A low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells and its application.

Abstract

This invention provides a low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells and its applications. It includes cell culture supplementary factors and a basal culture medium; the cell culture supplementary factors include: zinc sulfate, L-ascorbic acid, bFGF, IGF-1, LIF, EGF, cortisol, HGF, α-tocopherol, PVA, β-cyclodextrin, methylcellulose, and yeast extract. By simplifying and optimizing the added factors, and incorporating specific types and concentrations of cell culture supplementary factors, efficient suspension expansion of porcine muscle stem cells under serum-free and carrier-free conditions is achieved. This invention's culture medium significantly reduces the cost of adding exogenous factors, avoids various potential risk substances, and while ensuring efficient cell growth and simplifying the process, improves the food safety and economy of the culture medium, providing an important technical and raw material foundation for the industrial production of cell-cultured meat.

Description

Technical Field

[0001] This invention belongs to the field of serum-free culture medium for suspension cells, especially in the field of stem cell and animal cell culture for meat. Specifically, it relates to a low-cost, safe serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells and its application. Background Technology

[0002] Meat, as an important food source for humans, is rich in protein, fat, and various micronutrients. With continuously rising demand for meat, traditional livestock farming methods are no longer sufficient to meet the widening supply gap. Therefore, developing green and efficient meat production technologies to partially replace traditional farming methods has become an urgent need to achieve a sustainable meat supply.

[0003] Cell-cultured meat technology, based on the principles of animal muscle growth, produces meat by culturing stem cells in vitro. It eliminates the need for the entire animal husbandry process and allows for large-scale production directly in cell factories. This disruptive production technology offers a new avenue for supplementing future meat supplies and promoting green production. Research shows that compared to traditional livestock farming, cell-cultured meat can reduce energy consumption by 35%–60%, save 98% of land use, and reduce greenhouse gas emissions by more than 80%.

[0004] Porcine muscle stem cells are considered key seed cells for cultured meat due to their excellent in vitro proliferation and differentiation potential. However, the commonly used two-dimensional adherent culture mode or in vitro culture system for these cells still faces significant challenges in terms of large-scale expansion efficiency and production cost. In existing technologies, although some studies have addressed these challenges with serum-free culture medium solutions with clearly defined chemical compositions, such as the previously developed and disclosed technology for suspension culture of porcine muscle stem cells (see patent ZL202510757736.1), such solutions usually rely on the addition of a wide variety of exogenous factors, resulting in complex culture medium preparation, high raw material costs, and some added components not fully meeting the regulatory requirements for food-grade raw materials, or posing potential food safety approval risks (the safe residue limit for foscorine is 485 mg / kg, and the residue limit for N-acetylcysteine ​​is 5.71 mg / kg, etc.). For example, the "Regulations for the Review of Safety of New Food Raw Materials," the "Guidelines for Application Materials for Safety Assessment Opinions of New Food Raw Materials (Trial)," and the "Guidelines for the Approval of New Food Raw Materials" all impose requirements on food safety. Therefore, its production is limited in terms of large-scale and compliant production of cell-cultured meat, a final food product, and needs further optimization.

[0005] Therefore, developing a serum-free culture medium that is simplified in composition, low in cost, highly safe, and can support efficient cell expansion for suspension culture of porcine muscle stem cells is of vital importance for promoting the industrialization of cell-cultured meat. Summary of the Invention

[0006] To address the problems of complex composition, high cost, and potential safety risks in existing culture media, the present invention aims to provide a novel serum-free culture medium that, through careful selection and optimization of key additives, reduces the number of additives compared to patent ZL202510757736.1. Figure 1 At the same time, it ensures efficient support for the suspension expansion of porcine muscle stem cells, thereby significantly reducing the cost of culture medium and improving its food safety.

[0007] The term "serum-free" as used in this invention refers to the absence of any animal-derived serum components, including but not limited to fetal bovine serum, bovine serum, equine serum, or human serum, in the culture medium.

[0008] The low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells is formulated by adding specific types and concentrations of cell culture supplementary factors to the basal culture medium, and is used to replace traditional serum-containing culture media or complex and expensive serum-free culture media.

