Introduction: Laminin is a major component of the basement membrane, containing multiple domains that bind integrin, collagen, nidogen, dystroglycan, and heparan sulfate. Laminin-221, expressed in skeletal and cardiac muscles, has strong affinity for the cell-surface receptor, integrin α7X2β1. The E8 domain of laminin-221, which is essential for cell integrin binding, is commercially available as a purified recombinant protein fragment. In this study, recombinant E8 fragment was used to purify primary rodent myoblasts. We established a facile and inexpensive method for primary myoblast culture exploiting the high affinity binding of integrin α7X2β1 to laminin-221.
Methods: Total cell populations from dissociated muscle tissue were enzymatically digested and seeded onto laminin-221 E8 fragment-coated dishes. The culture medium containing non-adherent floating cells was removed after 2-hour culture at 37 °C. The adherent cells were subjected to immunofluorescence staining of desmin, differentiation experiments, and gene expression analysis.
Results: The cells obtained were 70.3 ± 5.49% (n = 5) desmin positive in mouse and 67.7 ± 1.65% (n = 3) in rat. Immunofluorescent staining and gene expression analyses of cultured cells showed phenotypic traits of myoblasts.
Conclusion: This study reports a novel facile method for primary culture of myoblasts obtained from mouse and rat skeletal muscle by exploiting the high affinity of integrin α7X2β1 to laminin-221.
Keywords: DM, differentiation medium; ECM, extracellular matrix; FACS, fluorescence-activated cell sorting; GM, growth medium; Integrinα7X2; Laminin-221; MACS, magnetic-activated cell sorting; Myoblast; PBS, Dulbecco's phosphate buffer saline; PFA, paraformaldehyde; Primary culture; Skeletal muscle.
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