Culture medium for improving synthesis of unsaturated fatty acid in cell culture fat

Abstract

The invention relates to the technical field of biological culture, and particularly discloses a culture medium for improving synthesis of unsaturated fatty acid in cell culture fat, which is used for solving the problem that the content of unsaturated fatty acid in the culture fat is not satisfactory. The differentiation culture medium comprises the following components: a basic culture medium, a specific porcine adipose-derived mesenchymal stem cell differentiation induction factor and an unsaturated fatty acid additive. Wherein the specific induction factors comprise but are not limited to factors such as insulin, dexamethasone, indometacin, 3-isobutyl-1-methylxanthine, rosiglitazone and the like. The unsaturated fatty acid additive comprises alpha-linolenic acid, and by adding the substances into the culture medium, the differentiation efficiency of the porcine adipose-derived mesenchymal stem cells and the directional synthesis of the unsaturated fatty acid in the cells can be improved. According to the improved differential culture medium, cell differentiation and unsaturated fatty acid synthesis are effectively promoted by optimizing components and proportions, and the improved differential culture medium has an important application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioculture technology, and in particular to a culture medium that enhances the synthesis of unsaturated fatty acids in cell culture fats. Background Technology

[0002] Fat determines the flavor, texture, nutrition, and visual appearance of meat, and also significantly affects its juiciness and mouthfeel. Therefore, cell-cultured fat plays a crucial role in cell-cultured meat. In the production of cell-cultured fat, improving the differentiation efficiency of adipocytes and the content of unsaturated fatty acids is of great significance for improving the quality of cultured meat. Unsaturated fatty acids (UFAs), such as n-3 and n-6 ​​fatty acids, are essential for human health, playing important roles in preventing cardiovascular disease, anti-inflammation, and promoting brain development. However, traditional culture media often fail to effectively promote adipocyte differentiation and the synthesis of unsaturated fatty acids.

[0003] The currently accepted culture medium formulation for inducing differentiation of adipose-derived mesenchymal stem cells (ADSCs) into mature adipocytes is DMEM / F12 basal medium supplemented with 10% fetal bovine serum and 1% penicillin and antibiotics, and inducing differentiation using a combination of five inducing agents: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, indomethacin, and rosiglitazone. This medium achieves good differentiation results around day 10. However, the differentiation efficiency of ADSCs induced by this medium and the content of unsaturated fatty acids in the cultured fat are not satisfactory.

[0004] Alpha-linolenic acid (ALA) is a widely available and readily accessible n-3 PUFA (anti-alpha-linolenic acid), an essential fatty acid that the body can utilize to synthesize other types of n-3 PUFAs, such as EPA and DHA. Studies have shown that the fatty acid composition in livestock and poultry is directly influenced by the fatty acid composition of their diet. The amount and duration of PUFA addition in the diet can regulate PUFA synthesis and metabolism in livestock and poultry, determining the amount of PUFA deposited in livestock and poultry products. Flaxseed is rich in ALA, and studies have found that adding flaxseed to the diets of finishing pigs can significantly increase the UFA content in pork.

[0005] Based on this, a culture medium that enhances the synthesis of unsaturated fatty acids in cell culture fat is now provided, which can eliminate the drawbacks of existing differentiation media. Summary of the Invention

[0006] The purpose of this invention is to provide a culture medium that enhances the synthesis of unsaturated fatty acids in cell culture fat, thereby solving the problem of unsatisfactory unsaturated fatty acid content in cultured fat in existing technologies.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A culture medium for enhancing the synthesis of unsaturated fatty acids in cell culture fat comprises the following components: an inducing factor that promotes adipogenic differentiation of stem cells and an exogenous unsaturated fatty acid additive, the remainder being a basal culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin antibiotics.

[0009] Based on the above technical solutions, the present invention also provides the following optional technical solutions:

[0010] In one alternative: the inducing factor includes one or more of insulin, dexamethasone, indomethacin, 3-isobutyl-1-methylxanthine, and rosiglitazone.

