A high-phospholipid cell culture fish oil and preparation method thereof

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a high-phospholipid cell culture fish oil and a preparation method thereof. The method comprises the following steps: firstly, amplifying cells under 2D conditions, then inoculating the cells on microcarriers for large-scale system culture, and inducing adipogenic transdifferentiation after the cells are fully grown. The adipogenic transdifferentiation induction of large yellow croaker muscle satellite cells can be achieved after 4 days of induction. The present invention achieves rapid adipogenic transdifferentiation of large yellow croaker muscle satellite cells by adjusting the adipogenic transdifferentiation culture medium, and prepares large yellow croaker fish oil with high phospholipid content by cell culture on a large scale, laying a foundation for further obtaining cell cultured fish meat. In addition, the present invention achieves the preparation of high-phospholipid content cell culture fish oil by adding oleic acid, glucose and vitamin C and physical regulation (culture system optimization), thereby further improving the nutritional quality of the fish oil.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to high-phospholipid cell culture fish oil and a preparation method thereof. Background Art

[0002] Fish oil, extracted from fish and their processing by-products, is rich in polyunsaturated fatty acids such as EPA, DHA, linolenic acid and linoleic acid, but has a low phospholipid content. Fish oil has special preventive and therapeutic effects on a variety of diseases, such as metabolic syndrome, cardiovascular and cerebrovascular diseases, inflammation, etc. However, fish oil is easily oxidized during processing, which not only affects the flavor, color and taste, but also reduces the nutritional value. With the increasing severity of marine pollution, fish oil directly extracted from marine fish often contains a variety of pollutants, which seriously affects the processing and safety of fish oil. Therefore, it is urgent to find new methods for producing high-phospholipid fish oil.

[0003] Cell cultured meat is a disruptive food manufacturing technology that has emerged in recent years. It aims to use stem cells and tissue engineering to culture cells in vitro on a large scale for the production of agricultural products. At present, cell cultured fat is mainly used for mammalian cells, such as chicken, pig and cattle, while there are few reports on the use of fish cells. The cell types currently used to culture fat include dedifferentiated adipocytes, adipose-derived stem cells, preadipocytes, pluripotent stem cells, etc. However, their adipogenic efficiency and proliferation ability decrease with the increase in the number of passages, which greatly limits their application in cell cultured fat and large-scale production. Therefore, the selection of stable cultured meat seed cell lines (such as muscle stem cells) has become the key to producing high-quality cell fish oil. Fish cell lines are easy to spontaneously immortalize, which is their biggest advantage as seed cells. Studies have shown that somatic cells and muscle satellite cells have the potential to transdifferentiate into adipocytes or adipocytes under specific conditions. Through transdifferentiation strategies, efficient and stable cell proliferation and precise differentiation control of cellular lipid composition can be achieved. Summary of the invention

[0004] The technical problem to be solved by the present invention is that there is currently a lack of suitable fish cell adipogenic differentiation culture medium and a method for large-scale preparation of cell culture fish oil with high phospholipid content, which seriously hinders the development of cell fish meat and its derivative products.

[0005] To solve the above problems, the present invention achieves rapid adipogenic transdifferentiation of large yellow croaker muscle satellite cells by adjusting the adipogenic transdifferentiation culture medium, and prepares large yellow croaker fish oil with high phospholipid content in cell culture on a large scale, laying a foundation for further obtaining cell cultured fish meat. In addition, the present invention achieves the preparation of high phospholipid content in cell cultured fish oil by adding oleic acid, glucose and vitamin C and physical regulation (culture system optimization), further improving the nutritional quality of fish oil.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present invention is: a method for preparing high-phospholipid cell culture fish oil, comprising the following steps: using complete culture medium to expand muscle cells, and then using adipogenic transdifferentiation medium to induce adipogenic transdifferentiation to obtain high-phospholipid cell culture fish oil;

[0007] Among them, the adipogenic differentiation medium consists of 85-94% ( v / v )DMEM / F12, 3-12%( v / v ) Horse serum, 1% ( v / v ) Penicillin-Streptomycin (100X), 1% ( v / v ) non-essential amino acids, 1% ( v / v ) GlutaMAX™, 0.25-1.5 μM dexamethasone, 5-25 μM rosiglitazone, 2.5-15 μg / mL insulin, 25-125 μM indomethacin, 125-750 μM IBMX, 50-500 μM oleic acid, 1-5 mM L-glutamine, 50-250 μM vitamin C, and 20-100 μM glucose; the differentiation time is 4-6 days.