[0009] The technical solution of this invention is as follows:

[0010] The first objective of this invention is to provide a low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells, comprising cell culture supplementation factors and a basal culture medium; wherein the serum-free culture medium contains no serum components of any animal origin;

[0011] The cell culture supplement factors include: zinc sulfate, L-ascorbic acid, bFGF, IGF-1, LIF, EGF, cortisol, HGF, α-tocopherol, PVA, β-cyclodextrin, methylcellulose, and yeast extract.

[0012] Furthermore, the basal culture medium is a basic proliferation medium; the serum-free culture medium is a serum-free culture medium used for in vitro batch suspension culture or passage (bleeding) culture of porcine muscle stem cells.

[0013] Furthermore, the basic proliferation medium is selected from one or more of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, and CD02 medium. The CD02 medium is based on the original CHO CD02 medium (e.g., Kang Sheng Biotechnology, QuaCell® CHO CD02 original medium, A12002), with 4-hydroxyethylpiperazine ethanesulfonic acid and dextran sulfate sodium removed, and the remaining dosages and preparation methods are prepared in the same manner as described in the original medium instructions.

[0014] Furthermore, in the serum-free culture medium, the total concentration of cell culture supplementary factors ranges from 0.02 to 40 mg / mL.

[0015] Furthermore, in the serum-free culture medium, the concentration of any single cell culture supplement factor ranges from 0.0000001 to 10 mg / mL.

[0016] Furthermore, the concentration range of any single cell culture supplement factor is 0.000001-3 mg / mL.

[0017] Furthermore, the concentration range of any single cell culture supplement factor is 0.00001-1 mg / mL.

[0018] Furthermore, the concentration range of any single cell culture supplement factor is 0.00002 - 0.5 mg / mL.

[0019] Furthermore, the concentrations of cell culture supplementary factors added to the serum-free culture medium are as follows: zinc sulfate 0.03 mg / mL, L-ascorbic acid 0.035 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, α-tocopherol 0.013 mg / mL, PVA 0.3 mg / mL, β-cyclodextrin 0.02 mg / mL, methylcellulose 0.0002 mg / mL, and yeast extract 0.5 mg / mL.

[0020] The second objective of this invention is to provide the application of the aforementioned low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells in the in vitro suspension expansion culture of porcine muscle stem cells, wherein the serum-free culture medium is used for batch suspension culture or passage culture of porcine muscle stem cells in vitro.

[0021] Furthermore, the application is to increase the cell expansion rate and / or shorten the cell doubling time in the in vitro suspension culture of porcine muscle stem cells and / or increase the final cell culture density and / or maintain a high cell viability, and to pass safety testing.

[0022] Furthermore, using the serum-free culture medium for batch suspension culture, porcine muscle stem cells can be expanded more than 10-fold within 2 days, and the cell doubling time is shortened to 13-14 hours.

[0023] Furthermore, culturing with the serum-free medium can support a peak density of porcine muscle stem cells of 1×10⁻⁶. 7Cells / mL or higher, and maintaining cell viability above 90%.

[0024] The beneficial effects achieved by the technical solution of this invention are as follows:

[0025] (1) Significantly Reduced Costs and Enhanced Safety: This invention significantly reduces the types and amounts of exogenous factors by simplifying and optimizing the composition of the culture medium. Compared to the complex serum-free culture medium formulations in existing technologies, the cost of added factors in the culture medium of this invention can be reduced from hundreds of yuan per liter to tens of yuan, a significant reduction. At the same time, the formulation avoids many potentially hazardous substances, such as 4-hydroxyethylpiperazine ethanesulfonic acid, dextran sulfate sodium, forscolin, and N-acetylcysteine. This improves the food safety of the culture medium and better meets the requirements of large-scale production of cell-cultured meat products for raw material costs and safety supervision.

