Expansion Of Rabbit Mesenchymal Stem Cells In Bioreactor

Objective: To establish a method to isolate and culture rabbit bone marrow-derived mesenchymal stem cells (MSCs) in vitro, and study large-scale expansion of MSCs from rabbit in rotating wall vessel (RWV)bioreactor.Methods: The rabbit bone marrow-derived MSCs were cultured with two different methods—direct culture and desity gradient centrifugation. Then the cells were examined under inverted microscope, and the growth curve was drawn via CCK-8 assay. Also CD44 and CD45 antigens were detected by FCM, and the osteogenic potential of the cells was investigated. MSCs were expanded in large scale in RWV by using microcarrier Cytodex-3. The histomorphology and biological functions of the expanded cells in RWV were compared with those in T-flasks.Results: For the rabbits of 4-7 weeks, MSCs isolated through density gradient centrifugation show better morphologies and higher viability. And the cells proliferate more rapidly in 4-7 passage. When the inoculative density of MSCs is l×l0~5cells/ml and the microcarrier Cytodex-3 concentration is 10mg/ml, MSCs cultivated in RWV for five days can expand more than nine times but only four times in T-fiask in one generation. Moreover, the culture parameters show that the media pH value in RWV is 7.4-7.6, and the glucose content is lower but the lactic acid content is higher in RWV than in T-flask.Conclusions: Density gradient centrifugation, a proper method, was estabished to isolate and cultivate bone marrow-derived mesenchymal stem cells. And the novel RWV can be taken as an ideal three-dimensional culture system for MSCs to be expanded in vitro.