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

Abstract

The present invention provides a serum-free culture medium for in vitro suspension culture of porcine muscle stem cells and its application, which can help porcine muscle stem cells expand and be suspended under serum-free and carrier-free conditions. Compared with traditional porcine muscle stem cell culture protocols and culture media, this serum-free suspension culture medium enables porcine muscle stem cells to be cultured continuously at high densities in both batch and fed-batch culture processes under serum-free and carrier-free suspension culture conditions, while maintaining high cell viability and achieving a peak cell density of 1×10 7 The beneficial effect of the present invention is to effectively solve the problem of serum-free and carrier-free efficient suspension expansion of cell cultured meat seed cells, and provide a technical and raw material basis for the industrial production of cell cultured meat.

Description

Technical Field

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

[0002] Meat is rich in protein, fat, and micronutrients and is widely consumed by humans. Fat provides a significant amount of energy, protein, and nutrients, giving meat its flavor, texture, and juiciness, making it highly sought after by consumers. However, with social development and population growth, meat consumption has increased annually. Traditional meat production methods, which rely on animal husbandry, are no longer able to meet this rapidly expanding demand and pose risks in resource utilization, environmental protection, animal welfare, and epidemic prevention. Therefore, to achieve sustainable and safe meat production, there is an urgent need to develop green and efficient meat production methods to partially replace traditional meat production.

[0003] Cell-cultured meat is derived from animal stem cells grown in vitro, mimicking the growth mechanisms of animal meat. It eliminates the need for animal husbandry and directly produces meat using cell-based factories. As a disruptive meat production method, cell-cultured meat offers a new path to replenish future meat supplies and achieve greener meat production. According to estimates, compared to traditional animal husbandry, the cell-cultured meat industry can reduce energy consumption by 35% to 60%, occupy 98% less land, and produce over 80% less greenhouse gases.

[0004] Porcine muscle stem cells are considered important seed cells for cell culture due to their excellent in vitro continuous expansion and differentiation capabilities. However, traditional in vitro culture methods rely on two-dimensional adherent culture, using a serum-containing medium supplemented with 10-20% fetal bovine serum (FBS). This high cost and the potential for the introduction of pathogens such as mycoplasma and viruses pose serious challenges for expanding cell culture.

[0005] Patent CN114574433A provides a culture medium with a defined chemical composition for the in vitro proliferation of myogenic cells, but it only maintains the normal adherence of myogenic cells. Conventional adherent culture models have a significant upper limit on unit density and are unable to meet the cost and efficiency requirements of large-scale culture.

[0006] At the same time, the culture medium used for adherent culture cannot be directly used for suspension culture. The reasons are: 1. The nutrient concentration in the serum-free culture medium used for adherent culture is low, and the ratio of nutrient factors is different, which cannot meet the requirements of high-density expansion culture of suspension cells; 2. The adhesion-promoting factors present in the serum-free culture medium used for adherent culture can easily cause suspension cells to grow in clusters and cannot proliferate normally; 3. The acclimation process of suspension cells generally has a corresponding suspension culture medium to match.

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

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

[0009] The serum-free component of the present invention refers to the component without any animal serum components such as horse serum, fetal bovine serum, bovine serum, and human serum.

[0010] The serum-free culture medium for in vitro suspension culture of porcine muscle stem cells is a serum-free suspension culture medium supplemented with cell culture supplement factors, and the serum components in traditional cell expansion culture medium are replaced by the cell culture supplement factors.

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

[0012] The cell culture supplement factors include 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 culture medium is a basal proliferation medium and / or a feed medium; the serum-free culture medium is:

[0014] Serum-free medium a: When the basal medium is a basal proliferation medium, the prepared medium is a serum-free medium a for proliferation culture in in vitro batch suspension culture or fed-batch suspension culture;

[0015] Serum-free medium b: When the basal medium is a basal proliferation medium and a feed medium, the prepared medium is a serum-free medium b for feeding in fed-batch suspension culture.

[0016] Furthermore, the basal proliferation medium is selected from one or more of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, and CD02 medium; and the feed medium is selected from Feed02 feed medium.

[0017] Furthermore, the total concentration range of the cell culture supplement factors added to the serum-free medium a is 0.03-50 mg / mL.

[0018] Furthermore, the total concentration range of the cell culture supplement factors added to the serum-free medium b is 0.03-50 mg / mL.

[0019] Furthermore, the concentration range of any cell culture supplement factor added to the serum-free medium a is 0.0000001-5 mg / mL.