[0026] (2) Supporting efficient cell expansion: This invention simplifies the culture medium composition and effectively supports the suspension growth of porcine muscle stem cells under serum-free and carrier-free conditions through the rational ratio of key growth factors (such as bFGF, IGF-1, etc.), anti-clustering factors (such as PVA), protectants, and nutritional supplements (such as yeast extract). Experiments show that using this culture medium for batch culture, compared with patent ZL202510757736.1, the cell doubling time is shortened from 15-16 hours to 13-14 hours, achieving more than 10-fold expansion within 2 days, and obtaining a high peak cell density. For example, a small-scale trial in a shake flask can reach approximately 9.17 × 10⁻⁶ cells / year. 6 The concentration of cells / mL can reach 1×10⁻⁶ in a 5L bioreactor. 7 The concentrations per mL were all above 5.0 × 10⁻⁶, exceeding those of patent ZL202510757736.1. 6 Cells / mL; while maintaining high cell viability, usually above 90%, higher than the 80% of patent ZL202510757736.1.

[0027] (3) Simplified process, conducive to scale-up: The simplification of the culture medium components makes the preparation process simpler and quality control easier to achieve, laying a good foundation for the stability of the production process and large-scale scale-up. Whether in shake flask or bioreactor system, the culture medium shows good compatibility and stable culture effect.

[0028] In summary, the low-cost, safe, serum-free culture medium provided by this invention effectively solves the bottleneck problem of balancing cost, safety, and efficient amplification in large-scale suspension culture of seed cells for cultured meat, providing important technical and raw material support for the industrial production of cultured meat. Attached Figure Description

[0029] Figure 1A comparison list of serum-free culture medium for in vitro suspension culture of porcine muscle stem cells in patent number ZL202510757736.1 and the culture medium formulation of the present invention.

[0030] Figure 2 The present invention provides a low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells, supporting cell culture density curves for each generation of continuous batch passage culture of porcine muscle stem cells.

[0031] Figure 3 The present invention provides a low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells, supporting cell viability curves for each generation in continuous batch passage culture of porcine muscle stem cells.

[0032] Figure 4 The present invention provides a low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells, supporting the statistical analysis of cell culture folds per generation in continuous batch passage culture of porcine muscle stem cells.

[0033] Figure 5 The present invention provides a low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells, showing the cell density change curves supporting continuous culture of porcine muscle stem cells in a 5 L bioreactor system.

[0034] Figure 6 The cell viability curve of the low-cost, safe, serum-free culture medium of the present invention for efficient in vitro suspension culture of porcine muscle stem cells supports continuous culture of porcine muscle stem cells in a 5 L bioreactor system.

[0035] Figure 7 Statistical chart showing the effect of supplemental factors on cell culture in promoting cell proliferation.

[0036] Figure 8 Cell culture density curves for batch passage culture of porcine muscle stem cells supported by serum-free culture medium with patent number ZL202510757736.1.

[0037] Figure 9 Chromatogram of residual Torrox in cultured meat in the culture medium formulation of this invention.

[0038] Figure 10 Chromatogram of AM2394 residue in cultured meat in the culture medium formulation of this invention.

[0039] Figure 11 Chromatogram of residual sodium dextran sulfate in cultured meat in the culture medium formulation of this invention.

[0040] Figure 12 Chromatogram of residual 4-hydroxyethylpiperazine ethanesulfonic acid in cultured meat in the culture medium formulation of the present invention.

[0041] Figure 13 Chromatogram of forsocolin residue in cultured meat in the culture medium formulation of this invention.

[0042] Figure 14 Chromatogram of N-acetylcysteine ​​residues in cultured meat in the culture medium formulation of this invention.

[0043] Figure 15 Chromatogram of thymidine residues in cultured meat in the culture medium formulation of this invention. Detailed Implementation

[0044] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0045] The core of this invention lies in the low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells, which is determined by the specific combination and concentration ratio of added cell culture supplementary factors in the basal culture medium. Apart from the culture medium formulation, other operating conditions for cell culture can be performed according to conventional suspension culture methods in the art.

