A low-serum culture medium and its application in the preparation of cell-cultured Pseudosciaena crocea

Abstract

The present invention discloses a low - serum medium and its application in the preparation of cultured meat from Larimichthys crocea cells, which is also applicable to other marine or freshwater fish, belonging to the field of bioengineering technology. The low - serum medium comprises a basal medium, fetal bovine serum accounting for 30 - 80 μL / mL, and exogenous additives. The exogenous additives include human serum albumin, ascorbic acid, basic fibroblast growth factor polypeptide, human holo - transferrin, lysophosphatidic acid, and human recombinant IGF - 1. Based on the DMEM / F12 medium, the low - serum medium provided by the present invention adds six substances that promote cell growth, reduces the dependence of satellite cells on fetal bovine serum, effectively reduces the usage amount of fetal bovine serum, and saves the culture cost. Compared with the general - type medium (DMEM high - glucose, adding 10% FBS), the low - serum medium provided by the present invention has an equivalent effect on the maintenance and proliferation ability of cell stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and specifically provides a low-serum medium for culturing large yellow croaker muscle satellite cells and its application in cultured meat. Background Art

[0002] With the development of the economic society, the demand for meat by humans is increasing. However, the traditional meat production method has been difficult to meet the needs of humans, and the traditional meat production method requires a large amount of grain and water resources, and will also cause serious environmental pollution. Cultured meat is produced by extracting animal stem cells or tissues that can proliferate efficiently and placing them in a culture dish to reproduce, and then differentiating into the original fibers of muscle tissue. Cultured meat involves processes such as the isolation and purification of stem cells, and the proliferation and differentiation of cells. During the production process, only a culture medium, a certain temperature and humidity, carbon dioxide, etc. are required to provide the nutrients and the environment required for growth, which will not cause environmental pollution, does not require the feed and water required in traditional meat production, and occupies a small area, saving space costs. It is the main research and development direction of future artificial meat.

[0003] In traditional cell culture, a certain amount of fetal bovine serum needs to be added to provide the nutritional components and biological factors required for cell attachment, proliferation, and growth maintenance. However, fetal bovine serum has certain disadvantages, mainly manifested in: (1) high price; (2) the components in the serum are uncertain; (3) it may contain risk contaminants such as fungi, bacteria, viruses, mycoplasma, etc.; (4) large batch-to-batch differences, and the sera obtained at different times and under different circumstances are inconsistent, resulting in instability in the quality of the cultured products.

[0004] Serum-free medium is a synthetic medium that can maintain the long-term growth and proliferation of cells in vitro without adding serum. For example, patent document CN 112210525 A discloses a serum-free medium, and all exogenous added components are chemically synthesized or biosynthesized. Since the artificial synthesis components are clear, the product batch is stable, the repeatability of cell culture is high, and at the same time, the risk of microbial contamination such as exogenous and endogenous viruses, bacteria, and mycoplasma brought about by the use of animal serum is reduced. Moreover, industrial production ensures sufficient supply, which is also beneficial to the downstream purification of biological products.

[0005] Although there are currently commercial serum-free media, their prices are also very expensive and not suitable for large-scale industrial applications. Therefore, how to reduce costs during large-scale culture is an urgent problem to be solved.

