Serum-free culture medium for in-vitro suspension culture of porcine muscle stem cells and application of serum-free culture medium

Abstract

The invention provides a serum-free culture medium for in-vitro suspension culture of porcine muscle stem cells and application of the serum-free culture medium. The serum-free culture medium can help the porcine muscle stem cells to be subjected to suspension enlarged culture under serum-free and carrier-free conditions. Compared with a traditional porcine muscle stem cell culture scheme and culture medium, the serum-free suspension culture medium has the advantages that high-density continuous culture of porcine muscle stem cells can be realized in a batch culture process and a fed-batch culture process under the serum-free carrier-free suspension culture condition, high cell viability is maintained, and the survival rate of the porcine muscle stem cells is increased. The peak density of the cells reaches 1 * 10 < 7 > cells / mL or above, and the cell viability is maintained to be 90% or above. The method has the beneficial effects that the problem of serum-free carrier-free efficient suspension amplification of the seed cells of the cell culture meat is effectively solved, and a technical and raw material basis is provided for industrial production of the cell culture meat.

Description

Technical Field

[0001] The present invention belongs to the technical field of serum-free media for suspension cells, especially the technical fields of stem cell and animal cell cultured meat, and specifically relates to a serum-free medium for in vitro suspension culture of porcine muscle stem cells and its application. Background Art

[0002] Meat is rich in protein, fat and micronutrients and is widely consumed by humans. Among them, fat can provide a large amount of energy, protein and nutrients, making meat flavorful, palatable and juicy, so it is highly favored by consumers. However, with the development of society and the growth of the population, the consumption of meat has increased year by year. The traditional meat production method relying on animal husbandry can no longer meet the rapidly expanding meat demand gap, and there are risks in aspects such as resource utilization, environmental protection, animal welfare, and epidemic control. Therefore, in order to achieve the sustainable and safe production of meat, it is urgent to develop a green and efficient meat production method to partially replace the traditional meat production.

[0003] Cultured meat is meat obtained by in vitro culture of its stem cells according to the growth mechanism of animal meat, and it does not require animal farming and directly produces meat using cell factories. As a subversive meat production method, cultured meat provides a new way to supplement future meat supply and achieve green meat production. According to calculations, compared with traditional animal husbandry, the cultured meat industry can reduce energy consumption by 35% - 60%, occupy 98% less land and generate more than 80% less greenhouse gas.

[0004] Porcine muscle stem cells are considered important seed cells for cultured meat due to their excellent in vitro continuous amplification ability and cell differentiation ability. However, the traditional in vitro culture method is two-dimensional adherent culture, and the medium used is a serum-containing medium with a basic medium supplemented with 10 - 20% fetal bovine serum. The high cost of serum and the serious problems such as the easy introduction of pathogens such as mycoplasma and viruses restrict the expansion culture of cells.

[0005] Patent CN114574433A provides a chemically defined medium for in vitro proliferation of myogenic cells, but it only maintains the normal adhesion of myogenic cells. And the traditional adherent culture mode has an obvious upper limit of unit density, and also cannot meet the cost and efficiency requirements for large-scale expansion culture.

[0006] Meanwhile, the culture medium for adherent culture cannot be directly applied to suspension culture. The reasons are as follows: First, the nutrient concentration in the serum-free medium for adherent culture is relatively low, and the proportion relationship of nutrient factors is different, which cannot meet the high-density amplification culture of suspension cells. Second, the adherent factors existing in the serum-free medium for adherent culture are likely to cause suspension cells to aggregate and grow, and they cannot proliferate normally. Third, during the domestication process of suspension cells, there is generally a corresponding suspension culture medium to match.

[0007] Therefore, for the suspension culture of muscle stem cells, it is particularly important to develop a serum-free medium for efficient in vitro suspension amplification of porcine muscle stem cells. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the purpose of the present invention is to develop a new serum-free medium to replace the fetal bovine serum component in the traditional cell culture method and medium, and to provide a set of serum-free medium that can support the efficient suspension amplification of porcine muscle stem cells and its application method.