[0046] In the following examples, the cells used were porcine muscle stem cell lines adapted to carrier-free and serum-free suspension culture. For specific acclimatization methods, please refer to patent CN116555171A. These cells are deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, China, with accession number CCTCC NO:C2022372 and deposit date of December 7, 2022.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] Example 1: Preparation of serum-free proliferation culture medium

[0049] Taking the preparation of 1L serum-free proliferation medium as an example, take 13.52 g of CD02 basal medium powder (Kangsheng Biotechnology, A12002, CHOCD02 original medium with 4-hydroxyethylpiperazine ethanesulfonic acid and dextran sodium sulfate removed, the remaining dosage and preparation method are the same as those in the original medium instructions) and prepare it according to the instructions: transfer it to 800 mL of purified water and stir for 10 minutes to mix well. 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 / L sodium bicarbonate powder, dissolve it and adjust the pH to 7.0. Make up the volume of the solution to 1 L with purified water. 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 CD02 basal medium.

[0050] The present invention provides a low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells, comprising a CD02 basal medium supplemented with cell culture supplementation factors.

[0051] Cell culture supplements were added to the prepared CD02 basal medium. The specific components and concentrations added were as follows: zinc sulfate 0.03 mg / mL, L-ascorbic acid 0.035 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, α-tocopherol 0.013 mg / mL, PVA 0.3 mg / mL, β-cyclodextrin 0.02 mg / mL, methylcellulose 0.0002 mg / mL, and yeast extract 0.5 mg / mL. After the cell culture supplements were added, the mixture was thoroughly mixed, filtered through a 0.22 μm filter membrane, and stored at 2–8°C to prepare serum-free proliferation medium.

[0052] Example 2: Detection of Batch Continuous Culture Capacity

[0053] This embodiment uses a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture. For specific domestication methods, please refer to patent CN116555171A. This cell line is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, China, with accession number CCTCC NO:C2022372 and deposit date of December 7, 2022.

[0054] Cells were cultured in the serum-free proliferation medium prepared in Example 1 at 8 × 10⁻⁶. 5 Cells were seeded at a density of 8 cells / mL in 30 mL flasks and cultured in a shaker at 37°C with 5% CO2 at 120 rpm. Cell density and viability were assessed every two days using trypan blue. Cells were then seeded again at a density of 8 × 10⁻⁶ cells / mL. 5 Re-inoculate at a density of cells / mL into 30 mL of serum-free proliferation medium and continue subculture.

[0055] The results showed that the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells provided by this invention can support continuous suspension culture and passage of porcine muscle stem cells. After 2 days of suspension culture, the highest supporting cell density reached 9.81 × 10⁻⁶. 6 The cell density reached 9.17 × 10⁶ cells / mL. 6 More than 1 / mL ( Figure 2 The cell viability remained above 90%. Figure 3 The cell doubling time is 13-14 hours, and the proliferation is 10-fold or more. Figure 4 ).

[0056] The above results demonstrate that the low-cost, safe, serum-free culture medium of the present invention for efficient in vitro suspension culture of porcine muscle stem cells can support efficient expansion of suspension cells in batch continuous culture.

[0057] Example 3: Test of Bleeding culture effect in bioreactor system

[0058] 5 L bioreactor passage Bleeding culture test

[0059] This embodiment uses a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture. For specific domestication methods, please refer to patent CN116555171A. This cell line is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, China, with accession number CCTCC NO:C2022372 and deposit date of December 7, 2022.

[0060] Using a 5 L bioreactor and the low-cost, safe, serum-free culture medium of the present invention for efficient in vitro suspension culture of porcine muscle stem cells prepared in Example 1, the culture was carried out at 8 × 10⁸ L. 5 Cells were seeded at a density of [number] cells / mL and cultured in a 5L bioreactor at a volume of 2500 mL. Cell density and viability were counted daily using trypan blue. After 48 hours, the density was reduced to 8 × 10⁶ cells / mL. 5 The culture was carried out at a rate of 10 cells / mL, and an equal volume of fresh culture medium was added to continue the culture. The experiment was terminated on the 10th day of culture.

[0061] The results showed that, in the Bleeding culture of a 5 L bioreactor, the low-cost, safe, serum-free culture medium of the present invention for efficient in vitro suspension culture of porcine muscle stem cells could achieve a peak cell density of 1 × 10⁻⁶ cells / year. 7 More than 1 / mL ( Figure 5 And maintain cell viability above 90% ( Figure 6 ).