[0020] Furthermore, the concentration range of any cell culture supplement factor added to the serum-free medium a is 0.000002-0.5 mg / mL.

[0021] The concentration of any cell culture supplement factor added to serum-free medium b is 0.0000005-10 mg / mL.

[0022] The concentration range of any cell culture supplement factor added to serum-free medium b is 0.00001-2.5 mg / mL.

[0023] The second object of the present invention is to provide the use of the aforementioned serum-free culture medium for in vitro suspension culture of porcine muscle stem cells in the in vitro suspension expansion culture of porcine muscle stem cells, and to use the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells to perform in vitro batch suspension culture or fed-batch suspension culture on porcine muscle stem cells.

[0024] In a specific embodiment, the serum-free medium used in the in vitro batch suspension culture is a serum-free medium a prepared from cell culture supplementary factors and a basal proliferation medium.

[0025] In a particular embodiment, the serum-free medium used in the fed-batch suspension culture is a serum-free medium a prepared by first using cell culture supplementary factors and a basal proliferation medium, and then serum-free medium b prepared by using cell culture supplementary factors, a basal proliferation medium, and a feed medium is regularly added for fed-batch suspension culture.

[0026] Furthermore, the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells can support increasing the cell culture density and / or maintaining the cell viability of porcine muscle stem cells in in vitro batch suspension culture or fed-batch suspension culture.

[0027] The beneficial effects achieved by the technical solution of the present invention are:

[0028] The serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention replaces the role of serum in cell culture by adding cell culture supplement factors, thus avoiding many problems associated with the use of serum. Furthermore, by utilizing a cell suspension culture process, porcine muscle stem suspension cells can be cultured in suspension and expanded under serum-free and carrier-free conditions. High-density continuous culture can be achieved in both batch and fed-batch culture processes, while maintaining high cell viability.

[0029] The serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention is not based on an adherent culture medium that simply increases the nutrient concentration to make it suitable for suspension culture. Instead, by adjusting the ratio of nutrient factors, anti-agglomeration factors and anti-shear force factors are increased and adhesion-promoting factors are reduced, so that the suspension culture effect of the culture medium is significantly better than that of existing conventional adherent serum-free culture media. The present invention provides a culture medium that can reduce costs while improving the suspension culture effect.

[0030] Specifically, from the specific experimental data, the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells provided by the present invention can support continuous suspension culture and subculture of porcine muscle stem cells. After 3 days of suspension culture, the cell density can reach up to 6.8×10 6 cells / mL, and the average cell density reached 5.0×10 6 The serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention can achieve a cell peak density of 1×10 cells / mL or more, maintain a cell viability of more than 80%, and support a cell expansion multiple of about 20-30 times. In a 5 L bioreactor, combined with a feed medium, the cell peak density can reach 1×10 7 In an 80 L bioreactor, the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention, combined with feed medium, can make the cell peak density reach 1×10 7The beneficial effect of the present invention is to effectively solve the problem of serum-free and carrier-free efficient suspension expansion of cell cultured meat seed cells, and provide a technical and raw material basis for the industrial production of cell cultured meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The serum-free culture medium for in vitro suspension culture of porcine muscle stem cells supports a cell culture density curve of each generation in continuous batch subculture of porcine muscle stem cells.

[0032] Figure 2 The serum-free culture medium for in vitro suspension culture of porcine muscle stem cells supports the cell viability curve of each generation in continuous batch subculture of porcine muscle stem cells.

[0033] Figure 3 The serum-free culture medium for in vitro suspension culture of porcine muscle stem cells supports statistics on cell culture proliferation times of each generation in continuous batch subculture of porcine muscle stem cells.

[0034] Figure 4 A cell density change curve diagram of the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention supporting fed-batch culture of porcine muscle stem cells in a 5 L bioreactor system.

[0035] Figure 5 A curve diagram showing changes in cell viability of the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells in a 5 L bioreactor system supporting fed-batch culture of porcine muscle stem cells.

[0036] Figure 6 A cell density change curve diagram of the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention supporting fed-batch culture of porcine muscle stem cells in an 80 L bioreactor system.

[0037] Figure 7 A curve diagram showing changes in cell viability of the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells in an 80 L bioreactor system supporting fed-batch culture of porcine muscle stem cells.

[0038] Figure 8 Statistical chart of the proliferation-promoting effects of some cell culture supplementary factors.

