal growth; when 5-azacytidine is lower than 50μg / ml, the expression of PPARγ is not significantly increased, and when it is higher than 100μg / ml, the expression of PPARγ is reduced.

[0011] Further, in the transdifferentiation medium, 5%-10% of bovine blood extract is added by volume, the content of rosiglitazone is 10μM-50μM, the content of pristanic acid is 20μM-50μM, the content of lecithin is 10μg / ml-50μg / ml, and the rest is DMEM / F12 basal medium. When rosiglitazone is below 10μM, the intracellular lipid accumulation is slow, and cells can quickly complete lipid accumulation in the range of 10μM-50μM. When it exceeds 50μM, rosiglitazone cannot be well dissolved in the culture medium; when lecithin is below 10μg / ml, intracellular lipid increases with the increase of lecithin, and when it exceeds 50μg / ml, cells begin to die on the second day; when pristanic acid is below 100μM, it cannot improve the problem of uneven cell lipid accumulation. When it is 100μM-500μM, it can effectively improve the uniformity of adipogenesis between cells, and when it exceeds 500μM, it will cause cell death.

[0012] Furthermore, the bovine blood extract is prepared by the following method:

[0013] (0-1) Centrifuge bovine whole blood containing sodium citrate, collect the supernatant plasma and add calcium chloride solution, and let it stand; the role of sodium citrate is to prevent the bovine whole blood from coagulation; the role of centrifugation is to remove blood cells and white blood cells from the bovine whole blood to prevent cell rupture and leakage of cell contents; the role of calcium chloride is to remove fibrinogen from the bovine blood. The calcium ions in the culture medium or nutritional supplements will cause fibrinogen to be converted into fibrin, causing the culture medium added with the bovine blood extract to coagulate.

[0014] (0-2) After standing, centrifuge again and collect the supernatant; then add H2O2 and vortex at room temperature to mix it thoroughly; the purpose of centrifugation again is to remove the fibrin that reacts with calcium chloride. After H2O2 treatment, mycoplasma and viruses in the bovine blood extract are effectively removed.

[0015] (0-3) The mixed solution was filtered through a 0.02 µm filter membrane and supplemented with bovine serum albumin to a final concentration of 500 µg / ml; the pH was adjusted to 6.8-7.2 to obtain a bovine blood extract for culture.

[0016] Furthermore, in step (0-1), sodium citrate is added in an amount of 3-10% by volume.

[0017] Furthermore, in step (0-1), bovine whole blood is centrifuged at 16-22°C and 1000 g / min for 20 min, the supernatant is collected and a 5%-15% calcium chloride solution is added by volume, and the mixture is allowed to stand for 12-48 h.

[0018] Furthermore, after standing in step (0-2), the mixture is centrifuged at 3000-12000 r / min for 10-20 min, the supernatant is collected and heated in a water bath at 56-65° C. for 0.5-1 h; then, 5%-15% H2O2 is added by volume, and the mixture is vortexed at room temperature for 10-30 s to be fully mixed.

[0019] Furthermore, the method comprises the following steps:

[0020] (1) Inoculate fish muscle stem cells into a cell culture plate so that the cell density reaches 50%;

[0021] (2) Remove the cell culture medium, rinse with PBS three times, add pretreatment culture medium for culture; replace with fresh culture medium every three days until the density of fish muscle stem cells reaches 100%; then replace with transdifferentiation culture medium, and replace with fresh culture medium every three days until obvious oil droplets can be observed in the cells. This method can enable rapid transdifferentiation of fish muscle stem cells in vitro, solving the problem of adipogenic transdifferentiation of muscle stem cells under serum-free conditions.

[0022] Application of the above method in fish muscle stem cell differentiation.

[0023] The beneficial effects of the present invention are as follows: by improving the formula, controversial ingredients in food safety are removed, the cost of cell culture to differentiation is effectively reduced, the cycle of cell adipogenic differentiation is shortened, and the cell is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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 work.

[0025] Figure 1The preparation of serum-free adipogenic differentiation pretreatment medium for fish muscle stem cells. The composition of the medium is determined by the degree of adipogenic differentiation of cells. The scale length in the figure is 20 μm. A is the case of differentiation after cells are treated with DM-1 pretreatment medium, and B is the case of differentiation after cells are treated with DM-2 pretreatment medium.

[0026] Figure 2 Preparation of serum-free adipogenic differentiation medium for fish muscle stem cells. The composition of the medium is determined by the state of cell adipogenic differentiation. The scale bar in the figure is 20 μm.