[0008] Among them, DMEM / F12 is the basic culture medium, which provides basic nutrition for cells; horse serum contains serum proteins, growth factors, amino acids, sugars, inorganic salts, etc., which can provide basic nutrition and energy for cells. Horse serum is not as nutritious as fetal bovine serum, and horse serum contains some hormones and factors that can guide cells to differentiate into specific cell types; penicillin-streptomycin (100X) can prevent microbial contamination; non-essential amino acids provide nutrition for cell proliferation and differentiation; GlutaMAX™ and L-glutamine can provide energy and nitrogen sources for cells and improve cell health; dexamethasone is a synthetic glucocorticoid that promotes C / EBPs expression; rosiglitazone is an insulin sensitizer that can activate PPARγ ; Insulin can activate CREB and a series of downstream transcriptional activators, and promote the synthesis of triglycerides from free fatty acids; Indomethacin can block the differentiation of cells into osteoclasts and osteoblasts, and is a PPARγ activator; IBMX can activate the cAMP-dependent protein kinase pathway to enhance the expression of C / EBPs and PPARγ; Oleic acid can serve as a natural activating ligand for PPARγ; Vitamin C can upregulate the transcriptional levels of adipogenesis-related genes such as PPARγ, retinoic acid X receptor α, and lipoprotein esterase, and can also promote collagen synthesis, upregulate the expression of LPL and GPDH, downregulate the expression of adiponectin, promote triglyceride accumulation, and accelerate the process of cell differentiation; Glucose provides energy and carbon source for cells through glycolysis.

[0009] Furthermore, the muscle cells are large yellow croaker muscle satellite cells. Compared with other cells used for adipogenesis, such as dedifferentiated adipocytes, adipose-derived stem cells, preadipocytes, and pluripotent stem cells, muscle satellite cells are easier to obtain, and their adipogenic efficiency and proliferation capacity will not decrease with the increase in the number of passages.

[0010] Furthermore, the complete culture medium is a DMEM / F12 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X). DMEM / F12 is a mixture of DMEM and F-12 in a 1:1 ratio. It combines the advantages of the two culture media, making the culture medium more comprehensive and providing basic energy and nutrition for cell growth and proliferation. Fetal bovine serum provides various growth factors, hormones, proteins, amino acids, vitamins, minerals and trace elements required for cell growth, which can promote cell proliferation, differentiation and maintain normal physiological functions. Cells can maintain normal cell morphology and proliferation efficiency in 10% fetal bovine serum. If the concentration of fetal bovine serum is too low, the cell proliferation rate will decrease, the morphology will deteriorate, and cavitation will occur easily. If the concentration is too high, the cost will increase and it will easily promote cell differentiation.

[0011] Furthermore, 2D adipogenic differentiation includes the following steps: inoculating muscle cells into a 6-well plate, adding complete culture medium, and replacing it with adipogenic differentiation medium after the cells are fully grown, and performing differentiation.

[0012] Furthermore, 3D adipogenic transdifferentiation includes the following steps:

[0013] (1) Add the microcarriers to a culture bottle with an internal impeller, add basal culture medium, and stir to swell;

[0014] (2) After standing, discard the upper basal culture medium, add the basal culture medium and stir again, and after standing, discard the upper basal culture medium;

[0015] (3) Add complete culture medium, inoculate cells at a certain density, and stir overnight; on the second day, add culture medium to 500 mL, adjust the stirring speed to a constant level, and change the medium half every other day;

[0016] (4) After the cells have grown all over the microcarriers, discard the complete medium and add the transdifferentiation medium. Change the medium every other day.

[0017] (5) Let the cells stand and discard the differentiation medium. Collect the samples, wash twice with PBS, add cell lysis buffer, centrifuge, and collect the supernatant.

[0018] Furthermore, in step (1), the addition ratio of the microcarrier is 2-5 mg / mL based on the volume of the total culture system; the swelling time is 12 h. Swelling the microcarrier in advance is beneficial to cell attachment and proliferation.

[0019] Furthermore, the basal culture medium in step (1) and step (2) is DMEM / F12 culture medium, and the addition amount is 50-100 mL; the stirring speed is 40 rpm. Substances that may be residual in the microcarriers and have an impact on the cells are washed away as much as possible. Keep it consistent with the basal culture medium in the complete culture medium to prevent the introduction of exogenous impurities.