[0039] Figure 9 Patent publication number CN114574433A discloses a culture medium with a defined chemical composition for in vitro proliferation of myogenic cells, supporting a cell culture density curve for batch subculture of porcine muscle stem cells. DETAILED DESCRIPTION

[0040] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0041] The serum-free culture 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 supplementary factors, and other aspects are consistent with the normal suspension cell culture method.

[0042] In the following examples, the cells were taken from a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture. The specific acclimation method can be referred to patent CN116555171A. The cells were deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, China, the deposit number is CCTCC NO: C2022372, and the deposit date is December 7, 2022.

[0043] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0044] Example 1 Preparation of serum-free proliferation medium

[0045] Taking the preparation of 1 L of proliferation medium as an example, take 15.78 g of CD02 basal medium powder (Kangsheng Bio, A12002) and prepare it according to the instructions: transfer it to 800 mL of pure water and stir for 10 minutes to mix thoroughly. Adjust the pH of the medium to 9.05 with 5 mol / L sodium hydroxide solution and stir thoroughly for 30 minutes. Then adjust the pH back to 7.0 with 6 mol / L dilute hydrochloric acid. Then add 1.9 g of sodium bicarbonate powder, dissolve it, adjust the pH to 7.0, and make up the solution volume to 1 L with pure water. Finally, adjust the solution osmotic pressure with sodium chloride powder to control it at 300 mOsmol / kg. Stir for 5 minutes to obtain CD02 basal medium.

[0046] The serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention comprises a CD02 basal culture medium supplemented with cell culture supplementary factors. Cell culture supplements were added to the prepared CD02 basal medium. The specific components and concentrations were 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.

[0047] After the cell culture supplement factors are added, the mixture is thoroughly mixed, filtered through a 0.22 μm filter membrane, and stored at 2-8° C. to prepare a serum-free proliferation medium, namely, serum-free medium a.

[0048] Example 2 Preparation of serum-free feed medium

[0049] Feed02 feed medium (Kangsheng Bio, A12902) was dissolved and prepared according to the product instructions provided by the manufacturer to obtain two feed packs, Feed A and Feed B. Cell culture supplement factors were then added according to the corresponding proportions. Taking the preparation of 1 L of feed medium as an example, the specific added components and concentrations were 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 oxamate 0.55 mg / mL, ammonium ferric citrate 0.025 mg / mL, and yeast extract 2.5 mg / mL to form a feed medium, i.e., serum-free medium b.

[0050] After the above materials are added, they are fully mixed, filtered with a 0.22 μm filter membrane, and stored at 2-8° C. to prepare a serum-free feed medium.

[0051] Example 3 Batch Continuous Culture Capacity Testing

[0052] This example uses a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture. The specific acclimation method can be referred to patent CN116555171A. The cells are deposited with the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, China, the deposit number is CCTCC NO: C2022372, and the deposit date is December 7, 2022.

[0053] The cells were cultured in the serum-free proliferation medium prepared in Example 1 at a concentration of 2×10 5 Cells were seeded at a density of 100 cells / mL and cultured in a 125 mL shake flask at a volume of 30 mL. The cells were placed in a shaker at 5% CO2, 37°C, and 120 rpm. Cell density and viability were counted using trypan blue every 3 days. 5 The cells were re-inoculated into 30 mL of serum-free proliferation medium at a density of 100 cells / mL and subcultured.

[0054] The results showed that the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells provided by the present invention can support continuous suspension culture and subculture of porcine muscle stem cells. After 3 days of suspension culture, the cell density reached a maximum of 6.8×10 6 cells / mL, and the average cell density reached 5.0×10 6 / mL or more ( Figure 1 ), the cell viability was maintained above 80% ( Figure 2 ), the supporting cell expansion multiple is about 20-30 times ( Figure 3 ).

[0055] The above results demonstrate that the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention can support efficient expansion of suspension cells in batch continuous culture.

[0056] Example 4: Test of fed-batch culture effect in a bioreactor system

[0057] 1) 5 L bioreactor fed-batch culture test

[0058] This example uses a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture. The specific acclimation method can be referred to patent CN116555171A. The cells are deposited with the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, China, the deposit number is CCTCC NO: C2022372, and the deposit date is December 7, 2022.

