Research Of The Effect Of Trypsin Digestion And Hepatocyte Growth Factor On The Proliferation Of Mesenchymal Stem Cells In Vitro

Mesenchymal stem cells(MSCs)are considered as one of the most important candidates in cell therapy and tissue engineering.Due to the limited number of MSCs obtained from body tissues,they need to be expanded in vitro.The expansion of MSCs in vitro is affected by many factors,such as the composition of culture medium and the operating parameters in the process of culture.The traditional serum-containing medium can not meet the requirements of clinical application,while the kinds of existing serum free medium(SFM)with clear chemical components were not insufficient to support the proliferation of MSCs.In view of the above problems,this paper takes human umbilical cord MSCs as the research object to investigate the effects of trypsin concentration and digestion time on cell harvest and subculture under the condition of serum-free culture.Moreover,in order to improve the cell proliferation ability of the self-developed SFM,the key component of culture medium,hepatocyte growth factor(HGF),on the proliferation effect of MSCs in vitro and its potential mechanism were also explored.The results showed that the proliferation and continuous passage ability of MSCs were weaker in commercial SFM and self-made SFM compared with serum-containing medium.Low concentration and short-time trypsin digestion were not conducive to cell harvest;while high concentration and long-time trypsin digestion destroyed the integrin ?1 on cell surface,leading to cell cycle delay,and in the subsequent generation of MSCs,the adhesion and proliferation of cultured cells had adverse effects.Therefore,0.25%trypsin digesting for 3 min was selected as the most appropriate parameters.Adding 10 ng/mL HGF to the self-made SFM could promote the proliferation of MSCs.Further research showed that adding HGF could enhance the expression of integrin ?1 and cyclin D1 by activating FAK-AKT-mTOR pathway,accelerate the process from G1 to S phase of cell cycle,and promote cell proliferation.The expression of surface phenotype and the differentiation potential of osteogenesis and adipogenesis of MSCs harvested from the optimized SFM were similar to those of the original SFM,but the expression of stemness genes,including Klf4 and Nanog were significantly increased.The results lay a foundation for further optimization of culture conditions,design of serum-free medium composition,regulation of MSCs proliferation and function in vitro,further contributing to the large-scale cultivation in SFM system and clinical application of MSC.