[0006] The large yellow croaker (Larimichthys crocea) is a fish species in the family Sciaenidae and the genus Larimichthys. The large yellow croaker has high economic value, with tender flesh and rich in protein, making it an excellent fresh food. However, due to overfishing, its resources have rapidly declined. Therefore, developing cell culture technology for large yellow croaker to produce cultured meat to meet human needs is a means to solve the resource decline, and providing a culture medium that can rapidly proliferate large yellow croaker muscle satellite cells at low cost is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] The object of the present invention is to provide a low-serum culture medium that can rapidly proliferate large yellow croaker muscle satellite cells and significantly reduce costs, providing a feasible method for subsequent large-scale production and the application of cultured meat.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a low-serum culture medium, which includes a basal medium, fetal bovine serum accounting for 30 - 80 μL / mL, and exogenous additives. The basal medium is DMEM high-glucose medium, DMEM / F12 medium or F10 medium. The exogenous additives include human serum albumin, ascorbic acid, basic fibroblast growth factor polypeptide, human holotransferrin, lysophosphatidic acid, and human recombinant IGF-1. The concentrations of each component in the culture medium are 0.1 - 8 mg / mL, 14.09 - 35.23 μg / mL, 1 - 10 ng / mL, 4 - 12 μg / mL, 0.23 - 0.69 μg / mL, and 5 - 15 ng / mL respectively.

[0010] Among the above exogenous additives, human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions, and many therapeutic molecules, etc.; ascorbic acid plays a regulatory role in cellular redox metabolism and can reduce cell death; basic fibroblast growth factor polypeptide is a member of the fibroblast growth factor family. Members of the FGF family bind to heparin and have extensive mitogenic and angiogenic activities; transferrin is a major iron-binding and cell delivery molecule present in serum. A serum-free cell culture system requires a form of iron delivery, and transferrin is the preferred form of iron delivery. Human holotransferrin is a high-affinity transferrin and can be used with a variety of cell types; lysophosphatidic acid has various effects on cell growth, proliferation, differentiation, and intracellular information transmission, and plays an important role in maintaining the normal physiological functions of the body and participating in the occurrence and development of various pathological processes; human recombinant IGF-1 is homologous to proinsulin in structure and plays a key role in cell proliferation.

[0011] The research of the present invention shows that when the low-serum medium is used for culturing Pseudosciaena crocea muscle satellite cells, normal cell growth can be achieved during the growth cycle, and the obtained cells have normal morphology, meeting the requirements of cell culture. Compared with the general medium (DMEM high glucose, 100 μL / mL FBS), the low-serum medium provided by the present invention reduces the serum dosage, saves the culture cost, and is suitable for large-scale industrial application.

[0012] In the present invention, each component in the above medium is a commercially available product.

[0013] Preferably, the basal medium is DMEM / F12 medium. Research shows that compared with DMEM high glucose medium and F10 medium, the medium composed of DMEM / F12 medium and the above exogenous added components is more conducive to the proliferation of Pseudosciaena crocea muscle satellite cells.

[0014] Preferably, the proportion of fetal bovine serum is 50 μL / mL. Reducing the addition amount of fetal bovine serum can reduce the cell culture cost. In the present invention, exogenous added components are added to supplement the nutrients and biological factors required for cell adhesion, proliferation, and growth maintenance.

[0015] Preferably, the concentrations of each component in the exogenous added components in the medium are 0.1 mg / mL, 35.23 μg / mL, 10 ng / mL, 4 μg / mL, 0.46 μg / mL, and 15 ng / mL respectively.

[0016] The present invention also provides the application of the low-serum medium in culturing animal stem cells or animal muscle cells in vitro.

[0017] Furthermore, the animal can be Pseudosciaena crocea or other marine fish, or can also be freshwater fish. Stem cells are isolated from adult or juvenile Pseudosciaena crocea for cell culture. Further, the low-serum medium is used as the medium for culturing Pseudosciaena crocea muscle satellite cells. Specifically, the Pseudosciaena crocea muscle satellite cells are obtained by isolation from the epaxial muscle of Pseudosciaena crocea.

[0018] Preferably, the seeding amount of cells during culture is 10 3 ~10 5 cells / mL of the low-serum medium.

[0019] Preferably, the culture conditions are culturing at 25 °C to 30 °C for 20 to 100 h.

[0020] The present invention also provides the application of the low-serum medium in the preparation of cultured meat. The application includes: inoculating muscle stem cells in the low-serum medium for culture.

