EPC Isolation and Culture

Experimental procedures involving animals as a source of porcine EPCs complied with the Guide for Care and Use of Agricultural Animals and were approved by the Institutional Agricultural Animal Care and Use Committee of Texas A&M University.

EPCs from blood can be isolated and identified based on in vitro adhesion characteristics and by cell surface markers. As EPCs differentiate in culture, they acquire endothelial cell markers such as VE-cadherin, PECAM-1 (CD31) and von Willebrand Factor (vWF) (Parant et al., 2009; Urbich, Dimmeler 2004).

Porcine EPCs (pEPCs) were isolated and cultured following a combination of two methods used for purifying EPCs from human blood (Brunt, et al. 2007, Hirschi, et al. 2008). Whole blood (50cc) was obtained from piglets within 6 h of birth, diluted 1:1 in PBS and layered over Ficoll-Paque (Invitrogen, Carlsbad, CA). Following centrifugation (400 × g, 20 min), the mononuclear cell layer was transferred to a new tube and washed twice with sterile phosphate-buffered saline (PBS) (1500 × g, 5 min). Cells were then resuspended in medium-199 (M199) containing 100 μg/ml heparin (Sigma, St. Louis, MO), 0.4 mg/ml lyophilized bovine hypothalamic extract (Pel-Freeze Biologicals, Rogers, AK) and 15% fetal bovine serum (Lonza, no. 14–471F, Walkersville, MD), and cultured in flasks coated with 5–10 μg/ml fibronectin (Invitrogen, Carlsbad, CA) in this medium. After 1 h, nonadherent cells were removed, the flasks were washed, and fibronectin-adherent cells were cultured for 14–21 days or until a confluent monolayer of cells was obtained. These cells appeared to be a pure culture as can be observed in the immunofluorescence characterization data shown in the results of the present study, although the possibility of other cell-types surviving in the cultures cannot be ruled out at this time. At this time, cells were passaged onto gelatin (Sigma-Aldrich, cat. no. G2500, St. Louis, MO) -coated flasks (1 mg/ml) and used for the experiments outlined below.