Human myoblast cell culture - a model for the study of metabolic myopathies

Abstract

Insufficiency of S-adenosyl-homocysteine hydrolase (SAHH; EC 1.1.3.3.) has been confirmed in nine patients so far. Although clinical symptoms are variable, myopathy is present from the birth in all patients who are affected by this multisystem disease. It is unclear why muscle tissue is affected in patients with SAHH insufficiency; therefore establishment of myoblast cell culture as a model has great value and presents the first step towards understanding the pathogenesis of the disease at the metabolite, protein and gene levels. Myoblasts were isolated from biopsy of skeletal muscle by enzymatic digestion. Myoblasts were cultivated in sterile conditions in a medium with growth factors. After achieving confluence of 70%-80%, myoblasts were divided by trypsinization into subcultures. A culture of myoblasts was established and purified by using an immune-magnetic sorting system and then stored in liquid nitrogen. Purity of myoblasts was confirmed by flow cytometry method using CD56+ and CD45- surface markers. Pure myoblasts were differentiated into myotubes (immature muscle fibers). In this report, optimized procedure of isolation, cultivation, differentiation and storage of myoblasts in vitro is presented.