[0062] Example 4: Detection of Hazardous Substances

[0063] In this embodiment, cells produced by the 5L bioreactor in Example 3 (20 batches in parallel) were used. Approximately 0.2g of cell culture biomass was weighed and added to 1 mL of acetonitrile-methanol-water mixture (1:1:1, v / v / v) containing 0.1% formic acid and 1mM Na2EDTA. The mixture was vortexed, ultrasonically extracted for 30 min, centrifuged (12000 rpm, 10 min), and the supernatant was collected. The supernatant was dried under nitrogen and reconstituted with the extraction solvent to 1 mL. After mixing, the supernatant was filtered through a 0.22 μm filter membrane and placed into a sample vial for analysis.

[0064] This method is used to detect Tolox, AM2394, 4-hydroxyethylpiperazine ethanesulfonic acid, forscorin, thymidine, and N-acetylcysteine.

[0065] Table 1: Detection parameters for various hazardous substances

[0066]

[0067] Meanwhile, in this embodiment, cells produced by the 5L bioreactor in Example 3 (20 batches in parallel) were used. Approximately 0.2g of cell culture biomass was weighed, 0.8 mL of distilled water was added, and the mixture was extracted by sonication for 30 min. After centrifugation (12000 rpm, 10 min), the supernatant was collected, and 50% trichloroacetic acid aqueous solution (200 mg / mL) was added. After centrifugation (12000 rpm, 10 min), the supernatant was collected, and the pH of the supernatant was adjusted to 6.7-7.0. 400 μL of the supernatant was collected, and 20 μL of 2-aminopyridine aqueous solution (20 ng / mL) was added. 332 μL of distilled water was added, and the mixture was incubated at 100℃ for 13 min. After cooling to room temperature, 48 μL of sodium cyanoborohydride aqueous solution (1 mg / mL) was added, and the mixture was incubated at 95℃ for 20 h. After centrifugation (12000 rpm, 10 min), the supernatant was filtered through a 0.22 μm filter membrane and placed into a sample vial for analysis. The liquid chromatography conditions were as follows: mobile phase consisted of 4.4 g / L disodium hydrogen phosphate dodecahydrate and 13.7 g / L sodium dihydrogen phosphate dihydrate, with isocratic elution; column temperature: 50℃; flow rate: 1.2 mL / min; injection volume: 40 μL; column: Sepax Zenix SEC-150 (7.8×300 mm); FLD detector: excitation wavelength: 320 nm; emission wavelength: 400 nm.

[0068] This method is used to detect sodium dextran sulfate.

[0069] Table 2: Detection Limits for Each Hazardous Substance

[0070]

[0071] The results showed that removing Tolox ( ) from this culture medium formulation Figure 9 AM2394 Figure 10 ), sodium dextran sulfate ( Figure 11 ), 4-hydroxyethylpiperazine ethanesulfonic acid ( Figure 12 ) and N-acetylcysteine ​​( Figure 14 After that, the residues of these substances were undetectable. However, fuscalid (… Figure 13 ) and thymidine ( Figure 15 The removal of sodium dextran sulfate, which increases cell permeability, also resulted in a significant decrease in its concentration within cells. All substances were within safety limits.

[0072] Comparative Example 1: Detection of the proliferation-promoting effect of additional cell culture supplementary factors.

[0073] Following the steps described in Example 1, CD02 basal culture medium was prepared and dissolved. Different combinations of cell culture supplementary factors were added to the CD02 basal culture medium to verify the role of some cell culture supplementary factors in this invention.

[0074] Group 1 was a culture medium supplemented with only a portion of cell culture supplementary factors. The specific formulation was as follows: 0.03 mg / mL zinc sulfate, 0.035 mg / mL L-ascorbic acid, 0.0001 mg / mL bFGF, 0.0002 mg / mL IGF-1, 0.00002 mg / mL LIF, 0.0001 mg / mL EGF, 0.000072 mg / mL cortisol, 0.00005 mg / mL HGF, 0.013 mg / mL α-tocopherol, and 0.5 mg / mL yeast extract were added to CD02 basal medium.

