Isolation, incubation and identification of bone marrow-derived mesenchymal stem cells

Objective: To establish a method for the isolation and cultivation of rabbit mesenchymal stem cells (MSCs) from bone marrow and to study their phenotypical properties to offer an experimental foundation for the further differentiation and actual application. Methods: MSCs were isolated from bone marrow and purified by density centrifuge and adhered to the cultural plastic in vitro. Then, the cells were expanded by subculture successively. The proliferation and growth characteristics were observed with phase contrast microscope in primary and passage culture. MSCs quality was evaluated with immunocytochemistry, FAC Scan flow cytometer, and transmission electronmicroscope (TEM). Results: MSCs had spindle shape and a typical morphological fibroblast. The primary cells were confluent in single layer after being plated for 7-10 days. The cells were noted to have a great expansive potential after subculture, and they uniformly expressed positive for CD90 and negative for CD34. Large number of villus on surface and rough surfaced endoplasmic reticuli and mitochondria in endoplasm were observed in cells through TEM. Conclusion: MSCs can be isolated and purified from postnatal bone marrow through density centrifuge, adherence ability and subculture. The cultured cells have basic phenotypical properties and characteristics in MSCs.