[0021] Specifically, the present invention provides a method for preparing cultured meat from large yellow croaker, and the method includes: inoculating large yellow croaker muscle satellite cells into the low-serum medium for culture.

[0022] Preferably, the inoculation amount of cells during culture is 1×10 4 ~2×10 4 cells / mL of the low-serum medium.

[0023] Preferably, the culture conditions are to culture at 25°C to 27°C for 24 to 72 hours, and change the medium about every 4 hours. The low-serum medium can enable the rapid proliferation of large yellow croaker muscle satellite cells.

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

[0025] (1) Based on the DMEM / F12 medium, the low-serum medium provided by the present invention adds six substances that promote cell growth, reduces the dependence of muscle satellite cells on fetal bovine serum, can effectively reduce the usage amount of fetal bovine serum, and saves costs.

[0026] (2) Compared with the general muscle satellite cell medium (DMEM high glucose, adding 100 μL / mL FBS), the low-serum medium provided by the present invention has an equivalent effect on maintaining and proliferating the cell stem cells. Description of the Drawings

[0027] Figure 1 It shows the cell viability after culturing large yellow croaker muscle satellite cells with basic exogenous added components in a serum-free medium for three days. Among them, (A) is cultured under different concentrations of human serum albumin (HSA), and the concentrations of the other two added components are fixed, which are 35.23 μg / mL ascorbic acid and 10 ng / mL basic fibroblast growth factor polypeptide respectively; (B) is cultured under different concentrations of ascorbic acid, and the concentrations of the other two added components are fixed, which are 0.1 mg / mL human serum albumin and 10 ng / mL basic fibroblast growth factor polypeptide respectively; (C) is cultured under different concentrations of basic fibroblast growth factor polypeptide, and the concentrations of the other two added components are fixed, which are 35.23 μg / mL ascorbic acid and 0.1 mg / mL human serum albumin respectively.

[0028] Figure 2 It shows the situation of culturing P19 generation large yellow croaker muscle satellite cells with different basic media in a serum-free medium. Among them, (A) is using DMEM / F12 medium; (B) is using DMEM high glucose medium; (C) is using F10 medium.

[0029] Figure 3Cell viability after culturing large yellow croaker muscle satellite cells for two days with different concentrations of each exogenous additive component in low-serum medium (all groups contain the same basic exogenous additive components).

[0030] Figure 4 Cell viability after culturing large yellow croaker muscle satellite cells for two days by combining the optimal concentration combinations of each exogenous additive component in low-serum medium and using the exclusion screening method (all groups contain the same basic exogenous additive components).

[0031] Figure 5 Comparison of cell viability after culturing large yellow croaker muscle satellite cells for one day with lysophosphatidic acid + human holotransferrin + IGF-1 (- insulin) and insulin + lysophosphatidic acid + IGF-1 (- human holotransferrin) added in low-serum medium (all groups contain the same basic exogenous additive components).

[0032] Figure 6 Comparison of the morphology of large yellow croaker muscle satellite cells at passage P13 cultured with a general medium and the low-serum medium of the present invention. Among them, (A) is the general medium (DMEM high glucose, 100 μL / mL FBS), and (B) is the low-serum medium of the present invention. Detailed implementation manners

[0033] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0034] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0035] DMEM high glucose medium ( BL304A); DMEM / F12 medium ( PM150312); F10 medium ( PM151113); FBS fetal bovine serum ( 086-150); human serum albumin ( BS921); ascorbic acid (Aladdin 50-81-7); basic fibroblast growth factor polypeptide ( bs-0217P); recombinant human insulin ( 11061-68-0); lysophosphatidic acid (Jiuding Chemistry 325465-93-8); human holotransferrin ( (11096-37-0); Recombinant human IGF-1( P00048).