[0009] The serum-free component described in the present invention means that no animal serum components including horse serum, fetal bovine serum, bovine serum, human serum, etc. are added.

[0010] The serum-free medium for in vitro suspension culture of porcine muscle stem cells is a medium for serum-free suspension culture added with cell culture supplementary factors, and by adding cell culture supplementary factors, the serum component in the traditional cell amplification medium is replaced.

[0011] The first object of the present invention is to provide a serum-free medium for in vitro suspension culture of porcine muscle stem cells. The serum-free medium for in vitro suspension culture of porcine muscle stem cells includes cell culture supplementary factors and a basal medium; the serum-free medium does not contain serum components.

[0012] The cell culture supplementary factors are zinc sulfate, L-ascorbic acid-2-phosphate trisodium salt, L-ascorbic acid, sodium selenite, bFGF, IGF-1, LIF, EGF, cortisol, HGF, Forskolin, α-tocopherol, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid, NAC (N-acetylcysteine), transferrin, Trolox, ethanolamine, AM2394, sodium oxamate, ammonium ferric citrate, and yeast extract.

[0013] Furthermore, the basal medium is a basal proliferation medium and / or a feeding medium; the serum-free medium is divided into: Serum-free medium a: When the basal medium is the basal proliferation medium, the prepared serum-free medium a is for proliferation culture in in vitro batch suspension culture or fed-batch suspension culture. Serum-free medium b: When the basal medium is the basal proliferation medium and the feeding medium, the prepared serum-free medium b is for feeding in fed-batch suspension culture.

[0014] Furthermore, the basal proliferation medium is selected from one or more of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, and CD02 medium; the feeding medium is selected from Feed02 feeding medium.

[0015] Furthermore, the total concentration range of cell culture supplement factors added in serum-free medium a is 0.03 - 50 mg / mL.

[0016] Furthermore, the total concentration range of cell culture supplement factors added in serum-free medium b is 0.03 - 50 mg / mL.

[0017] Furthermore, the concentration range of any added cell culture supplement factor in serum-free medium a is 0.0000001 - 5 mg / mL.

[0018] Furthermore, the concentration range of any added cell culture supplement factor in serum-free medium a is 0.000002 - 0.5 mg / mL.

[0019] The concentration of any added cell culture supplement factor in serum-free medium b is 0.0000005 - 10 mg / mL.

[0020] The concentration range of any added cell culture supplement factor in serum-free medium b is 0.00001 - 2.5 mg / mL.

[0021] The second object of the present invention is to provide the application of the aforementioned serum-free medium for in vitro suspension culture of porcine muscle stem cells in the in vitro suspension expansion culture of porcine muscle stem cells, and the serum-free medium for in vitro suspension culture of porcine muscle stem cells is used for in vitro batch suspension culture or fed-batch suspension culture of porcine muscle stem cells.

[0022] In a particular embodiment, the serum-free medium used for in vitro batch suspension culture is serum-free medium a prepared from cell culture supplement factors and the basal proliferation medium.

[0023] In a particular embodiment, the serum-free medium used in fed-batch suspension culture is first a serum-free medium a prepared from cell culture supplement factors and a basal proliferation medium, and then a serum-free medium b prepared from cell culture supplement factors, a basal proliferation medium, and a feeding medium is periodically added for fed-batch suspension culture.

[0024] Furthermore, the serum-free medium for in vitro suspension culture of porcine muscle stem cells can support the improvement of cell culture density and / or maintenance of cell viability in in vitro batch suspension culture or fed-batch suspension culture of porcine muscle stem cells.

[0025] The beneficial effects achieved by the technical solution of the present invention are as follows: The serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention, by adding cell culture supplement factors, replaces the role of serum in cell culture and avoids many problems existing in the use of serum. At the same time, combined with the application of the cell suspension culture process, porcine muscle stem suspension cells can be expanded in suspension under serum-free and carrier-free conditions, and high-density continuous culture can be achieved in both batch culture process and fed-batch culture process, and high cell viability can be maintained.

