Food-grade adipogenic differentiation inducer for cell-cultured meat

Abstract

The present invention discloses a food-grade adipogenic differentiation inducer used in cell cultured meat, and belongs to the technical field of animal cell culture and cell cultured meat. By adding insulin and vegetable oil (sunflower oil, peanut oil and tea seed oil) to the DMEM basal medium containing 2% FBS, a differentiation level and efficiency higher than that of the traditional adipogenic differentiation medium can be achieved, and adipogenic differentiation can be successfully induced to proceed smoothly. This application provides important technical support for the research on adipogenic differentiation of adipose-derived mesenchymal stem cells in the cell cultured meat industry and the efficient and economical production of high-quality fat that meets food grade requirements.

Description

Technical Field

[0001] The invention relates to a food-grade adipogenic differentiation inducer used for cell cultured meat, belonging to the technical field of animal cell culture and cell cultured meat. Background Art

[0002] Cell-cultured meat is a meat product made by culturing animal cells on a large scale in vitro, obtaining muscle tissue through three-dimensional shaping, and then processing it into food. It has the advantages of being safe, efficient, green and sustainable. It is one of the most promising solutions to the future difficulties in meat production and consumption.

[0003] As an important component of meat products, fat plays an important role in the nutrition, flavor, and taste of meat products, and is an indispensable component of cell-cultured meat. Mesenchymal stem cells are a type of adult stem cells that are widely present in body tissues and have the characteristics of self-renewal and cross-germ layer differentiation. Among them, adipose-derived mesenchymal stem cells have stronger in vitro proliferation and adipogenic differentiation abilities than other mesenchymal stem cells, and can be used as ideal seed cells for producing adipose tissue. At present, inducers such as insulin, dexamethasone, 3-isobutyl-1-methylxanthine, rosiglitazone, and indomethacin are commonly used to induce mesenchymal stem cells to differentiate into adipocytes. The combined use of insulin and other 1-3 compounds can activate specific signal transduction pathways (such as the PI3K / Akt pathway) to promote the expression of adipogenic differentiation-related transcription factors such as PPARγ and C / EBPα, so that mesenchymal stem cells differentiate into adipocytes. Furthermore, by increasing the expression of enzymes such as fatty acid synthase (FAS) and glycerol 3-phosphate acyltransferase (GPAT), the synthesis of fatty acids and triglycerides and the accumulation of lipid droplets are promoted. However, the above-mentioned inducers are expensive and not suitable for daily food use. Therefore, it is urgent to develop food-grade, low-cost inducers that promote the differentiation of mesenchymal stem cells into fat cells for the application scenarios of cell-cultured meat. Summary of the invention

[0004] The purpose of the present invention is to address the problem of food safety risks posed by the current small molecule chemical inducers that induce mesenchymal stem cells to differentiate into fat, and to provide a class of low-cost food-grade adipogenic differentiation inducers, which can be used in combination with insulin to efficiently induce mesenchymal stem cells to differentiate into fat cells, thereby ensuring efficient and economical production of fat cells that meets food safety standards for the preparation of cell-cultured meat.

[0005] The first object of the present invention is to provide a food-grade adipogenic differentiation inducer, which can be compounded with insulin to efficiently induce mesenchymal stem cells to differentiate into fat cells in vitro, and the food-grade adipogenic differentiation inducer is vegetable oil.

[0006] In one embodiment of the present invention, the vegetable oil comprises sunflower seed oil, peanut oil and / or tea seed oil.

[0007] In one embodiment of the present invention, the mesenchymal stem cells include but are not limited to adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

[0008] In one embodiment of the present invention, the sources of the mesenchymal stem cells include but are not limited to cattle, pigs, rabbits or fish.

[0009] The second object of the present invention is to provide an adipogenic differentiation medium, wherein the medium contains the adipogenic differentiation inducer.

[0010] In one embodiment of the present invention, the culture medium contains insulin, fetal bovine serum and basal culture medium.

[0011] In one embodiment of the present invention, the concentration of insulin is 50-200 nM.

[0012] In one embodiment of the present invention, the added amount of the fetal bovine serum is 2-10%.

[0013] In one embodiment of the present invention, the basal culture medium comprises DMEM, MEM or F10.

