Experimental study of humam umbilical cord mesenchymal stem cell isolation,culture and biological characteristics

Objective:To establish a method for isolation and culture of umbilical cord mesenchymal stem cells (UC-MSCs) from human umbilical cord in vitro,and explore its biological characterstics.Methods:Human UC-MSCs were isolated and expanded in culture with explantation technique and enzyme digestion (collagenase Ⅱand trypsin),respectively;the cell morphology was observed by Wright-staining;after the third passage,the surface markers of UC-MSCs were detected and idntified by flow cytometry,and the seventh passage,the cell proliferation was analyzed by MTT assay.After UC-MSCs were co-cultured with peripheral blood mononuclear cells,the impact on the T cells were observed by Hoechst and PI double staining.Results:About one week after explantation,the fibroblast-like cells sprawled out from the edge of the tissue,growing in cluster;3-5 days after enzyme digestion,cells were watched growing fiber-shaped.After passaged,UC-MSCs showed the status of long,spindle-shaped and circinate growth;they had large nucleus and clear nucleolus.Immunophenotype analysis showed that they had strong positive expression for CD29 (95.71±2.23)%,but week expression for CD31,and CD34 were (2.47±0.54)% and (3.24±0.34)%,respectively;the seventh passage UC-MSCs still had strong proliferation ability;UC-MSCs suppressed T cells proliferation in a dose-dependent manner;lymphocytes did not affect the growth of UC-MSCs.Conclusions:The cells isolated successfully from the umbilical cord have biological characteristics of the mesenchymal stem cell.