Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are not rich in bone marrow, and are difficult to culture in vitro. It is crucial to tissue engineering and to the cell experiments in vivo or in vitro that isolating and culturing in vitro BMSCs with high purity, strong vitality and homogeneous biological characteristics.
OBJECTIVE: To establish a method of isolation, cultivation and purification of rat BMSCs in vitro, to observe cell morphology, and to assess surface markers and multi-directional differentiation capacity.
METHODS: BMSCs from rats were isolated, cultured and purified by the whole bone marrow adherence method, for morphology observations, the growth curve was drawn, cell cycle was analyzed, cell surface markers were assessed by flow cytometry, and BMSCs were induced to differentiate into osteoblasts and adipocytes.
RESULTS AND CONCLUSION: BMSCs from rats were spindle cell-based, showing radial colony arrangement. Cells kept strong growth and could passage in continuous and stable manner over 10 passages. The growth curve and cell cycle demonstrated that BMSCs were consistent with the growth characteristics and good activity of normal cells. At the third passage, BMSCs were negative for CD34 and CD45, but positive for CD44, CD90 and CD105. Following induction, oil red O staining, alkaline phosphatase staining, von Kossa staining and alizarin red staining produced a strong reaction in cells. Whole bone marrow adherence method is simple and can isolate, purify and amplify BMSCs in vitro. The obtained cells have general biological characteristics of mesenchymal stem cells, and also have potentiality of multi-directional differentiation. This experimental method has important practical significance to provide adequate source of seed cells for tissue engineering.