[0014] In one embodiment of the present invention, an appropriate amount of antibiotics is added to the adipogenic differentiation medium.

[0015] The third object of the present invention is to provide a method for promoting adipogenic differentiation of mesenchymal stem cells in vitro, wherein the method comprises contacting the mesenchymal stem cells with the adipogenic differentiation inducer or the adipogenic differentiation medium.

[0016] In one embodiment of the invention, the contacting is performed for 7-10 days.

[0017] In one embodiment of the present invention, the method is to first culture the mesenchymal stem cells until they adhere to the wall and the confluence reaches 80% to 90%, and then replace the culture medium with the adipogenic differentiation medium and continue to culture for 7 to 10 days.

[0018] In one embodiment of the present invention, the culture conditions are culturing at 37° C. and 5% CO 2 .

[0019] The present invention also provides the method or the application of the culture medium in the field of cell culture and cell cultured meat.

[0020] In one embodiment of the present invention, the application is to prepare cell-cultured meat using the adipogenic differentiation medium.

[0021] Beneficial effects:

[0022] 1. Existing adipogenic differentiation inducers are expensive, and the amount of fetal bovine serum added during adipogenic differentiation induction is relatively high. The present invention finds that compounding vegetable oil (sunflower oil, peanut oil and / or tea seed oil) and insulin can effectively induce mesenchymal stem cells to differentiate into mature adipocytes. The present invention optimizes the concentration of fetal bovine serum, insulin concentration and adipogenic differentiation inducer in the culture medium, and the results show that when 2% FBS is added to the culture medium, the adipogenic differentiation effect of the cells is significantly better than 5% FBS and 10% FBS; further, when the insulin concentration is 100 nM and the amount of vegetable oil (peanut oil) added is 0.2 μL / mL, the cells have the best adipogenic differentiation effect, and the unit cell adipogenic area is 1.164 times that of the traditional chemical induction method.

[0023] 2. The present invention found that in the process of inducing mesenchymal stem cells to differentiate into mature adipocytes by combining vegetable oils (sunflower oil, peanut oil and tea seed oil) and insulin, the expression levels of acetyl coenzyme A (ACE) and perilipin 1 (PLIN1) proteins that characterize adipogenic differentiation were higher than those in the control group, indicating that the formation and aggregation of lipid droplets were promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The cell viability results of mesenchymal stem cells were measured by CCK-8 after adding different plant oils to the growth medium for 3 days.

[0025] Figure 2 (A) Mesenchymal stem cells were treated with no differentiation induction, vegetable oil alone, insulin alone, traditional culture medium, and vegetable oil and insulin combination for 8 days. The adipogenic differentiation status of the cells was observed under a 40× field of view using an inverted microscope (scale bar = 20 μm). Figure 2 (B) Statistical analysis of the lipid droplet area generated by mesenchymal stem cells under different conditions.

[0026] Figure 3 (A) The culture medium was prepared with 10% FBS, 5% FBS and 2% FBS with insulin and peanut oil, and differentiation was induced for 8 days. The adipogenic differentiation status of cells was observed under an inverted microscope at 40× (scale bar = 20 μm). Figure 3 (B) Statistical analysis of the lipid droplet area generated by mesenchymal stem cells under different serum concentrations.

[0027] Figure 4 (A) Mesenchymal stem cells were treated with different concentrations of insulin / vegetable oil and differentiated for 8 days. The adipogenic differentiation state of the cells was observed under an inverted microscope at 40× (scale bar = 50 μm). Figure 4(B) and (C) are statistical analyses of the lipid droplet area generated after mesenchymal stem cells were differentiated using different concentrations of insulin / vegetable oil.

[0028] Figure 5 (A) Western blot results of acetyl-coenzyme A (ACE) and perilipin 1 (PLIN1) in mesenchymal stem cells differentiated for 8 days using traditional culture medium and sunflower oil, peanut oil or tea seed oil combined with insulin. Figure 5 (B) and (C) are the statistical results of PLIN1 and ACE expression levels after normalization with GAPDH. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below by examples.

[0030] However, the following examples are only exemplary, and the content of the present invention is not limited to the following examples. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products or can be prepared by known methods.

