Objective: To establish a method for isolating and cultivating mesenchymal stem cells (MSCs) from SD rat bone marrow and to study their phenotypical and functional properties.
Methods: MSCs from rat bone marrow were separated and purified by gradient centrifugation and adherence to the culture plastic; then the cells were expanded by subculture successively. The growth curves were drawn, and the morphology was observed. In an attempt to analyze immuno- and adhesive-phenotype and differentiation properties, the MSCs were evaluated with cytochemical and immunocytochemical methods.
Results: MSCs belonged to the mononuclear cells of marrow, they could be isolated and purified by gradient centrifugation and adherence to the culture plastic; their living behavior was quite stable in Dulbecco's Modified Eagle's Medium with low glucose (L-DMEM) containing 100 ml/L newborn bovine serum; the growth curves of passage 1, 3 and 5 were much similar, exhibiting a 2.2-fold increase in cell number after each passage. The cells were noted to have a large expansive potential and a typical fibroblast-like morphology, and they uniformly expressed CD44, CD54, fibronectin and collagen I. When incubated in medium supplemented with dexamethasone, beta-glycerophosphate and ascorbic acid, the MSCs underwent differentiation into osteoblasts, showing positive stain of alkaline phosphatase activity and mineralized nodules.
Conclusion: The cells obtained in this experiment possess phenotypical and functional properties of mesenchymal stem cells, and the method for isolating and culturing rat marrow-derived MSCs has been established.