[0036] The primary cells of large yellow croaker muscle satellite cells were prepared by conventional methods in the laboratory. Muscle tissues were isolated from the upper axial part of the muscles of large yellow croaker larvae, and then digested with 0.1% type IV collagenase solution and 0.1% trypsin solution respectively. The digested tissues were filtered through 70μm and 40μm cell sieves respectively, centrifuged at 300g for 5 minutes, and the cell pellet was resuspended with complete medium. Then, the cells were inoculated in a 6-well plate at a concentration of 1×10 6 cells / mL and cultured in an incubator with the culture conditions of 27°C and 5% CO2.

[0037] Example 1: Exploration of Exogenous Additive Components in Low-Serum Medium

[0038] Approximately 2×10 4 muscle satellite cells were inoculated into each well of a 100μL 96-well plate and cultured aseptically at 27°C for 72h. The cell viability was measured using a cck-8 kit to evaluate the exogenous additive components in the serum-free medium.

[0039] The basal medium was DMEM high-glucose. First, the concentrations of different components of the exogenous additive components were set. Among them, human serum albumin: 0.1mg / mL, 1mg / mL, 8mg / mL; ascorbic acid: 14.09μg / mL, 21.14μg / mL, 35.23μg / mL; basic fibroblast growth factor polypeptide: 1ng / mL, 3ng / mL, 10ng / mL. The results are as Figure 1 shown.

[0040] The optimal concentration of each component was determined, and then the optimal concentration combinations of each component were combined to select the exogenous additive components in the serum-free medium, as shown in Table 1.

[0041] Table 1 Exogenous Additive Components

[0042]

[0043]

[0044] Under the culture condition of 27°C, the cells harvested from each well of the 96-well plate with the exogenous additive components in the serum-free medium had normal cell morphology. The cell density was 2.2×10 5 cells / mL, and the cell growth amount was about 1 / 3 of that of the control group medium (F10, added with 100μl / mL FBS). The cell density harvested from the control group medium (F10, added with 100μl / mL FBS) was 7.4×10 5cells / mL (The effects of different basal media on the proliferation of Pseudosciaena crocea muscle satellite cells were explored in the preliminary stage. The results showed that F10 had the best effect, so F10 was selected as the control medium here).

[0045] Therefore, 0.1 mg / mL human serum albumin, 35.23 μg / mL ascorbic acid, and 10 ng / mL basic fibroblast growth factor polypeptide were selected as the basic exogenous additives in the serum-free medium.

[0046] Example 2: Selection of basal medium for low-serum medium

[0047] Approximately 2×10 4 muscle satellite cells were inoculated into each well of a 96-well plate with 100 μL and cultured aseptically at 27°C for 24 h. The cell morphology and cell growth amount were observed by a microscope to evaluate the effect of the basal medium of the serum-free medium on the proliferation of muscle satellite cells.

[0048] The basal media were DMEM high glucose, DMEM / F12, and F10, respectively. The basic exogenous additives were the same, all being 0.1 mg / mL human serum albumin, 35.23 μg / mL ascorbic acid, and 10 ng / mL basic fibroblast growth factor polypeptide.

[0049] The results were as Figure 2 shown. After aseptic culture at 27°C for 24 h and observation by a microscope, compared with the basal media DMEM high glucose and F10, the cell morphology and cell growth amount of DMEM / F12 were the best.

[0050] By comparing the effects of the basal media of the serum-free medium on the proliferation of muscle satellite cells, DMEM / F12 had the best effect. Therefore, DMEM / F12 was selected as the basal medium for the serum-free medium.

[0051] Example 3: Preparation of low-serum medium

[0052] Approximately 2×10 4 muscle satellite cells were inoculated into each well of a 96-well plate with 100 μL and cultured aseptically at 27°C for 48 h. The cell viability was measured using a cck-8 kit to evaluate the low-serum medium.