[0011] In one alternative: unsaturated fatty acid additives include alpha-linolenic acid.

[0012] In one alternative: the basal medium comprises one of DMEM high glucose medium, DMEM low glucose medium, MEM medium, DMEM / F12 medium, and F10 medium;

[0013] In one alternative: the inducing factor includes one or more of insulin (5–50 μg / mL), dexamethasone (1–10 μM), indomethacin (0.1–10 mM), 3-isobutyl-1-methylxanthine (0.1–10 mM), and rosiglitazone (1–10 μM).

[0014] In one alternative: the added unsaturated fatty acid additive is 10 μM to 10 mM α-linolenic acid.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This invention can improve the differentiation efficiency of ADSCs and the directed synthesis of intracellular unsaturated fatty acids. The improved differentiation medium of this invention, through optimized composition and proportions, effectively promotes cell differentiation and unsaturated fatty acid synthesis, and has significant application prospects. Attached Figure Description

[0017] Figure 1 The diagram shows the effect of α-linolenic acid on adipogenic differentiation of ADSCs (A: Oil Red O staining; B: Quantitative analysis of adipogenic differentiation; C: Expression levels of adipogenic differentiation-related genes. ***P<0.001).

[0018] Figure 2 This is a diagram illustrating the PCA analysis of α-linolenic acid on intracellular fatty acid synthesis in ADSCs according to the present invention.

[0019] Figure 3 The diagram shows the effect of α-linolenic acid on the intracellular fatty acid content of ADSCs (A: total fatty acid content analysis, B: n-6 / n-3 PUFA ratio analysis). Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Isolation and culture of porcine adipose-derived mesenchymal stem cells:

[0022] Subcutaneous adipose tissue was aseptically collected from 3-day-old piglets. First, the subcutaneous adipose tissue was rinsed with 75% alcohol for 10 seconds, then rinsed with PBS containing 3% penicillin and antibiotics for 30 seconds. The rinsed subcutaneous adipose tissue was then cut into 1–2 mm pieces using curved scissors. 3 Small pieces of minced subcutaneous adipose tissue were mixed with collagenase digestion solution at a volume ratio of 2:1. The mixture was placed on a shaker at 120 rpm and incubated at 37°C for 90 minutes until completely digested. Growth medium was then added to neutralize the digestion. The mixture was filtered through a 100 μm filter and centrifuged at 330 g for 5 minutes, discarding the supernatant. 5 mL of erythrocyte lysis buffer was added, and the mixture was lysed on ice for 10 minutes. 30 mL of PBS was added, and the mixture was centrifuged at 330 g for 5 minutes, discarding the supernatant. 30 mL of PBS was added, and the mixture was gently resuspended and counted. PBS was added again, and the mixture was gently resuspended and centrifuged at 330 g for 5 minutes, discarding the supernatant. Growth medium was added, and the cells were gently resuspended and counted. The mixture was then centrifuged according to a 10-fold... 6 10 cells per 10 cm culture dish were seeded into cell culture dishes containing 8 mL of growth medium and incubated in an incubator at 37°C and 5% CO2.

[0023] Adipogenic differentiation induced by porcine ADSCs:

[0024] 5×10 4P3 generation ADSCs were seeded into 6-well plates containing 2 mL of proliferation medium, which was replaced every 2 days until the cell density reached 90%. The proliferation medium was discarded, and adipogenic differentiation induction medium was added, which was replaced every 2 days. Differentiation was induced until day 5, at which point the adipogenic differentiation medium was replaced with maintenance differentiation medium. Differentiation continued until day 7, at which point the maintenance differentiation medium was replaced with growth medium, and differentiation continued until day 10. During the adipogenic differentiation induction process, the experimental group ADSCs received 200 μM α-linolenic acid, while the control group received no α-linolenic acid.