[0020] Furthermore, in step (3), 200 mL of complete culture medium is added, and the density of inoculating muscle cells is 50,000-200,000 / mL. If the cell inoculation density is too low, the cell proliferation is slow, the state is poor, and the culture time is long; if the inoculation density is too high, the cell growth is restricted, the competition between cells is intensified, the cell growth and proliferation are affected, and the utilization efficiency of the microcarrier is reduced.

[0021] Furthermore, in step (3), the gradient stirring program is 0 rpm, 2 h, 40 rpm, 5 min, and cycle overnight; the constant stirring speed is 40 rpm. The attachment time of muscle satellite cells is about 2 h, and standing for 2 h can promote the attachment of cells to the microcarriers; stirring at 40 rpm has basically no shear stress damage to the cells, and stirring can make the cells attach more evenly.

[0022] Furthermore, in step (5), the cell lysis time is 2-3 min at room temperature; the centrifugation time is 5 min at 5000 rpm. The microcarriers are treated in the lysis solution for 2-3 min to separate the cells from the microcarriers. The microcarriers are then precipitated to the bottom by centrifugation, while the cells remain in the supernatant for easy collection.

[0023] A high-phospholipid cell culture fish oil prepared by the above method. The total lipid content in the obtained cell culture fish oil is 768.8 mg / g, of which triglycerides account for 46.35% and phospholipids account for 52.38%, while the total lipid content in the actual extracted fish oil is 680.37 mg / g, of which phospholipids account for 29.17% and triglycerides account for 66.75%. Compared with the two, the phospholipid content in the cell culture fish oil increased by 1.80 times.

[0024] The beneficial effects of the present invention are:

[0025] (1) Rapid adipogenic transdifferentiation of large yellow croaker muscle satellite cells can be achieved by optimizing the transdifferentiation culture medium, and large-scale cell culture fish oil preparation can be achieved using microcarriers, laying the foundation for the subsequent preparation of cell culture fat and cell culture meat.

[0026] (2) The present invention achieves the preparation of cell cultured fish oil with high phospholipid content by adding oleic acid, glucose and vitamin C as well as physical regulation (culture system optimization), thereby further improving the nutritional quality of the fish oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Comparative Example 1, 2D adipogenic transdifferentiation effect diagram of large yellow croaker muscle satellite cells (before optimization).

[0028] Figure 2 :The effect of adding different concentrations of oleic acid (50, 100, 200 μM) on adipogenic transdifferentiation in Example 1; wherein A is a graph showing the effect of adding 50 μM oleic acid to induce adipogenic transdifferentiation for 7 days; B is a graph showing the effect of adding 100 μM oleic acid to induce adipogenic transdifferentiation for 7 days; C is a graph showing the effect of adding 150 μM oleic acid to induce adipogenic transdifferentiation for 7 days.

[0029] Figure 3 : The effect diagram of adding 100 μM oleic acid and 50 μM glucose to induce adipogenic transdifferentiation for 7 days in Example 1.

[0030] Figure 4 : The effect diagram of adding 100 μM oleic acid, 50 μM glucose and 100 μM vitamin C in Example 1 to induce adipogenic transdifferentiation for 7 days.

[0031] Figure 5 : Graph showing the effect of 2D adipogenic transdifferentiation of large yellow croaker muscle satellite cells in Example 1 (after optimization).

[0032] Figure 6 : Diagram of the proliferation effect of 3D culture of large yellow croaker muscle satellite cells in Example 2.

[0033] Figure 7 : Schematic diagram of the 3D adipogenic transdifferentiation effect of large yellow croaker muscle satellite cells in Example 2.

[0034] Figure 8 : Analysis of lipid composition of 2D adipogenic transdifferentiation samples of large yellow croaker muscle satellite cells in Comparative Example 1.

[0035] Fig. 9 : Analysis of lipid composition of 2D adipogenic transdifferentiation samples of large yellow croaker muscle satellite cells in Example 1.

[0036] Fig.10 : Analysis of lipid composition of 3D adipogenic transdifferentiation samples of large yellow croaker muscle satellite cells in Example 2.