[0026] The serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention is not simply increasing the nutrient concentration of an adherent medium to make it suitable for suspension culture, but by adjusting the ratio of nutrient factors, increasing anti-clumping factors and anti-shearing force factors, and reducing pro-adherent factors, so that the suspension culture effect of this medium is significantly better than that of existing conventional adherent serum-free media. The present invention provides a medium that can improve the suspension culture effect while reducing costs.

[0027] Specifically, from specific experimental data, the serum-free medium for in vitro suspension culture of porcine muscle stem cells provided by the present invention can support the continuous suspension culture and passage of porcine muscle stem cells. After 3 days of suspension culture, it supports a maximum cell density of up to 6.8×10 6 cells / mL, and the average cell density reaches 5.0×10 6 cells / mL or more, the cell viability is maintained above 80%, and the support cell expansion multiple is about 20-30 times. The serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention, in a 5 L bioreactor, in combination with a feeding medium, can make the peak cell density reach 1×10 7 cells / mL or more, and maintain the cell viability above 90%. In an 80 L bioreactor, the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention, in combination with a feeding medium, can make the peak cell density reach 1×10 7Above [X] cells / mL, and maintaining the cell viability above 90%. The beneficial effects of the present invention effectively solve the problem of high-efficiency suspension amplification of serum-free and carrier-free seed cells for cultured meat, providing a technical and raw material basis for the industrial production of cultured meat. Description of the Drawings

[0028] Figure 1 The cell culture density curve of each generation of porcine muscle stem cells supported by the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention during continuous batch subculture.

[0029] Figure 2 The cell viability curve of each generation of porcine muscle stem cells supported by the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention during continuous batch subculture.

[0030] Figure 3 The statistical chart of the proliferation multiple of each generation of porcine muscle stem cells supported by the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention during continuous batch subculture.

[0031] Figure 4 The curve graph of cell density during fed-batch culture of porcine muscle stem cells supported by the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention in a 5 L bioreactor system.

[0032] Figure 5 The curve graph of cell viability during fed-batch culture of porcine muscle stem cells supported by the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention in a 5 L bioreactor system.

[0033] Figure 6 The curve graph of cell density during fed-batch culture of porcine muscle stem cells supported by the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention in an 80 L bioreactor system.

[0034] Figure 7 The curve graph of cell viability during fed-batch culture of porcine muscle stem cells supported by the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention in an 80 L bioreactor system.

[0035] Figure 8 The statistical chart for detecting the proliferation-promoting effect of some cell culture supplement factors.

[0036] Figure 9 The cell culture density curve of porcine muscle stem cells supported by a chemically defined medium for in vitro proliferation of myogenic cells with the patent publication number CN114574433A during batch subculture. Detailed Embodiments

[0037] The present invention will be further explained below in conjunction with embodiments, but the embodiments do not limit the present invention in any form.

[0038] The serum-free medium for in vitro suspension culture of porcine muscle stem cells proposed by the present invention is a cell culture medium supplemented with cell culture supplement factors, and other aspects are the same as the normal suspension cell culture method.

[0039] In the following embodiments, porcine muscle stem cell lines adapted to carrier-free and serum-free suspension culture are used. The specific domestication method can refer to Patent CN116555171A. These cells are preserved in the China Center for Type Culture Collection (CCTCC), with the preservation address being Wuhan University, China, the preservation number being CCTCC NO: C2022372, and the preservation date being December 7, 2022.