[0053] The basal medium was DMEM / F12 supplemented with 50 μL / mL fetal bovine serum. First, the concentrations of different components of the exogenous additives were set, where recombinant human insulin: 5.81 μg / mL, 8.72 μg / mL, 11.62 μg / mL; lysophosphatidic acid: 0.23 μg / mL, 0.46 μg / mL, 0.69 μg / mL; human holotransferrin: 4 μg / mL, 8 μg / mL, 12 μg / mL; recombinant human IGF-1: 5 ng / mL, 10 ng / mL, 15 ng / mL. The results are as Figure 3 shown. However, the cell morphology cultured under single-factor components mostly presented an oval shape, and the cell death rate was relatively high.

[0054] The optimal concentration of each component was determined, and then the optimal concentrations of each component were combined. The exclusion screening method (Table 2) was adopted. The specific method was as follows: In the first round of experiments, recombinant human insulin + lysophosphatidic acid + human holotransferrin + recombinant human IGF-1, lysophosphatidic acid + human holotransferrin + recombinant IGF-1 (- recombinant human insulin), insulin + human holotransferrin + recombinant human IGF-1 (- lysophosphatidic acid), recombinant human insulin + lysophosphatidic acid + recombinant human IGF-1 (- human holotransferrin), and recombinant human insulin + lysophosphatidic acid + human holotransferrin (- recombinant human IGF-1) were added to the low-serum basal medium respectively. The results are as Figure 4 shown. The best cell proliferation effect was achieved when recombinant human insulin and human holotransferrin were not added to the medium. To further confirm which one had a better cell proliferation effect between not adding recombinant human insulin and not adding human holotransferrin, in the second round of experiments, lysophosphatidic acid + human holotransferrin + IGF-1 (- insulin) and insulin + lysophosphatidic acid + IGF-1 (- human holotransferrin) were added to the low-serum basal medium respectively. The results are as Figure 5 shown. The cell proliferation effect was better when recombinant human insulin was not added to the medium compared to when human holotransferrin was not added. Finally, the exogenous additives were selected as lysophosphatidic acid, human holotransferrin, and recombinant human IGF-1. The cell morphology cultured under multi-factor combination conditions presented a long fusiform shape, with normal morphology and a low death rate.

[0055] Table 2 Confirmation of exogenous additives using the exclusion screening method

[0056]

[0057] The low-serum medium included the basal medium DMEM / F12, 50 μL / mL fetal bovine serum, and exogenous additives (Table 3), where the exogenous additives included the basic exogenous additives selected in the serum-free medium exploration and 3 cytokines explored later: human holotransferrin, lysophosphatidic acid, and recombinant human IGF-1.

[0058] Table 3 Added Components

[0059] Component Concentration Human holotransferrin 4 μg / mL Lysophosphatidic acid 0.46 μg / mL Recombinant human IGF-1 15 ng / mL Basic fibroblast growth factor polypeptide 10 ng / mL Ascorbic acid 35.23 μg / mL Human serum albumin 0.1 μg / mL

[0060] The results are as Figure 6 shown. When cultured aseptically at 27°C for 24 h, using the above low-serum medium formulation, the cell morphology is normal, and the harvested cell quantity is 4.8×10 5 cells / mL. For the control group using a general medium (DMEM high glucose supplemented with 100 μL / mL FBS), the harvested cell quantity is 5.0×10 5 cells / mL. This indicates that using the above low-serum medium formulation can achieve normal cell growth during the required growth cycle.

[0061] Example 4: Application of Low-Serum Medium in Cultured Meat

[0062] In this example, a small amount of fetal bovine serum and exogenous added components such as human serum albumin, ascorbic acid, basic fibroblast growth factor polypeptide, human holotransferrin, lysophosphatidic acid, recombinant human IGF-1, etc. are added to the basic medium DMEM / F12 to form a low-serum medium. This medium can rapidly proliferate large yellow croaker muscle satellite cells. The medium is changed about once every 4 h, and the cells are cultured at 27°C for 48 h, showing the same effect as the general medium (DMEM high glucose, 100 μL / mL FBS), with normal cell morphology, providing sufficient cells for the preparation of cultured meat.