[0027] Figure 3 The effect of adding pristanic acid on the accumulation of cellular lipids. The scale bar in the figure is 20 μm. A is the control group and B is the experimental group.

[0028] Figure 4 This is the advantage of using the pretreatment medium in combination with the differentiation medium. The scale in the figure is 20 μm. A is the case of cell differentiation using only the pretreatment medium, B is the case of cell differentiation using only the transdifferentiation medium, and C is the case of cell differentiation using the differentiation medium after treatment with the pretreatment medium.

[0029] Figure 5 This is an immunofluorescence image of fish muscle stem cells after adipogenic transdifferentiation. The scale bar in the figure is 50 μm. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] 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.

[0036] Example 1: Preparation of serum-free adipogenic differentiation pretreatment medium:

[0037] Two pretreatment culture media were prepared: DM-1: 50 ng / ml-100 ng / ml BMP-4, 5%-10% bovine blood extract and DMEM / F-12 medium; DM-2: 50 ng / ml-100 ng / ml BMP-4, 50μg / ml-100μg / ml 5-azacytidine, 5%-10% bovine blood extract and DMEM / F-12 medium. Adipogenic differentiation was performed using the published adipogenic differentiation method (10% FBS, 10μg / mL insulin, 0.5μM IBMX, 0.4μM dexamethasone, 1% concentrated lipid mixture) and oil red staining was performed.

[0038] The bovine blood extract is prepared by the following steps:

[0039] (1) Centrifuge bovine whole blood containing sodium citrate, wherein sodium citrate is added at a volume ratio of 3%-10%, and the bovine whole blood is centrifuged at 16-22°C and 1000-12000 r / min for 10-30 minutes; collect the supernatant plasma and add 5%-15% calcium chloride solution, and let it stand for 12-48 hours;

[0040] (2) After standing, centrifuge at 3000-12000 r / min for 10-20 min, collect the supernatant and heat in a 56-65°C water bath for 0.5-1 h; then add 5-15% H2O2 and vortex at room temperature for 10-30 s to mix thoroughly;

[0041] (3) The mixed solution is filtered through a 0.02 μm filter membrane and supplemented with bovine serum albumin to a final concentration of 500 μg / ml; the pH is adjusted to 6.8-7.2 to obtain a bovine plasma extract for culture.

[0042] The results are as follows Figure 1 As shown, A is the case of differentiation after the cells were treated with DM-1 pretreatment medium. B is the case of differentiation after the cells were treated with DM-2 pretreatment medium. As can be seen from the figure, the adipogenic differentiation of cells is more obvious when BMF-4 and 5-azacytidine are added at the same time. Therefore, BMF-4 and 5-azacytidine are selected to pretreat muscle stem cells for adipogenic differentiation.

[0043] Example 2: Preparation of serum-free adipogenic differentiation medium for fish muscle stem cells:

[0044] Three groups of experiments were set up based on DMEM / F-12 medium containing 5%-10% bovine blood extract: (1) adding 5, 10, 20, and 50 μM rosiglitazone; (2) adding 5, 10, 20, and 50 μg / ml phosphatidylcholine; (3) adding 10, 20, and 50 μg / ml phosphatidylcholine on the basis of adding 10 μM rosiglitazone. The composition of the medium was determined by the degree of cell adipogenic transdifferentiation.

[0045] The results are as follows Figure 2 As shown, A1-4 is the cell differentiation when rosiglitazone is added alone on a serum-free basis. It can be seen that when the rosiglitazone content is 5μM, the cells show no signs of differentiation; when it is 10-50μM, the cells show signs of differentiation, and the degree of differentiation is not dependent on the rosiglitazone content. B1-4 is the cell differentiation when lecithin is added alone on a serum-free basis. It can be seen that when the lecithin content is 5-50μg / ml, the cells show signs of differentiation, and the degree of cell differentiation increases with the increase in lecithin content. C1-3 is the cell differentiation after adding 10, 20, and 50μg / ml of lecithin, respectively, on the basis of adding 10μM rosiglitazone. It can be seen from the figure that the adipogenic differentiation of cells is more obvious when these two substances are added at the same time. Therefore, 10-50 μM rosiglitazone, 10-50 μg / ml lecithin, 5-10% bovine blood extract and DMEM / F-12 are selected to participate in the adipogenic transdifferentiation of muscle stem cells.

[0046] Example 3: Effect of pristanic acid on cellular lipid accumulation:

[0047] The formula in Example 2 was used as a control group, and 20 μM to 50 μM pristanic acid was additionally added to the formula in Example 2 as an experimental group.

