Study of the Isolation,Culture,and Biological Properties of Human Bone Marrow Mesenchymal Stem Cells

Objective To study a method for the isolation and culture of human bone marrow mesenchymal stem cells(MSCs) in vitro and explore their biological properties.Methods Bone marrow aspiration samples were obtained from 7 adult human donors 32 to 65 years old without severe hematopietic diseases.Mononuclear cells were acquired after density gradient centrifugation of the collected bone marrow aspiration.Then they were seeded at 1×10~6 cells/ml in DMEM/F12 supplemented with 10% fetal bovine serum(FBS) in a hole of 12 holes culture plate.The medium was changed first time after 48 hours,and once every 3 days thereafter.When the cultures reached nearly 90% of confluence,cells were passaged with routine methods.Cell surface antigens which contain CD29,CD44,CD166,CD105,CD34,CD45,HLA-DR were detected with flow cytometry.MSCs were treated with adipocyte culture fluid,then they were detected with oil red stain to ascertain whether MSCs could be induced to adipocyte or not.Results A small percentage of isolated cells were adherent to the flasks and grew as typically fibroblastic shape 48 hours after plating.Primary cells derived from adult human bone marrow reached 90% of confluence in 20 to 25 days.Passaged cells reached 90% of confluence in 10 to 15 days.The cultured adherent cells typically expressed CD29,CD44,CD166 and CD105,while CD34,CD45,HLA-DR of them were negative.The result of oil red stain showed that MSCs were able to be induced to adipocyte.Conclusion The isolated and cultured MSCs by the method of density gradient centrifugation are of singular configuration,stable growth and rapid proliferation.This method is a good one to isolate and culture MSCs.MSCs can be induced to adipocyte.