[0075] Group 2 was a culture medium with additional PVA, which was based on Group 1 with the addition of 0.3 mg / mL of PVA.

[0076] Group 3 was a culture medium with additional β-cyclodextrin, which was based on Group 1 with the addition of 0.02 mg / mL of β-cyclodextrin.

[0077] Group 4 was a culture medium with additional methylcellulose, which was based on Group 1 with the addition of 0.0002 mg / mL of methylcellulose.

[0078] Group 5 is the low-cost, safe, serum-free culture medium for efficient in vitro suspension culture of porcine muscle stem cells of the present invention, with the same factor addition as in Example 1.

[0079] After adding all the above materials, mix them thoroughly and evenly, filter them through a 0.22 μm filter membrane, and store them at 2-8℃ to prepare serum-free culture media for each group.

[0080] This embodiment uses a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture. For specific domestication methods, please refer to patent CN116555171A. This cell line is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, China, with accession number CCTCC NO:C2022372 and deposit date of December 7, 2022.

[0081] With 8×10 5 Cells were seeded at a density of 1 cell / mL and cultured in 30 mL volumes in 125 mL shake flasks. The cells were placed in a shaker at 37°C with 5% CO2 concentration and a rotation speed of 120 rpm. On the second day, cell density and viability were assessed using trypan blue.

[0082] The results showed that groups 1 to 4 were culture media with only partial cell culture supplements. Each group's culture media could only support the survival of suspended cells, but the cell proliferation capacity was significantly weaker than that of group 5 of the present invention. Specifically, in group 1, the cell proliferation was only 3.70-fold after 2 days compared to the number of inoculated cells. Culture media with additional PVA (group 2) helped suspended cells proliferate 4.77-fold after 2 days; culture media with additional β-cyclodextrin (group 3) helped suspended cells proliferate 5.68-fold after 2 days; and culture media with additional methylcellulose (group 4) helped suspended cells proliferate 5.18-fold after 2 days. In contrast, the serum-free culture medium of the present invention for in vitro suspension culture of porcine muscle stem cells (group 5), which combines all cell culture supplements, supported a 12.08-fold increase in suspended cells after 2 days, and the doubling time was shortened from 20-30 hours in groups 1 to 4 to 13-14 hours, significantly better than other formulations with only partial cell culture supplements. This shows that the combined formulation of the present invention has a significant synergistic effect, and that not all ratios can achieve the effects of the present invention. Figure 7 ).

[0083] Comparative Example 2

[0084] To compare and demonstrate the effectiveness of the low-cost, safe, and serum-free culture medium for efficient in vitro suspension of porcine muscle stem cells of the present invention, this comparative example uses a serum-free culture medium for in vitro suspension culture of porcine muscle stem cells (patent number ZL202510757736.1) and the low-cost, safe, and serum-free culture medium for efficient in vitro suspension of porcine muscle stem cells of the present invention as a control experiment. The porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture was cultured according to the operation procedure of Example 2.

[0085] With 8×10 5Cells were seeded at a density of 8 cells / mL in 30 mL flasks and cultured in a shaker at 37°C with 5% CO2 at 120 rpm. Cell density and viability were assessed every two days using trypan blue. Cells were then seeded again at a density of 8 × 10⁻⁶ cells / mL. 5 Re-inoculate at a density of cells / mL into 30 mL of serum-free proliferation medium and continue subculture.

[0086] The results showed that, compared with the serum-free culture medium for in vitro suspension culture of porcine muscle stem cells (patent number ZL202510757736.1), the average proliferation density was 5.78 × 10⁻⁶ after 2 days of culture. 6 Cells / mL, cell proliferation 7.23-fold ( Figure 8 The low-cost, safe, serum-free culture medium of the present invention for efficient in vitro suspension of porcine muscle stem cells can support continuous suspension culture and passage of porcine muscle stem cells. After 2 days of suspension culture, the supporting cell density reached a maximum of 9.8 × 10⁻⁶ cells / year. 6 The cell density reached 9.17 × 10⁶ cells / mL. 6 The cell count / mL was above 90%, supporting cell expansion at approximately 10-12 times, with the doubling time shortened to 13-14 hours, and a significantly improved harvest density (Example 2). It can be seen that although both methods are applied to the in vitro proliferation and culture of porcine muscle stem cells, the suspension culture medium of ZL202510757736.1 cannot achieve the technical effects of the present invention applied to efficient suspension culture.

