The biological characteristics of rat bone marrow-derived mesenchymal stem cells isolated and cultured with attachment method

Objective To establish a simple and effective method of isolation,purification and cultivation of rat bone marrow-derived mesenchymal stem cells(MSC) in vitro,and explore their biological characteristics.Methods MSC were isolated and purified for culture in vitro and serial passage by the attachment culture method.The morphological changes of the MSC were continuously observed under the inverted microscope.The cell growth curve was measured by MTT method,and membrane antigens were detected with flow cytometry(FCM).Results The MSC in primary culture which were adhered to plastic surface within 48 h after displacement took the oval,asteroid,short and fusiform shapes.12 days after culture in DMEM-F12 medium containing 10% FBS,the cells reached 90% confluence in single layers,taking a long and fusiform shape.Further purification was achieved by expansion at serial passages.MSC were in latency for 2 days,converted into growth period on the 3rd day and entered the stationary phase on the 7 th day.FCM results indicated that the positive rate of CD45 and CD90 was 19.60% and 95.38%,respectively.Conclusion The attachment culture method can be effectively used to isolate and purify rat MSC.The cultured MSC are stable in growth with active proliferation and share the general biological characteristics of MSC,which will further make them ideal seed cells for tissue engineering.