Effects Of Vitamin C On Porcine Muscle Stem Cells Proliferation Differentiation And Its Applications In Cultured Meat

Cultured meat,as a novel meat product,can effectively alleviate the negative effects of traditional animal husbandry on the environment,public health,and animal welfare.Muscle stem cells are the main seed cells for cultured meat.However,the in vitro growth of muscle stem cells requires high concentrations of fetal bovine serum and it is difficult to maintain the myogenic differentiation potential during proliferation,which severely limits the large-scale and low-cost production of cultured meat.In this study,we found that Vitamin C,an inexpensive food-grade compound,significantly enhanced the proliferation and differentiation of primary porcine muscle stem cells in vitro,and further analyzed the related mechanism,which will provide a practical method and theoretical basis for the efficient and low-cost manufacture of cultured meat.The main contents and results are as follows.（1）Investigation of the effect of Vitamin C on the proliferation of porcine muscle stem cells in vitro.First,the optimal concentration of Vitamin C for promoting proliferation turned out to be 100μM through thiazole blue（MTT）cell proliferation assay with freshly isolated primary porcine muscle stem cells.Then,the long-term in vitro culture experiments revealed that porcine muscle stem cells expanded by 2.8×10⁷±0.8×10⁷ folds with Vitamin C treatment in 29 days,which was approximately 360 times that of the control group.At the same time,Vitamin C treatment increased the proliferation rate of porcine muscle stem cells,accelerated the cell cycle,and increased the expression level of proliferation-related genes.It illustrated that Vitamin C can promote the proliferation of porcine muscle stem cells in vitro.（2）Investigation of the mechanism of Vitamin C promoting the proliferation of porcine muscle stem cells.Firstly,by comparing Vitamin C with other antioxidants（acetylcysteine,glutathione,vitamin E）and pro-collagen synthesis agents（transforming growth factor-β）,it was found that the pro-proliferative effect of Vitamin C on porcine muscle stem cells was not dependent on antioxidant activity and collagen synthesis activity.Subsequently,signaling pathway inhibition assays and core protein phosphorylation levels showed that Vitamin C activated the PI3K/Akt/m TOR/P70S6K signaling pathway by increasing insulin-like growth factor-1（IGF-1）expression,which caused a pro-proliferative effect on porcine muscle stem cells.（3）Investigation of the effect of Vitamin C on the myogenic potential and myogenic differentiation of porcine muscle stem cells.The differentiation rate and fusion rate of porcine muscle stem cells were dramatically raised after Vitamin C treatment with different days during the proliferation phase and then induced to differentiation.After 15 days of Vitamin C treatment,myosin My HC expression increased to 6.52 times of the control group.Furthermore,the addition of 200～400μM Vitamin C during the differentiation process of porcine muscle stem cells increased the cell fusion rate by 1.2～2.0 times and improved the expression level of My HC by 2～3 times,indicating that Vitamin C can maintain the myogenic potential of porcine muscle stem cells and promote myogenic differentiation.（4）Investigation of the mechanism of Vitamin C promoting myogenic differentiation of porcine muscle stem cells.First,Western Blot assays demonstrated that Vitamin C elevated intracellular IGF-1 expression during differentiation and activated the PI3K/Akt/m TOR pathway with a 1.3～2.0 fold increase in core protein phosphorylation levels.Subsequent molecular docking and surface plasmon resonance experiments demonstrated a strong interaction between Vitamin C and the IGF-1 receptor,with an interaction energy of-26.2728kcal/mol.（5）Investigation of the application of Vitamin C in the large-scale manufacturing of cultured meat.Firstly,the formulation of the serum-free medium was optimized with Vitamin C.The differentiation efficiency and differentiation-related proteins were examined by immunofluorescence and Western Blot,which proved that the serum-free medium was comparable to the serum-containing medium.Simultaneously,the microcarrier coating methods and cell seeding density were also optimized.The results showed that the cell adhesion and survival rates were significantly increased when coating microcarriers with Matrigel and Vitamin C at an inoculation density of 1.6×10⁴ cm^-2,which laid the foundation for the large-scale expansion of porcine muscle stem cells for cultured meat production.