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Objective To establish a novel and simple method for isolation and culture of mouse gingival mesenchymal stem cells （GMSCs） in vitro. Methods The gingival tissues from C57BL/6 mice were separated and digested with type IV collagenase, cultured in a dish and passaged. The proliferation pattern of mouse GMSCs was investigated by Cell Counting Kit-8 （CCK-8） assay. The surface markers of GMSCs were detected by flow cytometry, and adipogenic and osteogenic differentiation experiments were carried out to explore the multi-directional differentiation potential of the extracted GMSCs. Results CCK8 assay showed that mouse GMSCs actively proliferated at 3 to 4 d after culture （all P < 0.05 between any two consecutive days from 2nd to 5th d）, and began to become steady on day 5 （all P > 0.05 between any two consecutive days from 5th to 8th d）. Flow cytometry showed that GMSCs highly expressed CD29, CD44, CD73, CD90 and CD105 （all P < 0.05）, but basically did not express CD34 and CD45 compared with mouse oral mucosal epithelial cells （both P > 0.05）. Multi-directional differentiation induction experiment demonstrated that GMSCs successfully differentiated into corresponding tissue cells, confirming the multi-directional differentiation potential of GMSCs. Conclusions In this study, mouse GMSCs are successfully isolated by a new and simple method for the first time, which causes slight damage to primary cells and yields high cell acquisition rate. The cell markers and differentiation function are also identified.