[0037] Fig.11 : Comparative Example 2: Analysis of lipid composition of abdominal fat of large yellow croaker. DETAILED DESCRIPTION

[0038] The following description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0039] The following examples can be understood as a part of the local structure or method of the present invention alone, and can also be understood as the combination of the examples to explain the connotation of the larger structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are obtained from commercial sales.

[0040] Comparative Example 1:

[0041] Large yellow croaker muscle satellite cells were inoculated into 6-well plates, and complete medium (DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X)) was added. After the cells were confluent, they were replaced with adipogenic differentiation medium (consisting of 87% DMEM / F12, 10% fetal bovine serum, 1% penicillin-streptomycin (100X), 1 μM dexamethasone, 20 μM rosiglitazone, 10 μg / mL insulin, 100 μM indomethacin, 500 μM IBMX, 2% lipid mixture, 2 mM L-glutamine, 100 μM vitamin C, and 25 μM glucose). After 8 days of differentiation, oil red staining was performed to observe the adipogenic differentiation effect. After differentiation, the cells were washed twice with PBS, and cell lysis buffer was added. The cells were lysed at room temperature for 3 min, and the lysate was collected for lipid composition determination.

[0042] like Figure 1 As shown in the figure, lipid droplets can be observed in the muscle satellite cells of yellow croaker after adipogenic differentiation by oil red staining, indicating that the adipogenic differentiation of muscle satellite cells was successfully induced, but the differentiation cycle was long and the efficiency was not high. Figure 8 As shown, the total lipid content in the obtained cell culture fish oil was 557.57 mg / g, of which TG content accounted for 60.32% and PL content accounted for 37.40%.

[0043] Embodiment 1:

[0044] Large yellow croaker muscle satellite cells were seeded into 6-well plates, and complete medium (DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X)) was added. After the cells were confluent, they were replaced with adipogenic differentiation medium (consisting of 87% DMEM / F12, 10% horse serum, 1% penicillin-streptomycin (100X), 1% non-essential amino acids, 1% GlutaMAX™, 1 μM dexamethasone, 20 μM rosiglitazone, 10 μg / mL insulin, 100 μM indomethacin, 500 μM IBMX, 2 mM L-glutamine). Oleic acid, vitamin C, and glucose were added to compare the adipogenic differentiation effect of large yellow croaker muscle satellite cells. After differentiation, the cells were washed twice with PBS, and cell lysis buffer was added. The cells were lysed at room temperature for 3 min, and the lysate was collected for lipid composition determination.

[0045] The present invention achieves the preparation of high phospholipid content cell culture fish oil by adding oleic acid, glucose and vitamin C and physical regulation (culture system optimization). Figure 2 As shown in A, B, and C, different concentrations of oleic acid were added to induce adipogenic differentiation for 7 days. The higher the concentration of oleic acid added, the more obvious the adipogenic differentiation effect was and the more lipids were produced. However, high concentrations of oleic acid led to apoptosis of most cells in the late differentiation stage. Adding 50 μM glucose to induce adipogenic differentiation for 7 days on the basis of 100 μM oleic acid can achieve the same differentiation effect as adding 200 μM oleic acid. Figure 3 As shown. Figure 4 As shown, compared with the group without vitamin C addition ( Figure 3 ), additionally add 100 μM vitamin C, induce transdifferentiation for 5 days to achieve a good differentiation effect. After optimization, the muscle satellite cells of yellow croaker can be rapidly transdifferentiated into adipocytes after 5-6 days of differentiation, with high differentiation efficiency and high lipid content. The differentiated cells were observed by oil red staining. Although many cells fell off and floated during the staining process, the remaining cells still showed that they contained a large number of lipid droplets, such as Figure 5 As shown. Fig. 9 As shown, after the transdifferentiation medium was optimized, the cells were differentiated under 2D conditions for 6 days, and the total lipid content in the obtained cell culture fish oil was 580.70 mg / g, of which TG content accounted for 50.79% and PL content accounted for 47.29%. Compared with before optimization, the total lipid content increased, the TG proportion decreased, and the PL proportion increased.