[0040] In the following embodiments, materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0041] Example 1 Preparation of Serum-Free Proliferation Medium

[0042] Taking the preparation of 1 L of proliferation medium as an example, take 15.78 g of CD02 basal medium powder (Kangsheng Biotech, A12002) and prepare it according to the method in the instruction manual: transfer it to 800 mL of pure water, stir for 10 minutes to mix evenly, adjust the pH of the medium to 9.05 with 5 moL / L sodium hydroxide solution, stir well for 30 minutes, then adjust the pH back to 7.0 with 6 moL / L dilute hydrochloric acid. Subsequently, add 1.9 g of sodium bicarbonate powder, dissolve it and adjust the pH to 7.0, make up the volume of the solution to 1 L with pure water, and finally adjust the osmotic pressure of the solution with sodium chloride powder to control it at 300 mOsmol / kg, and stir for 5 minutes to obtain the CD02 basal medium.

[0043] A serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention comprises a CD02 basal medium supplemented with cell culture supplement factors. Cell culture supplement factors are added to the prepared CD02 basal medium. The specific components and concentrations added are as follows: zinc sulfate 0.03 mg / mL, L-ascorbic acid-2-phosphate trisodium salt 0.064 mg / mL, L-ascorbic acid 0.035 mg / mL, sodium selenite 0.00005 mg / mL, bFGF 0.0001 mg / mL, IGF-1 0.0002 mg / mL, LIF 0.00002 mg / mL, EGF 0.0001 mg / mL, cortisol 0.000072 mg / mL, HGF 0.00005 mg / mL, Forskolin 0.012 mg / mL, α-tocopherol 0.013 mg / mL, glycine 0.0075 mg / mL, L-alanine 0.0089 mg / mL, L-asparagine 0.0132 mg / mL, L-aspartic acid 0.0133 mg / mL, L-glutamic acid 0.0147 mg / mL, L-proline 0.0115 mg / mL, L-serine 0.0105 mg / mL, arachidonic acid 0.000002 mg / mL, cholesterol 0.00022 mg / mL, vitamin E acetate 0.00007 mg / mL, linoleic acid 0.00001 mg / mL, linolenic acid 0.00001 mg / mL, myristic acid 0.00001 mg / mL, oleic acid 0.00001 mg / mL, palmitic acid 0.00001 mg / mL, palmitoleic acid 0.00001 mg / mL, stearic acid 0.00001 mg / mL, N-acetylcysteine 0.163 mg / mL, transferrin 0.02 mg / mL, Trolox 0.013 mg / mL, ethanolamine 0.00458 mg / mL, AM2394 0.000021 mg / mL, sodium oxamate 0.11 mg / mL, ammonium ferric citrate 0.005 mg / mL, yeast extract 0.5 mg / mL.

[0044] After the addition of the cell culture supplement factors, mix well, filter with a 0.22 μm filter membrane and store at 2-8 °C to obtain a serum-free proliferation medium, that is, serum-free medium a.

[0045] Example 2 Preparation of serum-free feeding medium

[0046] Dissolve and prepare the Feed02 feeding medium (Kangsheng Bio, A12902) according to the product manual provided by the manufacturer to obtain two feeding packages, Feed A and Feed B. Then, add cell culture supplements according to the corresponding ratios. Taking the preparation of 1 L of feeding medium as an example, the specific components and concentrations added are as follows: Feed A 450 mL, Feed B 50 mL, CD02 500 mL, zinc sulfate 0.15 mg / mL, L-ascorbic acid-2-phosphate trisodium salt 0.32 mg / mL, L-ascorbic acid 0.175 mg / mL, sodium selenite 0.00025 mg / mL, bFGF 0.0005 mg / mL, IGF-1 0.001 mg / mL, LIF 0.0001 mg / mL, EGF 0.0005 mg / mL, cortisol 0.00036 mg / mL, HGF 0.00025 mg / mL, Forskolin 0.06 mg / mL, α-tocopherol 0.065 mg / mL, glycine 0.0375 mg / mL, L-alanine 0.0445 mg / mL, L-asparagine 0.066 mg / mL, L-aspartic acid 0.0665 mg / mL, L-glutamic acid 0.0735 mg / mL, L-proline 0.0575 mg / mL, L-serine 0.0525 mg / mL, arachidonic acid 0.00001 mg / mL, cholesterol 0.0011 mg / mL, vitamin E acetate 0.00035 mg / mL, linoleic acid 0.00005 mg / mL, linolenic acid 0.00005 mg / mL, myristic acid 0.00005 mg / mL, oleic acid 0.00005 mg / mL, palmitic acid 0.00005 mg / mL, palmitoleic acid 0.00005 mg / mL, stearic acid 0.00005 mg / mL, N-acetylcysteine 0.815 mg / mL, transferrin 0.1 mg / mL, Trolox 0.065 mg / mL, ethanolamine 0.0229 mg / mL, AM2394 0.000105 mg / mL, sodium oxalate 0.55 mg / mL, ammonium ferric citrate 0.025 mg / mL, yeast extract 2.5 mg / mL to form the feeding medium, that is, serum-free medium b.

