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.