[0087] Comparative Example 3

[0088] To compare the food safety of the low-cost, safe, serum-free culture medium for efficient in vitro suspension of porcine muscle stem cells of the present invention, this comparative example compares the concentrations of the risky substances Forskolin, N-acetylcysteine ​​(NAC), Trolox, sodium dextran sulfate, and 4-hydroxyethylpiperazine ethanesulfonic acid in the ZL202510757736.1 formulation with those in cells harvested using the present formulation. 4-hydroxyethylpiperazine ethanesulfonic acid and sodium dextran sulfate are present in the CD02 culture medium of the ZL202510757736.1 patent. Simultaneously, the concentration of the endogenous product thymidine in cultured meat obtained from the formulations of the present invention and ZL202510757736.1 is compared.

[0089] Different methods were used for residue detection for different substances. Pretreatment of forcoline, N-acetylcysteine, toloxetine, AM2394, thymidine, and 4-hydroxyethylpiperazine ethanesulfonic acid: Cell culture meat samples were extracted using a mixed solution of acetonitrile:methanol:water (volume ratio 1:1:1) containing 0.1% formic acid and 1mM Na2EDTA. The samples were then sonicated, centrifuged, and filtered to obtain the test samples, which were then detected using LC-MS / MS. The detection of sodium dextran sulfate was performed according to method CN119827646A.

[0090] The results showed that the contents of cellular hazardous substances in the formulation of this invention were as follows: fuscalin 17.32 μg / kg, N-acetylcysteine ​​<3.79 μg / kg, toloxetine <0.27 μg / kg, AM2394 <0.21 μg / kg, thymidine 71.21 μg / kg, sodium dextran sulfate <1000.00 μg / kg, and 4-hydroxyethylpiperazine ethanesulfonic acid <0.45 μg / kg. Compared with patent ZL202510757736.1, the concentrations of all hazardous substances were significantly reduced.

[0091] Table 3: Comparison of concentrations of various hazardous substances in cultured meat with different formulations

[0092]

[0093] The examples disclosed above are for illustrative purposes only and should not be construed as limitations of the invention. This document lists many different cell culture supplementary factors and their concentration ranges. Many different combinations are possible without departing from the scope and spirit of the invention. Therefore, the invention is not limited to the disclosed embodiments. In fact, all modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A serum-free medium for efficient suspension culture of porcine muscle stem cells in vitro, characterized by, The culture medium consists of cell culture supplements and a basal medium; the serum-free medium contains no animal-derived serum components; the cell culture supplements are: zinc sulfate, L-ascorbic acid, bFGF, IGF-1, LIF, EGF, cortisol, HGF, α-tocopherol, PVA, β-cyclodextrin, methylcellulose, and yeast extract; the concentration range of any single cell culture supplement is 0.00002-0.5 mg / mL; the basal proliferation medium is CD02 medium, which is based on the original CHOCD02 medium with 4-hydroxyethylpiperazine ethanesulfonic acid and dextran sulfate sodium removed.

2. The serum-free medium according to claim 1, characterized in that, The serum-free culture medium contains the following cell culture supplements at the following concentrations: zinc sulfate 0.03 mg / mL, L-ascorbic acid 0.035 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, α-tocopherol 0.013 mg / mL, PVA 0.3 mg / mL, β-cyclodextrin 0.02 mg / mL, methylcellulose 0.0002 mg / mL, and yeast extract 0.5 mg / mL.

3. Use of the serum-free medium according to claim 1 for the in vitro expansion of porcine muscle stem cells in suspension, characterized in that, The serum-free culture medium was used to perform in vitro batch suspension culture or passage culture of porcine muscle stem cells.

4. Use according to claim 3, characterized in that, The application is to increase the cell expansion rate and / or shorten the cell doubling time in the in vitro suspension culture of porcine muscle stem cells and / or increase the final cell culture density and / or maintain a high cell viability.

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