Human Endothelial Cell Cultures from Progenitor Cells Obtained by Leukapheresis

Abstract

Although improved prosthetic graft patency with endothelial cell (EC) seeding has been shown in animal models, the clinical application of this technique requires a convenient source of ECs. We have evaluated EC cultures derived from the mononuclear cell (MNC) fraction obtained during large-volume leukapheresis and compared this with cultures grown from peripheral blood cells obtained by phlebotomy. Leukapheresis was performed in healthy adult volunteers (n = 7) using software designed to increase the percentage of MNCs harvested. Blood (40–293 mL) was drawn from a peripheral vein in healthy adult volunteers (n = 13), and the MNCs were obtained by differential centrifugation using a Lymphoprep gradient. Significantly more MNCs were obtained by leukapheresis than by phlebotomy. Each leukapheresis procedure yielded 12.5 to 23 mL, which contained 2.29 ± 0.35 x 10⁹ MNCs, compared with 2.16 ± 0.50 x 10⁸ MNCs, for each phlebotomy (P < 0.001). EC colonies developed in significantly more cultures from leukapheresis-derived MNCs (6 of 7) than phlebotomy-derived MNCs (4 of 13; P = 0.008). Leukapheresis-derived cells developed EC morphology at 15.5 ± 2 days compared with 21 ± 3.4 days for cells obtained by phlebotomy (P = not significant). EC were identified by positive factor VIII and vascular endothelial growth factor receptor immunostaining. Leukapheresis significantly increases the number of progenitor cells available for differentiation into EC compared with phlebotomy and avoids the need for any surgical procedure to harvest a peripheral vein as a direct source of ECs.