Primary cultivation and identification of satellite cells of mice skeletal muscles / 解放军医学杂志

ABSTRACT

Objective To explore the reliable method for primary cultivation and identification of satellite cells of mice skeletal muscles in vitro.Methods Cell suspension was obtained from posterior limb skeletal muscles of newborn mice by mixed enzymatic digestion,and the muscle satellite cells were separated by Percoll-density gradient centrifugation and purified with differential adherence method.Cells growth was observed under an inverted microscope and the growth curve was drawn.Satellite cells were identified with monoclonal antibody against desmin and ?-actin.Results The primary satellite cells were round in shape,and began on adherence one day after cultivation,and the cells distributed uniformly,most of the cells were roundish.After cultured for 48 hours,the cells began on proliferation,and then became bigger and started to division,bubble cell arrangements with 2-3 or more cells were observed.At the third or fourth day of cultivation,the cells were in exponential phase of growth,and the round cells predominated,many clusters of cell clones were found.The satellite cells formed monolayer sheet(50%-70%) 7-10 days after cultivation,the fusiform cells predominated,but there were still some round cells in this phase.The cell proliferation decreased after cultured for 10-12 days.Immunocytochemical staining showed that the obtained cells expressed myogenic marker desmin and skeletal muscle-specific protein ?-actin.Conclusions High purity muscle satellite cells could be obtained by mixed enzymatic digestion,Percoll-density gradient centrifugation and differential adherence method.Immunocytochemical staining of desmin and ?-actin is the effective method to identify muscle satellite cells.