[0047] After adding the above materials, mix them thoroughly, filter through a 0.22 μm filter membrane, and store at 2 - 8°C to prepare the serum-free feeding medium formulation.

[0048] Example 3 Detection of batch continuous culture ability

[0049] In this example, a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture was used. The specific domestication method can be referred to in Patent CN116555171A. This cell is preserved in the China Center for Type Culture Collection (CCTCC), with the preservation address being Wuhan University, China, the preservation number being CCTCC NO: C2022372, and the preservation date being December 7, 2022.

[0050] The cells were cultured using the serum-free proliferation medium prepared in Example 1. The cells were inoculated at an inoculation density of 2×10 5 cells / mL, cultured in a 125 mL shake flask with a volume of 30 mL, placed in a shaking incubator with a carbon dioxide concentration of 5% and a temperature of 37°C, and the rotation speed was 120 rpm. Every 3 days, trypan blue was used to count the cell density and viability. Then, the cells were re-inoculated at a density of 2×10 5 cells / mL into 30 mL of serum-free proliferation medium and continued to be passaged and cultured.

[0051] The results showed that the serum-free medium provided by the present invention for in vitro suspension culture of porcine muscle stem cells could support the continuous suspension culture and passage of porcine muscle stem cells. After 3 days of suspension culture, the highest cell density supported reached 6.8×10 6 cells / mL, and the average cell density reached more than 5.0×10 6 cells / mL ( Figure 1 ), the cell viability was maintained above 80% ( Figure 2 ), and the cell expansion multiple was about 20 - 30 times ( Figure 3 ).

[0052] The above results proved that the serum-free medium of the present invention for in vitro suspension culture of porcine muscle stem cells could support the efficient expansion of suspended cells in batch continuous culture.

[0053] Example 4: Evaluation of the fed-batch culture effect in a bioreactor system

[0054] 1) Fed-batch culture test in a 5 L bioreactor In this example, a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture was used. The specific domestication method can be referred to in Patent CN116555171A. This cell is preserved in the China Center for Type Culture Collection (CCTCC), with the preservation address being Wuhan University, China, the preservation number being CCTCC NO: C2022372, and the preservation date being December 7, 2022.

[0055] A 5 L bioreactor and the serum-free medium a of the present invention for in vitro suspension culture of porcine muscle stem cells prepared in Example 1 were used. At 2×10 5Cells were inoculated at an inoculation density of [X] cells / mL and cultured in a 5 L bioreactor with a volume of 2500 mL. Starting from the 3rd day of culture, 10% of the feeding medium prepared in Example 2, namely serum-free medium b, was supplemented every other day to the culture volume. Trypan blue was used to count the cell density and viability every day, and the experiment was terminated on the 8th day of culture.

[0056] 2) Fed-batch culture test in an 80 L bioreactor An 80 L bioreactor and the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention were used. Cells were inoculated at an inoculation density of 2×10 5 cells / mL and cultured in an 80 L bioreactor with a volume of 25 L. Starting from the 3rd day of culture, 10% of the feeding medium was supplemented every other day to the culture volume. Trypan blue was used to count the cell density and viability every day, and the experiment was terminated on the 8th day of culture.

