A method for isolation, culture and identification of gingival mesenchymal stem cells from mice

Abstract

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.

Keywords

• |gingival mesenchymal stem cell|isolation and culture|induction differentiation|identification