Polylactic acid sheets seeded with genetically modified ovine diaphragmatic myoblasts for myocardial regeneration

F.D. OLEA; M. PENA; G.A. ABRAHAM; A. CROTTOGINI; C.S. GIMENEZ ; A. HNATIUK; F. MONTINI BALLARIN; L. CUNIBERTI; P. LOCATELLI; R. DEWEY; A. ORLOWSKI

Buenos Aires

Congreso; XXII International Society for Heart Research ISHR World Congress 2016; 2016

International Society for Heart Research

Our aim was to isolate, culture and characterize ovine diaphragmatic myoblasts (DMs), transduce them with connexin 43 gene (Cx43) to induce connection between cells, and finally grow them on scaffolds made from different materials to generate DMs-carrying sheets for later application on infarcted areas of sheep hearts with coronary artery ligation. Sheep diaphragm biopsies were digested with collagenase. The extracted DMs were cultured on a feeder layer of autologous activated macrophages (MFD) and characterized with antibodies against desmin, sarcomeric á-actin, SERCA-2 ATPase and Ki-67. To promote intercell connections, DMs were transduced with a lentivirus encoding connexin-43 after testing transduction efficiency of diverse multiplicities of infection (MOI) using lentivirus-GFP. DMs were satisfactorily grown on MFD, were positive for all antibodies and were able to differentiate into myotubes expressing myo-D and myosin heavy chain. With MOI=100, transduction efficiency was 70.8% and Cx43 was extensively expressed. Finally, in order to select the most adequate material to build up DMs-carrying sheets, we seeded DMs on scaffolds made from ovine pericardium (OP, n=8), pig bladder extracellular matrix (ECM, n=8) and polylactic acid (PLA, n=8), and tested DMs confluence (C) at 4 days using a score in which 0=0% C, 1=1-20% C, 2=21-40% C, 3=41-60% C, 4=61-80% C, and 5=81-100% C. C was 0 with PB, 2.6±1.1 with ECM and 4.75±0.5 with PLA (p