Culturing bone marrow mesenchymal stem cells from Sprague-Dawley rats using whole bone marrow adherence method / 中国组织工程研究

ABSTRACT

BACKGROUND:Tissue and cellimplantation entails high-quality seed cells. In order to satisfy this requirement, it is crucial to produce adequate wel-conditioned, high-purity and strong proliferation ability bone marrow-derived mesenchymal stem cells. OBJECTIVE:To establish a simple, rapid and effective in vitro isolation and culture method of bone marrow-derived mesenchymal stem cells, and to define the biological features of bone marrow mesenchymal stem cells. METHODS:Rat bone marrow mesenchymal stem cells were isolated from the bilateral tibial and femoral bones by the method of whole bone marrow, then purified and passaged by attachment method. The morphology and features of bone marrow mesenchymal stem cells were observed, the growth curve was drawn and the cellsurface antigen was detected by flow cytometry. The bone marrow mesenchymal stem cells were induced to differentiate along the osteogenic, chondrogenic and adipogenic lineages. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells isolated by the whole bone marrow adherence method grew vigorously and were highly purified. The cultured cells were spindle-shaped. The growth curve was S-shaped and the population doubling time was 29 hours. The cells stil maintained a strong proliferative capacity after they were passaged for 10 generations. The surface markers such as CD44, CD29, CD90 were positive, while CD45, CD34, CD11b were negative. At the third passage, bone marrow mesenchymal stem cells were induced to differentiate along the osteogenic, chondrogenic and adipogenic lineages, respectively. Fol owing induction, Alizarin red staining, alkaline phosphatase staining, von-kossa mineralized nodules staining, toluidine blue staining, and oil red O staining were al positive. This shows that the whole bone marrow adherence method is a simple and reliable method for the in vitro isolation, culture and proliferation of bone marrow mesenchymal stem cells. Moreover, they have multi-lineage differentiation capacity under different inducers. The third passage bone marrow mesenchymal stem cells have the highest biological activity and can act as the ideal seed cells for subsequent experiments.