[0048] The results are as follows Figure 3 As shown, A is the control group and B is the experimental group. After adding pristanic acid, the lipid accumulation in the cells became more uniform and the lipid accumulation degree was significantly improved. Thus, a complete adipogenic differentiation medium was prepared.

[0049] Example 4: Advantages of using pretreatment medium in combination with differentiation medium:

[0050] The cells were subjected to differentiation experiments using the above-mentioned pretreatment medium and differentiation medium, respectively. The intracellular lipid droplets were stained with Oil Red to observe the intracellular adipogenesis, which was compared with the differentiation method given in this study.

[0051] The results are as follows Figure 4 As shown, A is the cell differentiation with only the pretreatment medium, and it can be seen that the cells cannot produce intracellular lipid droplets; B is the cell differentiated with only the transdifferentiation medium, and there are obvious lipid droplets in the cell; C is the case of differentiation with the differentiation medium after treatment with the pretreatment medium. It can be seen that the pretreatment medium has no differentiation effect and is used as a differentiation enhancer. After pretreatment, the cells produce significantly more lipid droplets than cells differentiated with only the differentiation medium. This is because in the pretreatment medium, 5-azacytidine is a DNA methylation inhibitor that can change the methylation of genes related to lipid differentiation, thereby making the promoter easier to activate, and BMP-4 can transmit differentiation signals through cell-to-cell contact after the cells reach a certain density, promoting cells to synthesize triglycerides and gene expression.

[0052] Example 5: Immunofluorescence images of adipogenic transdifferentiation of fish muscle stem cells:

[0053] After differentiation with the above-mentioned transdifferentiation medium, immunofluorescence staining of PPARγ, a marker gene for adipogenic differentiation, was performed.

[0054] The results are as follows Figure 5 As shown, when muscle stem cells are subjected to adipogenic transdifferentiation by the method of the present invention, PPARγ expression can be clearly observed.

[0055] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0056] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A serum-free method for inducing adipogenic transdifferentiation of fish muscle stem cells, characterized in that: A pretreatment medium and a transdifferentiation medium are used in combination; wherein the pretreatment medium comprises DMEM / F12 basal medium, bovine blood extract, BMP-4 and 5-azacytidine; the transdifferentiation medium comprises DMEM / F12 basal medium, bovine blood extract, rosiglitazone, pristanic acid and lecithin; the bovine blood extract in the transdifferentiation medium is added at a volume ratio of 5%-10%, the content of rosiglitazone is 10μM-50μM, the content of pristanic acid is 20μM-50μM, the content of lecithin is 10μg / ml-50μg / ml, and the rest is DMEM / F12 basal medium; The bovine blood extract is prepared by the following method: (0-1) Centrifuge bovine whole blood containing sodium citrate, collect the supernatant plasma and add calcium chloride solution, and let it stand; (0-2) After standing, centrifuge again and collect the supernatant; then add H2O2 and vortex at room temperature to mix thoroughly; (0-3) The mixed solution was filtered through a 0.02 µm filter membrane and supplemented with bovine serum albumin to a final concentration of 500 µg / ml; the pH was adjusted to 6.8-7.2 to obtain a bovine blood extract for culture.

2. The method according to claim 1, characterized in that: The pretreatment medium contains 5%-10% bovine blood extract by volume, 50 ng / ml-100 ng / ml of BMP-4, 50 μg / ml-100 μg / ml of 5-azacytidine, and the rest is DMEM / F12 basic medium.

3. The method according to claim 1, characterized in that: In step (0-1), sodium citrate is added in an amount of 3-10% by volume.

4. The method according to claim 1, characterized in that: Step (0-1) Centrifuge bovine whole blood at 16-22°C and 1000 g / min for 20 min, collect the supernatant and add 5%-15% calcium chloride solution by volume, and let stand for 12-48 h.

5. The method according to claim 1, characterized in that: Step (0-2) After standing, centrifuge at 3000-12000 r / min for 10-20 min, collect the supernatant and heat in a 56-65°C water bath for 0.5-1 h; then add 5%-15% H2O2 by volume, and vortex at room temperature for 10-30 s to mix thoroughly.

6. The method according to claim 1, characterized in that The following steps are involved: (1) Inoculate fish muscle stem cells into a cell culture plate so that the cell density reaches 50%; (2) Aspirate the cell culture medium, rinse with PBS three times, and add pretreatment medium for culture; replace the medium with fresh one every three days until the density of fish muscle stem cells reaches 100%; then replace it with transdifferentiation medium and replace it with fresh one every three days until obvious oil droplets can be observed in the cells.

7. Use of the method according to any one of claims 1 to 6 in the differentiation of fish muscle stem cells.

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