[0046] α-Glycerol phosphate is the key raw material for the synthesis of phospholipids, but fat and muscle cells lack glycerol kinase and cannot directly use glycerol to produce α-Glycerol phosphate. Glucose is the starting material for glycolysis, and its intermediate product dihydroxyacetone phosphate can produce α-Glycerol phosphate under the action of α-Glycerol phosphate dehydrogenase, thereby providing raw materials for phospholipid synthesis. In addition, acetyl coenzyme A, an intermediate product of glycolysis, is also a precursor for the synthesis of fatty acids, providing a carbon source for lipid production. High sugar can stimulate the expression of adipogenesis-related genes such as C / EBPα, ACC, AdipoQ, etc. in muscle satellite cells, regulate the de novo synthesis of fatty acids in cells, and promote lipid accumulation. Oleic acid is a monounsaturated ω-9 fatty acid that can promote the expression of genes related to fat synthesis such as PPARγ, C / EBPα, SREBP1, etc., and can also be used for the synthesis of phospholipids to provide a source of fatty acids. Vitamin C can also increase the expression of adipogenesis-related genes in the early and middle stages of differentiation, thereby promoting the process of cell differentiation. Vitamin C inhibits glycolysis by targeting the activity of GAPDH, but its metabolite 2,3-diketo-L-gulonic acid (DKG) can activate glycolysis. DKG activates an atypical tricarboxylic acid cycle characterized by an increase in succinate, fumarate, and malate, prompting a shift in cellular metabolism from oxidative phosphorylation to glycolysis.

[0047] Comparative Example 2:

[0048] About 1 kg of large yellow croaker was taken, anesthetized and killed, and the abdominal fat tissue was taken after laparotomy. The fat tissue was homogenized, and an appropriate amount of the homogenate was weighed, and the total lipid was extracted by the Folch method, and the lipid composition was determined by UPLC-QE-MS.

[0049] like Fig.11 As shown in the figure, the total lipid content in the abdominal fat of large yellow croaker was 690.37 mg / mL, of which triglycerides (TG) accounted for 66.75% and phospholipids (PL) accounted for 20.17%.

[0050] Embodiment 2:

[0051] 1 g of microcarriers were dissolved in 200 mL of DMEM / F12 basal medium and transferred to a 500 mL impeller culture flask, stirred at 40 rpm for 12 h; after standing, the upper basal medium was discarded, and 100 mL of basal medium was added, stirred at 40 rpm for 5 min, and the upper basal medium was discarded after standing; 200 mL of complete medium (DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X)) was added, 50 million cells were inoculated, and the culture was allowed to stand for 2 h, 40 rpm for 5 min, and gradient circulation was performed overnight; the next day, the culture medium was supplemented to 500 mL, and stirred at a constant speed of 40 rpm; after the cells were fully grown, the complete medium was removed after standing, and the transdifferentiation medium (consisting of 87% DMEM / F12, 10% horse serum, 1% penicillin-streptomycin (100X), 1% non-essential amino acids, 1% GlutaMAX™, 1 μM dexamethasone, 20 μM rosiglitazone, 10 The cells were induced to differentiate for 4 days with a medium containing 100 μg / mL insulin, 100 μM indomethacin, 500 μM IBMX, 100 μM oleic acid, 2 mM L-glutamine, 100 μM vitamin C, and 50 μM glucose. The cells were allowed to stand for 4 days and the differentiation medium was discarded. The samples were collected and washed twice with PBS. Cell lysis buffer was added and the cells were lysed at room temperature for 3 min. The cells were centrifuged at 5000 rpm for 5 min and the supernatant was collected for lipid composition determination.

[0052] like Figure 6 As shown, large yellow croaker muscle satellite cells attached and proliferated on the microcarriers. After about 6 days of culture, the cells were fully grown and the cell density could reach 700,000 / mL.

[0053] like Figure 7 As shown, large yellow croaker muscle satellite cells can achieve a good differentiation effect after 3-4 days of transdifferentiation induction on microcarriers, which is two days shorter than the differentiation time under 2D culture conditions, and the differentiation efficiency is further improved.

