Culturing Endothelial Progenitor Cells from PBMNCs

PBMNCs were harvested at a concentration of 1 × 10⁵ cells/ml and resuspended with 30% FBS/PBS 200 µl. The following recombinant human cytokines were then added to the cells: human SCF (#300-07; PeproTech) at a concentration of 66.7 ng/ml; human VEGF (#100-20; PeproTech) at a concentration of 33.3 ng/ml; human IL3 (#200-03; PeproTech) at a concentration of 13.3 ng/ml; human IGF-1 (#100-11; PeproTech) at a concentration of 33.3 ng/ml; human FGF Basic (#100-18B; PeproTech) at a concentration of 33.3 ng/ml; and, human EGF (#100-15; PeproTech) at a concentration of 33.3 ng/ml. The cell mixture was resuspended with complete MethoCult™ media (#04236; STEMCELL Technologies, Inc., Vancouver, British Columbia, Canada) at a final volume of 2 ml, and then cultured in a 37 °C environment for 14 days [11 (link)]. Endothelial progenitor cell (EPC) colony forming cells (EPC-CFCs) were assessed under phase-contrast light microscopy (Eclipse TE300; Nikon Instruments, Tokyo, Japan). A colony was defined as the presence of at least 50 cells [15 (link)]. All experiments were performed in triplicate. The numbers of colonies of PBMNCs cultured in QQ culture media and in standard culture media were compared.