J L Lequerica, V Mirabet, J A Montero, C Hurtado, S Piquer, F Carbonell
Ann Transplant 1999; 4(3-4): 103-108
Objectives: In vitro culture of myoblasts and subsequent grafting into injured myocardium represents a new therapeutic approach for the treatment of myocardial infarct. A major limitation to developing enough myoblasts to engrafting purpose is the isolation and purification. In the present work we purified myoblast from primary culture using an immunomagnetic bead technique. Methods: Primary culture was obtained by trypsin-EDT A digestion of human muscle biopsies. Cells were cultured in DMEM growth medium containing 10% FBS, 2 mM L-glutamine and antibiotics. Immunotechniques using both monoclonal anti-myosin heavy chain (skeletal fast) and 5.IH II antibody combining with flow cytometry did identification of myoblasts. Positive selection was on myoblasts bound to 5.I.H II incubating with human antimouse IgG coated magnetic beads (Dynabead) and subsequent isolation by magnet. releasing cells from beads with DNAse. Results: More than 59% of primary cell culture are positive to 5.tH II and decreasing with passage. The coating of culture dish surface increased specific growth rate of myoblast clones twice. Positive selection allows to increasing concentration of myoblasts from 8.4% in mixed culture to more than 90% without affecting neither viability nor platting efficiency. Conclusion: Purification procedure reported here is easy. efficient and requires small amount of sample, which will facilitate the purpose of autologous implant.
Keywords: human skeletal muscle, myoblast, Flow Cytometry