Research Of Serum-Free Medium For In Vitro Expansion Of Mesenchymal Stem Cells

Mesenchymal stem cells(MSCs)are considered as one of the most attractive candidates of regenerative medicine due to their dif-f'erention potential and imunoregulatory function.The number of MSCs derived from tissue is limited,and the existing serum-containing medium for MSCs expansion can not used in clinical treatments,meanwhile,the commercial serum-free medium also has some problems,such as poor ability to maintain the functional characteristics of MSCs,which leads to great obstacles to the clinical application of MSCs.The main reason is that the effect and related mechanisms of key factors on cell proliferation and function are still unclear,which brings difficulties for accurately regulating cell behavior.However,the composition of serum and the commercial serum-free medium is unclear,making it difficult to carry out further research.To address these issues,a serum-free medium which suitable for bone marrow-derived MSCs(BMSCs)was developed by statistical methods in the present study.On the basis of the serum-free medium,the underlying mechanism of the key effector(Transforming growth factor-(31,TGF-(31)on stimulating BMSCs proliferation was investigated.19 different factors were selected according to the characteristics of MSCs,then all the factors were combined randomly by Plackett-Burman design.It was found that TGF-?1,epidermal growth factor(EGF),platelet-derived growth factor-BB(PDGF-BB)and insulin can promote BMSCs proliferation,and a serum-free medium(OptM)was obtained to maintain the growth of BMSCs in vitro,but its proliferative ability is still inferior to that of serum-containing medium and commercial serum-free medium.To further improve the adherence and proliferation of BMSCs in OptM,the culture plates were pretreated with collagen I.Then,steepest ascent design and response surface methodology were used to optimize the concentrations of TGF-?1,EGF and insulin and a serum-free medium(SM)that can support the growth and function of BMSCs in vitro was finally obtained.BMSCs cultured in SM maintain its specific surface antigen,osteogenic and adipogenic diff'erentiation potential,and the expression rate of sternness gene is better than that of commercial serum-free medium.On this basis,the impacts of TGF-?1 on the proliferation of BMSCs and its molecular mechanism were investigated in vitro in the present study.In effect,TGF-?1 can enhance BMSCs proliferation and the activation of focal adhesion kinase(FAK)is required for accelerating G1-S cell cycle by TGF-?1,potentially via Akt-mTOR-S6K1 pathway.These findings lead to a better understanding of how TGF-?1 influences BMSCs proliferation and provide more evidence for supporting of the addition of TGF-?1 into SFM in expansion of clinical-grade BMSCs for regenerative medicine.