Efficient Proliferation Of Porcine Muscle Stem Cells Regulated By Cytokine Combination

Cultured meat is a novel protein resource and meat product that can effectively alleviate the negative impact of traditional animal husbandry on the environment,public health,and animal welfare.It provides an efficient,safe,and sustainable animal protein supply solution.Muscle stem cells,as the main seed cells for producing cultured meat,have the potential to differentiate to form muscle fibers.However,during ex vivo culture,muscle stem cells require fetal bovine serum（FBS）to maintain their growth,and their proliferation and differentiation ability decreases significantly with the extension of culture time,which makes it difficult to realize large-scale and low-cost culture of muscle stem cells ex vivo,and this has seriously limited the industrialized production of cultured meat.Therefore,there is an urgent need to develop a culture system that can effectively regulate the proliferation and differentiation of muscle stem cells,obtain more cells with myogenic differentiation potential within a limited time,and establish a large-scale and low-cost culture system for muscle stem cells.This study focuses on porcine muscle stem cells.A cytokine combination that can efficiently promote the proliferation of porcine muscle stem cells ex vivo was screened based on the serial elimination screening approach.The cytokine combination was co-expressed in the yeast genome using Saccharomyces cerevisiae and applied in the culture of porcine muscle stem cells at low cost.Meanwhile,the intracellular signaling pathways and cellular energy metabolism pathways through which the cytokine combination functions were explored.Further,a low-cost and serum-free medium was developed and a suspension culture system for porcine muscle stem cells was constructed based on a WAVE reactor,laying the foundation for the industrial production of cultured meat.The main research results are as follows:（1）A cytokine combination that efficiently promotes the proliferation of muscle stem cells was obtained based on the serial elimination screening approach,and its function was verified.First,high-purity porcine muscle stem cells were isolated from young pig muscle fibers,and an optimal cytokine combination was identified through 5 rounds of screening based on the serial elimination screening approach from 12 cytokines:basic fibroblast growth factor（b FGF）,long-chain human insulin growth factor-1（LR³-IGF-1）,epidermal growth factor（EGF）and platelet-derived growth factor BB（PDGF-BB）,which can effectively promote the long-term and efficient proliferation of muscle stem cells ex vivo under the effect of cytokine combination.Under the condition of reducing the FBS usage by half in the conventional porcine muscle stem cell proliferation medium（from 10%FBS to 5%FBS）,when the cytokine combination was added,porcine muscle stem cells were continuously cultured ex vivo for 28 days.The cell number reached 3.15×10¹¹,and the expansion fold reached 6.31×10⁷,which was approximately 3.01×10⁴ times higher than the 5%FBS group,the number of divisions increased by more than 2.42 times,and the doubling time was reduced by more than half.Compared to the conventional porcine muscle stem cell proliferation medium（10%FBS）,the expansion fold increased by about 6.12 times,and the number of divisions increased by 1.14times.Meanwhile,the cytokine combination also maintained the myogenic properties of the porcine muscle stem cells,and more cells with myogenic differentiation ability could be obtained after 28 days of culture,which was about 4.58×10⁴-fold higher than that of the 5%FBS group and about 2.11-fold higher than that of the 10%FBS group.（2）The co-expression of cytokine combination in the genome was realized by using S.cerevisiae as the chassis strain,and the yeast lysate containing the recombinant cytokine combination was obtained by lysis to effectively promote the proliferation of porcine muscle stem cells ex vivo.First,a cytokine expression system was established using the S.cerevisiae C800 as a starter strain to achieve episomal expression and stable expression of the cytokines b FGF,EGF,LR³-IGF-1,and PDGF-BB.The cytokine yields were further improved by promoter optimization,yeast endogenous protease knockout,etc.The cytokine yields of b FGF,EGF,LR³-IGF-1,and PDGF-BB could reach up to 1108.07μg/L,63.82μg/L,941.79μg/L,and86.32μg/L,respectively.Second,the co-expression of the four cytokines at the multi-copy site Ty2 of the S.cerevisiae genome was realized using the Cre-lox P system.A recombinant S.cerevisiae strain CPK2B2 capable of co-expressing four cytokines was obtained,and the cytokine yield was further increased to 18.35 mg/L by optimizing the gene order of the cytokine expression frame and fed-batch fermentation to the 3 L fermenter.Finally,the CPK2B2 lysate was obtained by physical lysis,filtered and added directly to the culture medium of porcine muscle stem cells,and only 1 g/L of CPK2B2 lysate could effectively promote the proliferation of porcine muscle stem cells,and significantly reduce the cost of cytokine application in porcine muscle stem cell culture.（3）The intracellular signaling pathways and cellular energy metabolism pathways of cytokine combination were analyzed.First,the gene and protein expression levels of key differential genes were verified by transcriptome analysis after the cytokine combination acted on porcine muscle stem cells.It was found that the cytokine combination accelerated the cell cycle,significantly shortened the cell doubling time and improved the cell proliferation efficiency by increasing the protein phosphorylation level and activating the PI3K/Akt/m TOR and MAPK/MEK signaling pathways after acting on porcine muscle stem cells.Meanwhile,by analyzing the level of cellular energy metabolism,it was found that the cytokine combination significantly increased the rate of ATP generated by oxidative phosphorylation and the rate of total ATP production in porcine muscle stem cells,which provided more energy for cell proliferation.（4）The optimal microcarrier for porcine muscle stem cells was screened and optimized,and the suspension culture system of porcine muscle stem cells was established based on the WAVE bioreactor and serum-free medium.First,the most suitable microcarrier HDMC for porcine muscle stem cells was screened,and the cell viability and harvesting rate could reach more than 90%,and the culture conditions of porcine muscle stem cells on HDMC were optimized,and the highest cell expansion multiplicity of 4.35-fold and the highest proliferation efficiency were obtained when the cell density was 5000 cells/cm²,and the best culture conditions were the intermittent cycling control conditions.Furthermore,the operating conditions of HDMC in WAVE bioreactor were optimized,and the functional relationship between mixing time（t）and reaction speed（v）and reaction angle（θ）of HDMC in WAVE bioreactor was established based on the Open CV computer vision algorithm,and the optimal operating speed of 8 rpm and angle of 6°were determined.The serum-free medium SFM for porcine muscle stem cells was developed and optimized,which can effectively support cell growth and proliferation and avoid the use of serum.Finally,the expansion culture of porcine muscle stem cells was realized on a 1.2 L WAVE bioreactor,and the final harvested cell number of porcine muscle stem cells was 3.05×10⁸ after 3 days of culture,with 12.19-fold cell expansion,which was 3.02 times the expansion efficiency of cells in two-dimensional culture.