[0059] The serum-free culture medium a for in vitro suspension culture of porcine muscle stem cells prepared in Example 1 was used in a 5 L bioreactor at a concentration of 2×10 5Cells were inoculated at an inoculation density of 100 μg / mL and cultured in a 5 L bioreactor at a volume of 2500 mL. Starting from the third day of culture, 10% of the culture volume of the feed medium prepared in Example 2, i.e., serum-free medium b, was supplemented every other day. Cell density and viability were counted daily using trypan blue. The experiment was terminated on the eighth day of culture.

[0060] 2) 80 L bioreactor fed-batch culture test

[0061] An 80 L bioreactor was used with the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells according to the present invention, and 2×10 5 Cells were inoculated at an inoculation density of 100 cells / mL and cultured in an 80-L bioreactor at a volume of 25 L. Starting from the third day of culture, feed medium was supplemented every other day at 10% of the culture volume. Cell density and viability were counted daily using trypan blue. The experiment was terminated on the eighth day of culture.

[0062] The results showed that in the fed-batch suspension culture of a 5 L bioreactor, the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention, in the form of serum-free proliferation medium combined with serum-free feed medium, could achieve a peak cell density of 1×10 7 / mL or more ( Figure 4 ), and maintain cell viability above 90% ( Figure 5 ).

[0063] Similarly, in the fed-batch suspension culture of an 80 L bioreactor, the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of the present invention, in the form of serum-free proliferation medium combined with serum-free feed medium, can achieve a peak cell density of 1×10 7 / mL or more ( Figure 6 ), and maintain cell viability above 90% ( Figure 7 ).

[0064] Comparative Example 1 Detection of the Proliferation-Promoting Effect of Additional Cell Culture Supplementary Factors

[0065] According to the steps described in Example 1, CD02 basal medium was prepared and dissolved, and different cell culture supplement factor combinations were added to the CD02 basal medium to verify the role of some cell culture supplement factors in the present invention.

[0066] Group 1 is a culture medium supplemented with only some cell culture supplement factors. The specific formula is to add L-ascorbic acid-2-phosphate trisodium salt 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.

[0067] Group 2 is a culture medium with additional zinc sulfate added, which is the culture medium of Group 1 with 0.03 mg / mL zinc sulfate added.

[0068] Group 3 is a culture medium supplemented with L-ascorbic acid, which is the culture medium of Group 1 with 0.035 mg / mL of L-ascorbic acid added.

[0069] Group 4 is a culture medium with additional sodium selenite added, which is the culture medium of Group 1 with 0.00005 mg / mL of sodium selenite added.

[0070] Group 5 is a culture medium supplemented with α-tocopherol, which is the culture medium of Group 1 supplemented with 0.013 mg / mL of α-tocopherol.

[0071] Group 6 is a culture medium with additional cholesterol added, which is the culture medium of Group 1 with 0.00022 mg / mL of cholesterol added.

[0072] Group 7 is a culture medium supplemented with N-acetylcysteine, which is the culture medium of Group 1 supplemented with 0.163 mg / mL N-acetylcysteine.

[0073] Group 8 is a culture medium with additional transferrin added, which is the culture medium of Group 1 with 0.02 mg / mL transferrin added.

[0074] Group 9 is a culture medium with additional sodium oxamate added, which is the culture medium of Group 1 with 0.11 mg / mL sodium oxamate added.

[0075] Group 10 is a culture medium with additional ferric ammonium citrate added, which is the culture medium of Group 1 with 0.005 mg / mL ferric ammonium citrate added.

[0076] Group 11 is a culture medium supplemented with yeast extract, which is the culture medium of Group 1 supplemented with 0.5 mg / mL yeast extract.

[0077] Group 12 is the serum-free culture 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.

[0078] After the above materials were added, they were fully mixed, filtered with a 0.22 μm filter membrane, and stored at 2-8°C to prepare serum-free culture media for each group.

[0079] This example uses a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture. The specific acclimation method can be referred to patent CN116555171A. The cells are deposited with the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, China, the deposit number is CCTCC NO: C2022372, and the deposit date is December 7, 2022.

[0080] 2×10 5 Inoculate cells at a density of 100 cells / mL and culture in a 30 mL volume in a 125 mL shake flask. Incubate the cells in a shaker at 5% CO2, 37°C, and 120 rpm. Count cell density and viability on day 3 using trypan blue.

