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

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.
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.
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.
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.