Abstract:Objective: To explore a method for separation and purification of satellite cells (SCs) from rats levator ani muscle (LAM), and to observe the proliferation and myogenic characteristics of SCs in vitro. Methods: The SCs from rat LAM were separated by enzyme digestion with collagenase type 1 and trypsin, and purified by differential wall adhesion method. From that, we obtained highly purified SCs from rat LAM. The morphology and growth of primary and passage cells were observed by inverted microscope. CCK-8 method was used to detect the growth of LAM SCs (cultured in vitro) and draw the growth curve. Immunofluorescence was used to identify SCs in different stages. Results: Microscopically, it was observed that SCs separated from rat LAM grew well. In CCK-8 test, the SCs grew slowly in the first to second days of in vitro culture, after 3 days, the SCs augmented multiply. On the 10th day, the cell growth rate decreased and some cells began to differentiate. Immunofluorescence and DAPI staining showed that the characteristic gene expression of protein was observed different in different stages of SCs. Conclusion: In this study, SCs were successfully separated from rat LAM tissue with good proliferation and differentiation ability in vitro, laying a preliminary foundation for exploring the mechanism of muscle injury and repair by LAM SCs.