[0057] The results showed that in the fed-batch suspension culture in a 5 L bioreactor, the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention, in the form of a serum-free proliferation medium combined with a serum-free feeding medium, could enable the peak cell density to reach above 1×10 7 cells / mL ( Figure 4 ), and maintain the cell viability above 90% ( Figure 5 ).

[0058] Similarly, in the fed-batch suspension culture in an 80 L bioreactor, the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention, in the form of a serum-free proliferation medium combined with a serum-free feeding medium, could enable the peak cell density to reach above 1×10 7 cells / mL ( Figure 6 ), and maintain the cell viability above 90% ( Figure 7 ).

[0059] Detection of the proliferation-promoting effect of additional cell culture supplement factors in Comparative Example 1

[0060] According to the steps described in Example 1, the CD02 basal medium was prepared and dissolved. Different combinations of cell culture supplement factors were added to the CD02 basal medium to verify the role of some cell culture supplement factors in the present invention.

[0061] Group 1 is a culture medium supplemented with only partial cell culture supplements. The specific formula is to add trisodium L-ascorbate-2-phosphate 0.064 mg / mL, bFGF 0.0001 mg / mL, IGF-1 0.0002 mg / mL, LIF 0.00002 mg / mL, EGF 0.0001 mg / mL, cortisol 0.000072 mg / mL, HGF 0.00005 mg / mL, Forskolin 0.012 mg / mL, glycine 0.0075 mg / mL, L-alanine 0.0089 mg / mL, L-asparagine 0.0132 mg / mL, L-aspartic acid 0.0133 mg / mL, L-glutamic acid 0.0147 mg / mL, L-proline 0.0115 mg / mL, L-serine 0.0105 mg / mL, arachidonic acid 0.000002 mg / mL, vitamin E acetate 0.00007 mg / mL, linoleic acid 0.00001 mg / mL, linolenic acid 0.00001 mg / mL, myristic acid 0.00001 mg / mL, oleic acid 0.00001 mg / mL, palmitic acid 0.00001 mg / mL, palmitoleic acid 0.00001 mg / mL, stearic acid 0.00001 mg / mL, Trolox 0.013 mg / mL, ethanolamine 0.00458 mg / mL, AM2394 0.000021 mg / mL to the CD02 basal medium.

[0062] Group 2 is a culture medium supplemented with zinc sulfate additionally. It is supplemented with 0.03 mg / mL of zinc sulfate on the basis of Group 1.

[0063] Group 3 is a culture medium supplemented with L-ascorbic acid additionally. It is supplemented with 0.035 mg / mL of L-ascorbic acid on the basis of Group 1.

[0064] Group 4 is a culture medium supplemented with sodium selenite additionally. It is supplemented with 0.00005 mg / mL of sodium selenite on the basis of Group 1.

[0065] Group 5 is a culture medium supplemented with α-tocopherol additionally. It is supplemented with 0.013 mg / mL of α-tocopherol on the basis of Group 1.

[0066] Group 6 is a culture medium supplemented with cholesterol additionally. It is supplemented with 0.00022 mg / mL of cholesterol on the basis of Group 1.

[0067] Group 7 is a culture medium supplemented with N-acetylcysteine additionally. It is supplemented with 0.163 mg / mL of N-acetylcysteine on the basis of Group 1.

[0068] Group 8 is the culture medium with additional transferrin, which is based on Group 1 and supplemented with 0.02 mg / mL of transferrin.

[0069] Group 9 is the culture medium with additional sodium oxalate, which is based on Group 1 and supplemented with 0.11 mg / mL of sodium oxalate.

[0070] Group 10 is the culture medium with additional ammonium ferric citrate, which is based on Group 1 and supplemented with 0.005 mg / mL of ammonium ferric citrate.