[0081] The results showed that groups 1 to 11 were all culture media with only some cell culture supplementary factors added. The culture media of each group 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. Specifically: in the culture condition of group 1, the number of cells expanded only 8.13 times in 3 days compared with the number of cells inoculated. The culture medium with additional zinc sulfate (Group 2) helped the suspension cells expand 3.75-fold in 3 days, the culture medium with additional L-ascorbic acid (Group 3) helped the suspension cells expand 4.25-fold in 3 days, the culture medium with additional sodium selenite (Group 4) helped the suspension cells expand 4-fold in 3 days, the culture medium with additional α-tocopherol (Group 5) helped the suspension cells expand 4.33-fold in 3 days, the culture medium with additional cholesterol (Group 6) helped the suspension cells expand 7.25-fold in 3 days, the culture medium with additional N-acetylcysteine ​​(Group 7) helped the suspension cells expand 11.67-fold in 3 days, the culture medium with additional transferrin (Group 8) helped the suspension cells expand 4.13-fold in 3 days, the culture medium with additional sodium oxamate (Group 9) helped the suspension cells expand 10.88-fold in 3 days, the culture medium with additional ammonium ferric citrate (Group 10) helped the suspension cells expand 12.25-fold in 3 days, and the culture medium with additional yeast extract (Group 11) helped the suspension cells expand 13.75-fold in 3 days. The serum-free medium for in vitro suspension culture of porcine muscle stem cells (Group 12) of the present invention, which combines all cell culture supplement factors, supports 30.42-fold expansion of suspension cells in 3 days, which is significantly better than other formulas that only add some cell culture supplement factors, indicating that the combination of the present invention has a significant synergistic effect ( Figure 8 ).

[0082] Comparative Example 2

[0083] In order to compare the effects of the serum-free culture medium for in vitro suspension of porcine muscle stem cells of the present invention, this comparative example uses a culture medium with a clear chemical composition for in vitro proliferation of myogenic cells with patent number CN114574433A and a serum-free culture medium for in vitro suspension culture of porcine muscle stem cells of this patent for control experiments, and cultured the porcine muscle stem cell line that has been adapted to carrier-free serum-free suspension culture according to the operating procedure of Example 3.

[0084] 2×10 5 Cells were seeded at a density of 100 cells / mL and cultured in a 125 mL shake flask at a volume of 30 mL. The cells were placed in a shaker at 5% CO2, 37°C, and 120 rpm. Cell density and viability were counted using trypan blue every 3 days. 5 The cells were re-inoculated into 30 mL of serum-free proliferation medium at a density of 100 cells / mL and subcultured.

[0085] The results showed that, compared with a patented culture medium with a clear chemical composition for in vitro proliferation of myogenic cells, it could not support the normal proliferation of a carrier-free, serum-free suspension culture of porcine muscle stem cells within 3 days of culture. The cells were severely clumped and the cell viability was only 30.21%. After counting, it was found that the cells had only expanded 1.48 times. After one passage, all cells died and could not be further passaged ( Figure 9 The patented serum-free culture medium for porcine muscle stem cells in vitro suspension culture can support continuous suspension culture of porcine muscle stem cells. After 3 days of suspension culture, the cell density can reach up to 6.8×10 6 cells / mL, and the average cell density reached 5.0×10 6 The cell viability was maintained at above 80%, and the cell expansion was supported at 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 achieve the technical effect of the technical solution of this patent applied to suspension culture.

[0086] The examples disclosed above are intended to illustrate embodiments of the present invention and are not to be construed as limiting the invention. Numerous different cell culture supplemental factors and their concentration ranges are listed herein, and many further combinations are possible without departing from the scope and spirit of the invention. Therefore, the present invention is not limited to the disclosed embodiments. Indeed, various modifications of the above-described inventions that would be apparent to one skilled in the art are intended to be encompassed within the scope of the present invention.

Claims

1. A serum-free culture medium for in vitro suspension culture of porcine muscle stem cells, characterized in that: It includes cell culture supplement factors and basal culture medium; the serum-free culture medium does not contain serum components; The cell culture supplement 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, transferrin, Trolox, ethanolamine, AM2394, sodium oxamate, ammonium ferric citrate and yeast extract; The concentrations of the cell culture supplement factors in the serum-free medium for in vitro suspension culture of porcine muscle stem cells 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, and 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.

2. The serum-free culture medium according to claim 1, wherein The basal culture medium is a basal proliferation medium and / or a feed medium; the serum-free culture medium is divided into: Serum-free medium a: When the basal medium is a basal proliferation medium, the prepared medium is a serum-free medium a 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 feed medium, the prepared medium is a serum-free medium b 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; and the feed medium is selected from Feed02 feed medium.

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

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