[0054] like Figure 8 As shown, before the optimization of the adipogenic transdifferentiation method (i.e., the method described in Comparative Example 1), the cells were differentiated under 2D conditions for 6 days, and the total lipid content in the obtained cell culture fish oil was 557.57 mg / g, of which the TG content accounted for 60.32% and the PL content accounted for 37.40%. Fig.10 As shown in the figure, after the optimization of the transdifferentiation medium, the cells were differentiated under 3D conditions for 4 days, and the total lipid content in the obtained cell culture fish oil was 768.8 mg / g, of which TG accounted for 46.35% and PL accounted for 52.38%. After optimization, the total lipid content and PL content were greatly increased. During the transdifferentiation induction process, it was found that under different culture systems, namely 2D and 3D conditions, the same transdifferentiation medium was used to induce cell adipogenic transdifferentiation, and the induction efficiency and lipid composition of the obtained cell culture fish oil were very different. Fig. 9 As shown, after 6 days of differentiation under 2D conditions, the total lipid content of the obtained cell culture fish oil was 580.70 mg / g, of which TG content accounted for 50.79% and PL content accounted for 47.29%. The total lipid and PL contents were lower than those of the cell culture fish oil obtained under 3D conditions ( Fig.10 ). By comparing the three, it can be found that the cell transdifferentiation effect is better under 3D conditions, and the PL content is higher, such as Fig.10 As shown in the figure, after the transdifferentiation medium was optimized, the cells were differentiated under 3D conditions for 4 days, and the total lipid content in the obtained cell culture fish oil was 768.8 mg / g, of which TG accounted for 46.35% and PL accounted for 52.38%, which may be related to the differentiation culture system and the microcarrier material (gelatin) used. The lipid composition of the fish oil cultured by large yellow croaker cells obtained by 2D or 3D culture is quite different from the fish oil extracted from the abdominal fat of large yellow croaker. The total lipid content of cell fish oil is higher than that of the actual extracted fish oil, and triglycerides (TG) are significantly lower than those of the actual extracted fish oil, but the phospholipid PL content is significantly higher than that of the actual extracted fish oil.

[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 method for preparing high phospholipid cell culture fish oil, characterized in that The following steps are involved: The muscle cells were expanded using complete medium, and then induced into adipogenic differentiation using adipogenic differentiation medium to obtain high-phospholipid cell culture fish oil; The complete culture medium was DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100X); The adipogenic differentiation medium consists of 85-94% DMEM / F12, 3-12% horse serum, 1% penicillin-streptomycin (100X), 1% non-essential amino acids, 1% GlutaMAX™, 0.25-1.5 μM dexamethasone, 5-25 μM rosiglitazone, 2.5-15 μg / mL insulin, 25-125 μM indomethacin, 125-750 μM IBMX, 50-500 μM oleic acid, 1-5 mM L-glutamine, 50-250 μM vitamin C, and 20-100 μM glucose; the differentiation time is 4-6 days.

2. The preparation method according to claim 1, characterized in that: The muscle cells are large yellow croaker muscle satellite cells.

3. The preparation method according to claim 1, characterized in that 2D adipogenic differentiation includes the following steps: inoculate muscle cells into a 6-well plate, add complete culture medium, and replace with adipogenic differentiation medium after the cells are fully grown for differentiation.

4. The preparation method according to claim 1, characterized in that 3D adipogenic transdifferentiation includes the following steps: (1) Add the microcarriers to a culture bottle with an internal impeller, add basal culture medium, and stir to swell; (2) After standing, discard the upper basal culture medium, add the basal culture medium and stir again, and after standing, discard the upper basal culture medium; (3) Add complete culture medium, inoculate cells, and stir overnight; on the second day, add culture medium to 500 mL, adjust stirring to constant speed, and change the medium half every other day; (4) After the cells have grown all over the microcarriers, discard the complete medium and add the transdifferentiation medium. Change the medium every other day. (5) Let the cells stand and discard the transdifferentiation medium. Collect the samples, wash twice with PBS, add cell lysis buffer, centrifuge, and collect the supernatant.

5. The preparation method according to claim 4, characterized in that: The addition ratio of the microcarrier in step (1) is 2-5 mg / mL based on the volume of the total culture system; the swelling time is 12 h; the basic culture medium in steps (1) and (2) is DMEM / F12 culture medium, and the stirring speed is 40 rpm.

6. The preparation method according to claim 4, characterized in that: Step (3) Add 200 mL of complete culture medium and inoculate muscle cells at a density of 50,000 to 200,000 cells / mL.

7. The preparation method according to claim 4, characterized in that: The gradient stirring program in step (3) was 0 rpm, 2 h, 40 rpm, 5 min, and cycled overnight; the constant stirring speed was 40 rpm.

8. The preparation method according to claim 4, characterized in that: In step (5), the cell lysis time is 2-3 min at room temperature; the centrifugation time is 5 min at 5000 rpm.

9. A high-phospholipid cell culture fish oil prepared by the preparation method according to any one of claims 1 to 8.

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