[0071] Group 11 is the culture medium with additional yeast extract, which is based on Group 1 and supplemented with 0.5 mg / mL of yeast extract.

[0072] Group 12 is the serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention, and the added factors are the same as those in Example 1.

[0073] After adding the above materials, mix them thoroughly, filter through a 0.22 μm filter membrane and store at 2-8°C to prepare the serum-free media for each group.

[0074] In this example, a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture is used. The specific domestication method can refer to Patent CN116555171A. The cells are preserved in the China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan University, China, the preservation number is CCTCC NO: C2022372, and the preservation date is December 7, 2022.

[0075] Inoculate the cells at an inoculation density of 2×10 5 cells / mL, culture them in a 125 mL shake flask with a volume of 30 mL, place the cells in a shaker at a carbon dioxide concentration of 5% and 37°C, and the rotation speed is 120 rpm. On the 3rd day, use trypan blue to count the cell density and viability.

[0076] The results showed that media in Groups 1 to 11 were media supplemented with only some cell culture supplements, and each group of media could only support the survival of suspended cells, but the cell proliferation ability was significantly weaker than that of Group 12 of the present invention's solution. Specifically: In the case of Group 1 culture, compared with the number of inoculated cells, it only expanded 8.13 times in 3 days. The medium supplemented with zinc sulfate (Group 2) helped the suspended cells expand 3.75 times in 3 days, the medium supplemented with L-ascorbic acid (Group 3) helped the suspended cells expand 4.25 times in 3 days, the medium supplemented with sodium selenite (Group 4) helped the suspended cells expand 4 times in 3 days, the medium supplemented with α-tocopherol (Group 5) helped the suspended cells expand 4.33 times in 3 days, the medium supplemented with cholesterol (Group 6) helped the suspended cells expand 7.25 times in 3 days, the medium supplemented with N-acetylcysteine (Group 7) helped the suspended cells expand 11.67 times in 3 days, the medium supplemented with transferrin (Group 8) helped the suspended cells expand 4.13 times in 3 days, the medium supplemented with sodium oxalate (Group 9) helped the suspended cells expand 10.88 times in 3 days, the medium supplemented with ammonium ferric citrate (Group 10) helped the suspended cells expand 12.25 times in 3 days, and the medium supplemented with yeast extract (Group 11) helped the suspended cells expand 13.75 times in 3 days. The serum-free medium for in vitro suspension culture of porcine muscle stem cells of the present invention that combines all cell culture supplements (Group 12) supported the suspended cells to expand 30.42 times in 3 days, which was significantly better than other formulations that only added some cell culture supplements, indicating that the combined solution of the present invention has a significant synergistic effect ( Figure 8 ).

[0077] Comparative Example 2

[0078] To compare and reflect the effect of the serum-free medium for in vitro suspension of porcine muscle stem cells of the present invention, in this comparative example, a medium with a defined chemical composition for in vitro proliferation of myogenic cells with the patent number CN114574433A and a serum-free medium for in vitro suspension culture of porcine muscle stem cells of this patent were used for a control experiment, and the porcine muscle stem cell line that had been adapted to carrier-free and serum-free suspension culture was cultured according to the operation process of Example 3.

[0079] Cells were inoculated at an inoculation density of 2×10 5 cells / mL, cultured in a 125 mL shake flask with a volume of 30 mL, placed the cells in a shaker at a carbon dioxide concentration of 5% and 37 °C, and the rotation speed was 120 rpm. The cell density and viability were counted using trypan blue every 3 days. Then, the cells were re-inoculated at a density of 2×10 5 cells / mL into 30 mL of serum-free proliferation medium and continued to be passaged and cultured.

