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

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

Buenoa Aires

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

International Society for Heart Research (ISHR)

Our aim was to isolate, culture and characterize ovine diaphragmatic myoblasts(DMs), transduce them with connexin 43 gene (Cx43) to induce connection betweencells, 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 α-actin, SERCA-2 ATPase and Ki-67. To promote inter- cell 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 myoDand myosin heavy chain. With MOI=100, transduction efficiency was 70.8% andCx43 was extensively expressed. Finally, in order to select the most adequatematerial to build up DMs-carrying sheets, we seeded DMs on scaffolds made fromovine pericardium (OP, n=8), pig bladder extracellular matrix (ECM, n=8) andpolylactic 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