Improvement Of In-vitro Culture Method Of Mesenchymal Stem Cells And Mechanism Of Inducing Tolerogenic Dendritic Cells

Objective:Mesenchymal stem cell is a kind of pluripotent cell, which is widely used in clinical practice and the treatment of GVHD and other diseases.In addition, it also has immunomodulatory effect, can induce the formation of "Regulatory dendritic cells", but the specific mechanism of which is inconclusive.The popular method used to culture mesenchymal stem cells leads to their degradation, apoptosis and senescence.Therefore, this study intends to find a way which can maintain MSC`s stemness, delaying it`s apoptosis and aging process,and to discuss the mechanism of interaction between dendritic cells and mesechymal stem cells.Methods:Chitosan was dissolved in acetic acid and coated on the surface of the culture plate.Then dried completely in the oven at 60℃.Mouse bone marrow mesenchymal stem cells were isolated and seeded in the culture plates coated with chitosan and ordinary untreated culture plates.Cells were collected 5 days later to detect stemness associated markers CD44 and Sca-1;analyze the situation of apoptosis by flow cytometry and fluorescence microscopy;analyse the aging situation under an optical microscope.We analysed the effects of three-dimensional spheroid culture system on biological characteristics of mouse mesenchymal stem cells by above methods.We collected the culture supernatan of good-conditioned mesenchymal stem cells,isolated exosome from which and added to the culture medium of dendritic cells.After 48 hours,we collected the culture supernatan of dendritic cells and detected the content of IL-10 by ELISA.We co-cultured the dendritic cells which had been cultured with MSC`s exosome for 48 hours with mouse lymphocytes for another 72 hours,then analysed the proportion of regulatory T cells and the proliferation ability of T lymphocytes.We investigate whether exosomes was involved in the regulation of mesenchymal stem cells to dendritic cell by analysing the content of IL-10 and DC`s ability to activate lymphocytes.Results:The mouse mesenchymal stem cells began to form spheroids on day 3. The stemness-related markers, including CD44 and Sca-1, expressed higher in spheroid mesenchymal stem cells than the MSCs under normal culturing. Compared with the normal culture group, the apoptosis and senescence of cells from spheroid culturewere lower. The results indicate that the formation of spheroids on chitosan films can increase the stemness, decrease the apoptosis and slow the senescence of mesenchymal stem cells.The content of IL-10 from dendritic cells which co-cultured with exosome for 48 hours was higher than the control group.After adding exosomes,the activation of T cell by dendritic cells was weakened, and induced more regulatory T cells.Conclusion:Mesenchymal stem cell has gained much attention in recent years, and are widely used in the field of tissue engineering and cell therapy.But the mesenchymal stem cells lost the stemness in current culture conditions,thereby seriously affecting its value.We adopt chitosan film to make mesenchymal stem cells grow in the three-dimensional sphere state,simulating cells growth pattern in vivo,by which not only maintain the stemness and slowing the apoptosis and aging process,so that mesenchymal stem cells can be better used in scientific research and clinical practice.Through the analysis of the secretion of IL-10 as well as the activation of T cells, it can be seen that exosomes join in the regulation of dendritic cells with cytokines, which probably by protein or nucleic acids it contains.Mesenchymal stem cells` immune regulation mechanism is very complex and needs further research.