[0031] Unless otherwise specified, the detection methods used in the following examples are all public experimental methods.

[0032] The following examples involve culture media:

[0033] The mesenchymal stem cell growth medium is: 10 vol% fetal bovine serum, 89 vol% DMEM medium, 1 vol% penicillin-streptomycin double antibody solution, wherein the penicillin content of the penicillin-streptomycin double antibody solution is 10000 U / mL, and the streptomycin content is 10 mg / mL.

[0034] The traditional mesenchymal stem cell adipogenic differentiation medium is: 10 vol% fetal bovine serum, 1 vol% penicillin-streptomycin double antibody solution, 89 vol% DMEM medium, 100 nM insulin, 5 μM rosiglitazone, 1 μM dexamethasone, and 50 μM IBMX.

[0035] The optimized mesenchymal stem cell adipogenic differentiation medium is: 2 vol% fetal bovine serum, 1 vol% penicillin-streptomycin double antibody solution, 97 vol% DMEM medium, 100 nM insulin, and 0.2 μL / mL vegetable oil (sunflower seed oil, peanut oil, tea seed oil).

[0036] The culture conditions used in the following examples are 5% (v / v) CO2 incubator, 37°C culture.

[0037] Example 1: Detection of the effect of vegetable oil on cell viability during adipogenic differentiation

[0038] (1) Culture of mesenchymal stem cells

[0039] Fresh bovine adipose tissue was isolated and digested with collagenase type I, followed by centrifugation and filtration. Primary bovine adipose mesenchymal stem cells were obtained. The cells were plated at 5×10 4 -1×10 5 The cells were inoculated at an equal density of 100 μg / mL in a 12-well plate, and 0.2 μL / mL of different plant oils (sunflower oil, peanut oil, or tea seed oil) were added to the growth medium (1.5 mL) for 3 days. The growth medium containing plant oil was replaced every day.

[0040] (2) Effects of vegetable oils on cell viability

[0041] CCK-8 method to determine cell viability: remove the original culture medium and add 500 mL DMEM basal culture medium to each well of a 12-well plate. Add 50 µL CCK-8 reagent, mix well and incubate at 37°C for 2 h. Detect the absorbance of the sample at 450 nm using an ELISA reader. The results are as follows: Figure 1 As shown, the addition of vegetable oil did not affect cell viability.

[0042] Example 2: Effect of vegetable oil on cell adipogenic differentiation

[0043] Bovine adipose-derived mesenchymal stem cells were digested and resuspended, and 5 × 10 4 -1.5×10 5 The cells were inoculated in a 12-well plate containing growth medium and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 90%, 1 mL of traditional mesenchymal stem cell adipogenic differentiation medium was added to induce cell adipogenesis, and the medium was replaced daily. The mesenchymal stem cell adipogenic differentiation medium optimization experimental group was set up, that is, on the basis of complete medium, 0.2 μL / mL vegetable oil (sunflower oil, peanut oil or tea seed oil) and / or 100 nM insulin were added, and the medium was replaced daily.

[0044] After 8 days of differentiation, it can be observed through a microscope that the cell morphology has changed from the initial long spindle to a round shape, the cell outline has become lighter, and oil droplets with strong light transmittance have been produced, indicating that the cells have differentiated and matured. Oil Red O staining was used to examine the formation of lipid droplets in each group. Oil Red O is a strong lipid solvent and lipid dye. It is easier to combine with triglycerides to form small lipid droplets, and its binding force with phospholipids is slightly weaker. Because its solubility in intracellular lipids is greater than that in the original solvent, fat staining can be performed.

[0045] The Oil Red O staining kit was used for the experiment. The specific steps are as follows:

[0046] (1) Aspirate the culture medium and wash the plate 2-3 times with PBS to remove dead cells.

[0047] (2) Mix the stock solution and diluent in a ratio of 5:2, filter through a 0.22 μm filter membrane to remove impurities. Add an appropriate amount of dye to just cover the cell layer, stain at 37°C for 15 min, and wash with PBS 2-3 times.

[0048] (3) Stain with counterstain for 5 min and wash 2-3 times with PBS.

[0049] (4) Use an inverted microscope to observe the adipogenic differentiation status of cells under a 40× field of view.

