Laminin-221-derived recombinant fragment facilitates isolation of cultured skeletal myoblasts

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 a7X2b1. 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 a7X2b1 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 a7X2b1 to laminin-221. ?? 2022, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

Automated summary

This study presents a novel and cost-effective method for the primary culture of myoblasts obtained from mouse and rat skeletal muscle, utilizing the high affinity of integrin a7X2b1 to laminin-221.