[0063] For cost calculation, taking 500 mL of complete medium as an example, the general medium (DMEM high glucose, 100 μL / mL FBS) costs about 305 yuan, while the low-serum medium of the present invention costs about 240 yuan. Therefore, using the above low-serum medium can reduce the culture cost, and can culture large yellow croaker muscle satellite cells on a large scale at low cost, which is beneficial to industrial transformation.

[0064] The above embodiments are preferred embodiments of the present invention, but they are not intended to limit the present invention. The low-serum medium provided by the present invention is also applicable to other marine fish or freshwater fish. Any changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should be regarded as equivalent replacement methods and should be included within the protection scope of the present invention.

Claims

1. Use of a low-serum medium in culturing large yellow croaker stem cells or large yellow croaker muscle cells in vitro, characterized in that, The culture medium comprises a basal medium, fetal bovine serum at a proportion of 30 - 80 μL / mL, and exogenous additives. The basal medium is DMEM / F12 medium, and the exogenous additives are human serum albumin, ascorbic acid, basic fibroblast growth factor polypeptide, human holotransferrin, lysophosphatidic acid, and human recombinant IGF-1. The concentrations of each component in the culture medium are 0.1 - 8 mg / mL, 14.09 - 35.23 μg / mL, 1 - 10 ng / mL, 4 - 12 μg / mL, 0.23 - 0.69 μg / mL, and 5 - 15 ng / mL respectively.

2. The application according to claim 1, characterized in that, The proportion of the fetal bovine serum is 50 μL / mL. The concentrations of human serum albumin, ascorbic acid, basic fibroblast growth factor polypeptide, human holotransferrin, lysophosphatidic acid, and human recombinant IGF-1 in the exogenous additives in the culture medium are 0.1 mg / mL, 35.23 μg / mL, 10 ng / mL, 4 μg / mL, 0.46 μg / mL, and 15 ng / mL respectively.

3. The application according to claim 1, characterized in that, The seeding density of cells during cultivation is 10 3 ~10 5 cells / mL of low-serum medium.

4. The application according to claim 1 or 3, characterized in that, The culture conditions are culturing at 25°C - 30°C for 20 - 100 h.

5. A method for preparing cell-cultured large yellow croaker meat, characterized in that, The method includes: inoculating large yellow croaker muscle satellite cells into a low-serum culture medium for culture. The low-serum culture medium comprises a basal medium, fetal bovine serum at a proportion of 30 - 80 μL / mL, and exogenous additives. The basal medium is DMEM / F12 medium, and the exogenous additives are human serum albumin, ascorbic acid, basic fibroblast growth factor polypeptide, human holotransferrin, lysophosphatidic acid, and human recombinant IGF-1. The concentrations of each component in the culture medium are 0.1 - 8 mg / mL, 14.09 - 35.23 μg / mL, 1 - 10 ng / mL, 4 - 12 μg / mL, 0.23 - 0.69 μg / mL, and 5 - 15 ng / mL respectively.

6. The method for preparing large yellow croaker cell-cultured meat according to claim 5, wherein The proportion of the fetal bovine serum in the low-serum culture medium is 50 μL / mL. The concentrations of human serum albumin, ascorbic acid, basic fibroblast growth factor polypeptide, human holotransferrin, lysophosphatidic acid, and human recombinant IGF-1 in the exogenous additives in the culture medium are 0.1 mg / mL, 35.23 μg / mL, 10 ng / mL, 4 μg / mL, 0.46 μg / mL, and 15 ng / mL respectively.

7. The method for preparing cultured Pseudosciaena crocea meat according to claim 5, wherein The seeding density of cells during cultivation is 1×10 4 ~2×10 4 cells / mL in low-serum medium.

8. The method for preparing cultured Pseudosciaena crocea meat according to claim 5, characterized in that, The culture conditions are culturing at 25°C - 27°C for 24 - 72 h.

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