Isolation and Identification of mesenchymal stem cells derived from human bone marrow in vitro

Objective To isolate and identify mesenchymal stem cells derived from human bone marrow in vitro.Methods Mononuclear cells were separated from human bone marrow by the method of density gradient centrifugation.To change medium frequently after seeding 3 hours.Cells were lift by incubation in 0.25% trypsin for 2 min at room temperature.Incubation was proceeding after subculturing.Surface markers of mesenchymal stem cells were detected with flow cytometry.The cells were induced for 14 days to differentiate into other lineage cells,and identified by staining with alizarin red,toluidine blue and oil red.Results Cell confluence is achieved at the 21th day and they were both like long shuttle,clone growth.Flow cytometry show that they have high expression of CD105,CD73 and CD90,while they have low expression of CD45,CD34 and CD14.Harvesting cells could be induced to differentiate into mineralizing cells,chondrocytic cells and adipocytes.Conclusions Mesenchymal stem cells could be separated successfully from human bone marrow by the method of adherent selection in combination with density gradient centrifugation.Hematopoietic lineage cells contamination were obviously dereased after frequent medium changing and limited trypsin action.