Abstract:

BACKGROUND: At present, there are two main methods of isolating and purifying bone marrow mesenchymal stem cells (BMSCs): density gradient centrifugation and the whole bone marrow adherent method. The former has complicated procedures, and the latter has simple operation, but the purified outcomes are not ideal.
OBJECTIVE: To establish a rat BMSCs isolated and cultured in vitro purification methods on the basis of the whole bone marrow adherence and isolation of BMSCs in combination of differential passage digestion method.
METHODS: By whole bone marrow adherent culturing to isolate and differential digestion and passage of rat BMSCs, the speed of MSCs in the process of digestion and passage was quicker than other bone marrow cells, as well as the characteristics of adherent speed, instead of density gradient centrifugation to separate and purify MSCs, and their morphological characteristics were observed. Cell growth and proliferation of two kinds of culture method were compared with the density gradient centrifugation separation. Alkaline phosphatase and oil red staining results were observed to verify BMSC differentiation capacity, to detect the cell surface markers, to validate immune properties and to test its purity.
RESULTS AND CONCLUSION: The whole bone marrow adherent culture method isolated and differentially subcultured rat BMSCs. Flow cytometry and osteogenic and adipogenic culture results displayed cytoimmunity property, purity and differential capacity, which have no significant difference compared with density gradient centrifugation. However, cell viability and reproductive activity are obviously elevated.