[0050] The results are as follows Figure 2 (A) shows that fat is red and cell nuclei are dark blue. The images were processed using Image J software to compare the area of ​​lipid droplets formed per cell. Figure 2 (B) As shown. In each group, only adding vegetable oil to culture cells basically did not cause adipogenic differentiation; using sunflower oil, peanut oil or tea seed oil in combination with insulin can effectively induce mesenchymal stem cells to differentiate into mature adipocytes, and the adipogenic differentiation rate is not significantly different from the traditional chemical induction method. It was preliminarily verified that sunflower oil, peanut oil and tea seed oil have the ability to act as adipogenic differentiation inducers, and can be used to induce adipogenic differentiation of mesenchymal stem cells after being combined with insulin.

[0051] Example 3: Optimization of serum, insulin and vegetable oil concentrations

[0052] In this example, the adipogenic differentiation medium was optimized based on the mesenchymal stem cell growth medium. The serum concentration was adjusted in the adipogenic differentiation medium, and the addition amounts of insulin and vegetable oil were optimized. The serum concentrations were 10%, 5% and 2%, respectively; the insulin concentrations were 0, 50 nM, 100 nM, 200 nM and 300 nM, respectively; and the addition amounts of vegetable oil were 0, 0.1 μL / mL, 0.2 μL / mL, 0.3 μL / mL and 0.5 μL / mL, respectively.

[0053] Bovine adipose-derived mesenchymal stem cells were digested and resuspended, and 5 × 10 4 -1.5×10 5 The cells were inoculated into a 12-well plate containing complete medium and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 90%, 1 mL of differentiation medium was added to induce cell adipogenesis, and the medium was changed daily.

[0054] After 8 days of differentiation, the cells were stained with Oil Red O using the same method as in Example 2.

[0055] The results are as follows Figure 3 (A) Figure 4(A) As shown. The images were processed using Image J software to compare the lipid droplet area formed per cell. Figure 3 (B) Figure 4 (B) Figure 4 (C) As shown. According to the results of Oil Red O staining, the use of 2% FBS, 100 nM insulin and 0.2 μL / mL vegetable oil has the best adipogenic differentiation effect. When the insulin concentration reaches 300 nM and the vegetable oil concentration reaches 0.5 μL / mL, some cells lose their ability to adhere to the wall and gradually fall off during the differentiation process. Therefore, high concentrations of insulin and vegetable oil have cytotoxic effects. For cells cultured at the optimal concentration, the unit cell adipogenic differentiation area is 1.164 times that of traditional adipogenic differentiation medium. And it effectively reduces the addition of fetal bovine serum, effectively reducing production costs.

[0056] Example 4: Effects of vegetable oil on the expression of ACE and PLIN1 proteins related to cell adipogenesis

[0057] After the bovine mesenchymal stem cells were digested and resuspended, 5×10 5 -1×10 6 The cells were inoculated in a 6-well plate containing complete medium and cultured in a 37°C, 5% CO2 incubator. When the cell density reached about 90%-95%, 2 mL of traditional adipogenic differentiation medium and optimized medium were added to induce cell adipogenesis. Among them, the optimized medium was set up in sunflower oil group (0.2 μL / mL sunflower oil + 100 nM insulin), peanut oil group (0.2 μL / mL peanut oil + 100nM insulin) and tea seed oil group (0.2 μL / mL tea seed oil + 100 nM insulin), and three parallels were set up in each group. The medium was replaced daily and continued to be cultured in a 37°C, 5% CO2 incubator. On the 8th day of differentiation, the cells in each treatment group were lysed to extract proteins for Western blot detection.

[0058] The specific steps are as follows:

[0059] (1) Protein extraction and concentration determination

[0060] ① Add 100 μL / well RIPA lysis buffer to a 6-well plate and incubate on ice for 20 min.

[0061] ②Take the protein lysate from ① and quantify it using the BCA kit to obtain the loading amount data.

[0062] ③ Add loading buffer to the protein sample at a ratio of 3:1 and place in a metal bath at 100°C for 10 min.

[0063] (2) SDS-PAGE

[0064] ①Take out the precast gel, tear off the gel strip and place it in the electrophoresis slot. After clamping, pour in the electrophoresis solution. Pull out the comb and load the sample according to the BCA test results.

