[Isolation and culturation, phenotype detection of rat bone marrow mesenchymal stem cells].

AIM: To investigate the more effective methods of isolation, culture of mesenchymal stem cell (MSC), and examine the surface phenotype of MSC. MSC were separated from rats bone marrow by plastic adherence methods and purified by controlling the time of digestion combied with adhesion separation, and proliferated in culture flasks that were coated with Ⅰcollogen. METHODS: The morphology of MSC were studyed with phase contrast microscope; cell cycles of the forth generation of MSC were tested by flow cytometry and the phenotype of MSC were identified by using immunocytochemical methods. RESULTS: Primary cultured MSC were oval, spindle-shaped or polygonal, and adhered to plastic surface within 24 h and reached 90% confluence within 7～8 days. After purification and proliferation, they were uniformly long spindle-shaped form and passaged every five days. The adhesion rate within 24 hours was all. The flow cytometry showed 80% cells of the forth generation of MSC were at G0/G1 phase. Immunocytochemistry showed MSC were positive for CD29, CD105, CD166, VLA-4 and P-selectin, while negative for CD34 and CD45. CONCLUSION: The MSC obtained from our experiments were more purified, and expanded more rapidly, expressed some cell adhesion molecules. The protocol should make it possible to undertake a large number of experiments with MSC in future application.