Ovine diaphragmatic myoblasts for myocardial regeneration: obtention and culture on a feeder layer of autologous macrophages. Preliminary results

GIMÉNEZ CARLOS SEBASTIÁN; RAMIREZ RODRIGO; OLEA FERNANDA DANIELA; LOCATELLI PAOLA; HNATIUK ANNA; SEPÚLVEDA DIANA; DULBECCO EDUARDO; LAGUENS RUBÉN; CROTTOGINI ALBERTO

Foz de Iguazú

Congreso; Congreso Panamericano de Fisiología; 2014

Sociedade Brasileira de Fisiologia

Diaphragmatic myoblasts (DM) are type I fibers and display high tolerance to exhaustion. Therefore, they may be more suitable than skeletal myoblasts from peripheral muscles for stem cell-mediated myocardial regeneration. To this end, successfully obtaining and growing these cells is mandatory. On the other hand, on account that activated macrophages secrete numerous growth factors such as fibroblast growth factor and insulin like growth factor, we hypothesized that a feeder layer of autologous activated macrophages could provide the factors needed for growing DM. Methods: Forty ml venous blood was taken from the antecubital vein of 3 adult sheep and monocytes were isolated by Ficoll-Paque gradient, plaqued and cultured at 37°C and 5% CO2 in high glucose DMEM + 1% antibiotic + 20% FBS. Culture medium was renewed after 24 and 72 hs. On day 7, sheep underwent a sterile right mini-thoracotomy and a 2 cm2 sample of the diaphragm muscle was excised, minced into small pieces and digested with type V collagenase. After successive purification cycles, DM were seeded on the macrophage feeder layer. From culture samples, macrophages and DM were characterized by immune fluorescence with specific antibodies. At day 7, DM reached 100% confluence, and macrophage-free DM cultures were achieved after 5 passages. Conclusion: DM can be characterized and successfully grown on a feeder layer of autologous macrophages.