[0065] ② Perform electrophoresis at a constant voltage of 120V for 70 min. Adjust the voltage and electrophoresis time according to the size of the target protein.

[0066] (3) Transfer

[0067] ① Preparation of transfer solution: Glycine (MW75.07): 15.11 g; Tris (MW121.14): 3.03 g, distilled water to 850 mL, add 150 mL of methanol, fully dissolve, and precool for 1 h.

[0068] ② Pry open the gel plate, cut out the corresponding gel containing the target protein band according to the protein marker, cut the PVDF membrane according to its size, and activate the membrane with methanol for 15-30 s.

[0069] ③ Make a "sandwich" interlayer: black well plate - black sponge - white filter paper - gel - PVDF membrane - white filter paper - black sponge - transparent well plate. Be careful not to make a mistake in the order, remove bubbles, and avoid touching the PVDF membrane with your hands;

[0070] ④ Transfer the membrane at a constant current of 300 mA for 3 h in an ice bath.

[0071] (4) Immune response and imaging analysis

[0072] ① Add blocking solution to block for 1 h and wash three times with TBST.

[0073] ②Incubate it in primary antibodies against ACE, PLIN1, and GAPDH (1:1000) at 4°C overnight.

[0074] ③Recover the primary antibody and wash three times with TBST.

[0075] ④ Add the corresponding secondary antibody (1:1000) according to the species of the primary antibody and react for 3 h.

[0076] ⑤Recover the secondary antibody and wash with TBST 3 times. Make sure to wash thoroughly in this step.

[0077] ⑥Add ECL color developing solution according to the kit (A solution: B solution = 1:1) and use a chemiluminescence instrument to expose and color.

[0078] (5) Result processing

[0079] Image J software was used to analyze the Western Blot protein bands, and the grayscale values ​​of the bands were used for quantitative analysis. The relative protein expression was the ratio of the grayscale value of the target protein band to that of the internal reference protein GAPDH.

[0080] Wentern Blot results are as follows Figure 5 As shown. After 8 days of inducing cell differentiation using sunflower oil, peanut oil or tea seed oil in combination with insulin, the expression of adipogenic differentiation-related proteins perilipin 1 (PLIN1) and acetyl coenzyme A (ACE) were slightly higher than those in the control group, among which the expression of peanut oil histones was higher, 1.28 times and 1.31 times that of the control group, respectively. This result shows that the use of sunflower oil, peanut oil or tea seed oil in combination with insulin during the in vitro differentiation of mesenchymal stem cells can further improve the level of adipogenic differentiation without using chemical inducers.

[0081] The present invention claims to protect the use of food-grade raw material vegetable oil to induce adipogenic differentiation of mesenchymal stem cells. This invention provides important technical support for the production of cell-cultured meat that meets food safety requirements.

[0082] 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. An adipogenic differentiation medium, characterized in that The culture medium contains an adipogenic differentiation inducer, insulin, fetal bovine serum and a basal culture medium; the adipogenic differentiation inducer is peanut oil; The concentration of the adipogenic differentiation inducer is 0.2 μL / mL, and the concentration of the insulin is 50 to 200 nM; The added amount of the fetal bovine serum is 2%; The basal culture medium is DMEM; The adipogenic differentiation medium is used for bovine mesenchymal stem cells.

2. The adipogenic differentiation medium according to claim 1, characterized in that Antibiotics are added to the adipogenic differentiation medium.

3. A method for promoting adipogenic differentiation of mesenchymal stem cells in vitro, characterized in that: The method comprises contacting mesenchymal stem cells with the adipogenic differentiation medium of claim 1; the mesenchymal stem cells are bovine mesenchymal stem cells.

4. The method according to claim 3, characterized in that The contacting is carried out for 7-10 days.

5. The method according to claim 4, characterized in that The method comprises the following steps: firstly culturing the mesenchymal stem cells until they adhere to the wall and the confluence reaches 80% to 90%, then replacing the culture medium with the adipogenic differentiation culture medium and continuing culturing for 7 to 10 days.

6. Use of the adipogenic differentiation medium according to claim 1 or 2 or the method according to any one of claims 3 to 5 in the field of cell culture or cell cultured meat.

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