[0025] Oil Red O staining and quantitative adipogenic differentiation:

[0026] (1) Remove cells that have differentiated into adipocytes on day 10, discard the culture medium, wash three times with PBS, discard the PBS, add 4% paraformaldehyde, and fix at room temperature for 15 minutes.

[0027] (2) Discard the fixative, carefully add 2 mL of PBS to rinse the cells, remove the PBS, repeat three times, add 2 mL of Oil Red O working solution, and incubate at room temperature for 15 minutes;

[0028] (3) Discard the Oil Red O working solution, add 2 mL of 60% isopropanol, rinse for 30 seconds to remove excess Oil Red O working solution from the culture dish;

[0029] (4) Discard 60% isopropanol, add 2 mL PBS to wash the cells, discard the PBS, repeat three times. After the third addition of PBS, it is not necessary to discard the PBS. Take pictures with an inverted phase contrast microscope, taking at least three fields of view for each sample;

[0030] (5) Take 1 mL of isopropanol into a 6-well plate, shake slowly on a shaker at 30 rpm for 5 minutes to extract Oil Red O from the cells, and detect the absorbance at 510 nm using an ELISA reader.

[0031] qPCR was used to detect the adipogenic differentiation ability of ADSCs;

[0032] mRNA extraction;

[0033] Add 2 mL of PBS gently to a cell culture dish, wash three times to remove as much PBS as possible, add 0.5 mL of lysis buffer, and lyse on ice for 5 minutes. Collect cells in a 1.5 mL centrifuge tube. Add 0.5 mL of ethanol, gently invert to mix, transfer to an adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube. Add 300 μL of RW elution buffer, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube. Remove the adsorption column and place it in a new 1.5 mL sterile, enzyme-free centrifuge tube. Air dry in a laminar flow hood for about 5 minutes, add 30 μL of DEPC water, and let stand for about 5 minutes until the RNA dissolves in the DEPC water. Centrifuge and discard the adsorption column. Measure the concentration and purity of the extracted RNA using a micro spectrophotometer. RNA purity is high when the OD260 / 280 value is within the range of 1.8–2.2.

[0034] mRNA is reverse transcribed into cDNA

[0035] According to the instructions of the reverse transcription kit from Nanjing Novizan Company, total RNA was reverse transcribed into cDNA. The reverse transcription reaction system was carried out in 20 μL, as shown in Table 1.

[0036] Table 1 Reverse Transcription System

[0037]

[0038] Reaction conditions: React at 37℃ for 15 minutes, inactivate the enzyme by heating at 85℃ for 5 seconds, and store at 4℃. After the reaction, remove the cDNA, mix the cDNA stock solution with DEPC water at a volume ratio of 1:3 to prepare the cDNA working solution for amplifying the target gene. Then, mix the cDNA working solution with DEPC water at a volume ratio of 1:5 for amplifying the internal control gene GAPDH, and store at -20℃ for later use.

[0039] (3) Primer sequences were designed based on the gene sequences provided by NCBI and synthesized by GenScript. The primer sequences are shown in Table 2.

[0040] Table 2 Primer sequences

[0041]

[0042] The qPCR reaction system is shown in Table 3:

[0043]

[0044] The qPCR reaction conditions were as follows: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 10 seconds and 60℃ for 30 seconds, repeated for 40 cycles; melting curve generation at 95℃ for 15 seconds, 60℃ for 1 minute, and 95℃ for 15 seconds. Each experiment was performed in triplicate, with three replicates per experiment.

[0045] Fatty acid content determination;

[0046] Samples were collected on day 10 of differentiation, flash-frozen in liquid nitrogen for 15 minutes, and then sent to Shanghai Paisennuo Biotechnology Co., Ltd. The fatty acid composition and content in the cells of the experimental group and the control group were determined by gas chromatography-tandem mass spectrometry (GC-MS).

