Isolation, replication and transfection of rabbit adipose stromal cells (ASCs) for cardiovascular regeneration studies

OLEA FD, PÉREZ SÁEZ JM, LOCATELLI P, BUSSMANN UA, BERCOVICH A, CROTTOGINI A

La Plata, Argentina

Congreso; XVII Annual Meeting of the International Society for Heart Research Latin American Section; 2010

Interantional Society for Heart Research Latin American Section

Rabbit bone marrow mesenchymal stromal cells (MSCs) are difficult to replicate in vitro. Adipose tissue has been proposed as an alternative source. Given our need to use genetically modified MSCs in rabbit models of cardiovascular disease, we aimed to: 1) identify and culture MSCs from adipose tissue (ASCs), and 2) assess the transfection condition that maximizes transfection efficiency (TE), for later transfection of ASCs with plasmids encoding growth factors. Abdominal fat was digested with colagenase. Plastic-adherent cells were incubated until p4 and then identified by their antigen phenotype and their differentiation capacity. Diverse ratios of cationic lipids (Lipofectamine 2000, GeneJuice and Lipofectamine LTX) to plasmidic DNA were tested. Results: Cells were positive for CD44, CD90 and CD105 and negative for CD34 and CD45, and differentiated into osteocytes and condrocytes, indicating their MSC nature. Luciferase activity was maximal with 1 µg DNA/cm2 (corresponding to a TE of 9% by GFP flow citometry) using 3 µl Lipofectamine 2000/µg DNA. Conclusion. Isolation, characterization and replication of rabbit ASCs is feasible. TE, as assessed by luciferase activity and GFP expression, is acceptable, suggesting that ASCs may be employed in cardiovascular regeneration studies.