Isolation and optimization of mice skeletal muscle satellite cells using preplating method and culture media substitution

Introduction: Satellite cells are known as the main regenerative cell type in skeletal muscles. Our study established a modified digestion and preplating method for the isolation of slow or weak adherent cells for the enrichment of satellite cells. Low-survival rate of these primary stem cells prompted us to address whether cell culture medium substitution might change cell viability status. Methods: Skeletal muscle from 10-day-NMRI mice was gently isolated, dissected and digested by collagenase type I, IV and dispases. The isolated cells were verified by cellular (immunocytochemistry and flow-cytometry) and molecular (real-time PCR) techniques and the results were compared with sub-cultured cells (non-preplated cells) to determine the efficiency of preplating technique as a common isolating procedure of satellite cells. All data were analyzed using SPSS 16 and One Away ANOVA test. Results: The isolated cells exhibited a close gene expression pattern with satellite cells for self-renewal and fusion phases. The findings revealed that Pax7 as a self-renewal marker was expressed ~ 201.4 times higher than sub-cultured-group. Moreover, the findings obviously indicated that substitution of α-MEM to DMEM cell culture medium improves the survival rates of the cells. Conclusion: Our results recommend that preplating technique is a useful procedure for the isolation of satellite cells. In addition, it seems that substitution of culture medium paves the way for investigators to seek various therapeutic methods for skeletal muscle-related disorders such as skeletal muscle atrophy (SMA), amyotrophic lateral sclerosis (ALS), sarcopenia, diabetes and aging.