[0047] Results and Analysis;

[0048] The effect of α-linolenic acid on adipogenic differentiation of ADSCs;

[0049] To investigate the effect of α-linolenic acid (ALA) on the differentiation capacity of ADSCs, in this study, P3 generation porcine ADSCs were induced to undergo adipogenic differentiation using differentiation media with and without ALA. Ten days after induction, Oil Red O staining was performed and Oil Red O was extracted. The results showed that the lipid quality produced by cells in the ALA group was significantly higher than that in the untreated ALA group (Ctr l group). Figure 1 AB). Subsequently, qPCR was used to detect the expression levels of adipogenic differentiation-related genes Plin1, FABP4, and Adipoq in the two groups of cells. The results showed that the expression levels of adipogenic differentiation-related genes in the ALA group were significantly higher than those in the Ctrl group (AB). Figure 1 C). In conclusion, adding α-linolenic acid to the differentiation induction medium can significantly improve the adipogenic differentiation ability of porcine ADSCs;

[0050] To investigate the effect of α-linolenic acid on fatty acid synthesis in ADSCs, adipogenic differentiation of P3 generation porcine ADSCs was induced using differentiation media with and without α-linolenic acid. Ten days after induction, the intracellular unsaturated fatty acid composition was measured. PCA results showed that the distribution within each group was relatively concentrated, and the samples were located in different regions, indicating small intra-group differences but significant inter-group differences, suggesting different fatty acid compositions in the two groups. Figure 2 Specifically, the addition of α-linolenic acid had no significant effect on the total intracellular saturated fatty acid (SFA) content. Figure 3 However, it can significantly increase the content of C10:0, C12:0, C14:0, and C20:0 fatty acids (Table 4); in addition, the addition of α-linolenic acid significantly increases the content of intracellular unsaturated fatty acids (UFA). Figure 3Examples of α-linolenic acid include C16-1, C18-2n6c, C18-3n3, C20-1(ci s-11), C20-1T, C20-2, and C20-3n3. In particular, the addition of α-linolenic acid significantly increases the intracellular content of polyunsaturated fatty acids (PUFAs). Figure 3 It significantly increased the intracellular content of n-3 PUFAs (C18-3n3 and C20-3n3) and significantly decreased the intracellular content of n-6 PUFAs (C20-4n6) and the ratio of n-6 / n-3 fatty acids. Figure 3 );

[0051] Table 4. Composition and content of fatty acids in adipose-derived mesenchymal stem cells;

[0052]

[0053]

[0054]

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium for enhancing the synthesis of unsaturated fatty acids in cell culture fat, characterized in that: It includes the following components: inducing factors that promote adipogenic differentiation of stem cells and exogenous unsaturated fatty acid additives, with the remainder being a basal culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin antibiotics.

2. The culture medium for enhancing the synthesis of unsaturated fatty acids in cultured fats according to claim 1, characterized in that, The inducing factors include one or more of insulin, dexamethasone, indomethacin, 3-isobutyl-1-methylxanthine, and rosiglitazone.

3. The culture medium for enhancing the synthesis of unsaturated fatty acids in cell culture fat according to claim 1, characterized in that, Unsaturated fatty acid additives include alpha-linolenic acid.

4. The culture medium for enhancing the synthesis of unsaturated fatty acids in cell culture fat according to claim 1, characterized in that, The basal culture medium includes one of the following: DMEM high glucose medium, DMEM low glucose medium, MEM medium, DMEM / F12 medium, and F10 medium.

5. The culture medium for enhancing the synthesis of unsaturated fatty acids in cultured fats according to claim 2, characterized in that, Inducing factors include one or more of the following: insulin at 5–50 μg / mL, dexamethasone at 1–10 μM, indomethacin at 0.1–10 mM, 3-isobutyl-1-methylxanthine at 0.1–10 mM, and rosiglitazone at 1–10 μM.

6. The culture medium for enhancing the synthesis of unsaturated fatty acids in cultured fats according to claim 3, characterized in that, The added unsaturated fatty acid additive is 10μM~10mM α-linolenic acid.

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