Objective To seek better methods of isolating and culturing bone mesenchymal stem cells(BMSCs)．Methods BMSCs were isolated from human bone marrow by density gradient centrifugation and adherence separation respectively. BMSCs cultured in normal culture flasks and flasks irradiated by 60Co were observed. The characteristics and morphology of BMSCs were observed with an inverted microscope each day. The surface markers of BMSCs were identified by immunocytochemical technique. The expression of surface molecules was examined by flow cytometry. Results BMSCs were in uniformly long spindle shaped and cell colonies were formed. Culture flasks irradiated by 60Co (5 Gy) were better for the adherence and growth of BMSCs. Immunocytochemistry and flow cytometric analysis indicated that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. The primary cells cultured by adherence separation with flasks irradiated by 60Co showed higher cytoactivity, faster proliferation, earlier colony confluence and a shorter time for passage. Conclusions BMSCs are obtained both by density gradient centrifugation and adherence separation. Adherence separation with flasks irradiated by 60Co is an efficient technique for the isolation and purification of BMSCs.
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