[0080] The results show that, compared with a culture medium with a clear chemical composition in the comparative patent for in vitro proliferation of myogenic cells, it cannot support the normal proliferation of a porcine muscle stem cell line cultured in a carrier-free and serum-free suspension within 3 days of culture. The cells form serious aggregates, and the cell viability is only 30.21%. After counting, it is found that the cells only expand 1.48 times. After one passage, all the cells die and cannot be further passaged ( Figure 9 ). The serum-free medium for the in vitro suspension culture of porcine muscle stem cells in this patent can support the continuous suspension culture and passage of porcine muscle stem cells. After 3 days of suspension culture, it supports the cell density to reach a maximum of 6.8×10 6 cells / mL, and the average cell density reaches more than 5.0×10 6 cells / mL. The cell viability is maintained above 80%, and it supports the cell expansion multiple to be about 20 - 30 times (Example 3). It can be seen that although both are applied to the in vitro proliferation culture of porcine muscle stem cells, the applicability of the culture medium in adherent culture and suspension culture is completely different. The adherent culture medium of CN114574433A cannot obtain the technical effect of the technical solution of this patent applied to suspension culture.

[0081] The examples disclosed above are for illustrating the implementation cases disclosed in the present invention, but cannot be construed as limitations of the present invention. Many different cell culture supplement factors and their concentration ranges are listed herein. Without departing from the scope and spirit of the present invention, there are still many different further combinations possible. Therefore, the present invention is not limited to the disclosed implementation cases. In fact, all obvious modifications to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A serum-free medium for in vitro suspension culture of porcine muscle stem cells, characterized in that, It includes cell culture supplements and a basal medium; the serum-free medium does not contain serum components; The cell culture supplements are zinc sulfate, L-ascorbic acid-2-phosphate trisodium salt, L-ascorbic acid, sodium selenite, bFGF, IGF-1, LIF, EGF, cortisol, HGF, Forskolin, α-tocopherol, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid, NAC, transferrin, Trolox, ethanolamine, AM2394, sodium oxamate, ammonium ferric citrate, and yeast extract.

2. The serum-free medium according to claim 1, wherein The basal medium is a basal proliferation medium and / or a feeding medium; the serum-free medium is divided into: Serum-free medium a: When the basal medium is a basal proliferation medium, the prepared serum-free medium a is for proliferation culture in in vitro batch suspension culture or fed-batch suspension culture; Serum-free medium b: When the basal medium is a basal proliferation medium and a feeding medium, the prepared serum-free medium b is for feeding in fed-batch suspension culture.

3. The serum-free medium according to claim 2, wherein The basal proliferation medium is selected from one or more of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, and CD02 medium; the feeding medium is selected from Feed02 feeding medium.

4. The serum-free medium according to claim 2, wherein The total concentration range of the added cell culture supplements in serum-free medium a is 0.03 - 50 mg / mL.

5. The serum-free medium according to claim 2, wherein The total concentration range of the added cell culture supplements in serum-free medium b is 0.03 - 50 mg / mL.

6. The serum-free medium according to claim 2, wherein The concentration of any added cell culture supplement in serum-free medium a is 0.0000001 - 5 mg / mL.

7. The serum-free medium according to claim 6, wherein The concentration range of any added cell culture supplement in serum-free medium a is 0.000002 - 0.5 mg / mL.

8. The serum-free medium according to claim 2, wherein The concentration of any added cell culture supplement in serum-free medium b is 0.0000005 - 10 mg / mL.

9. The serum-free medium according to claim 8, wherein, The concentration range of any added cell culture supplement in serum-free medium b is 0.00001 - 2.5 mg / mL.

10. Use of the serum-free medium for in vitro suspension culture of porcine muscle stem cells in in vitro suspension expansion culture of porcine muscle stem cells, characterized in that, Use the serum-free medium for in vitro suspension culture of porcine muscle stem cells according to claim 1 to perform in vitro batch suspension culture or fed-batch suspension culture on porcine muscle stem cells.

11. The application according to claim 10, characterized in that, The serum-free medium for in vitro suspension culture of the porcine muscle stem cells can support the porcine muscle stem cells to increase the cell culture density and / or maintain the cell viability in in vitro batch suspension culture or fed-batch suspension culture.

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