Optimized method for culturing outgrowth endothelial progenitor cells

Abstract

Outgrowth endothelial progenitor cells (OECs) are expected to be a valuable source of blood vessels for regenerative medicine. Their properties and functions have been intensively studied, but the feasibility of reliable acquisition remains unclear. The aim of this study was to determine an optimized method for obtaining OECs from human umbilical cord blood (hUCB). We isolated mononuclear cells from hUCB and determined the OEC colony formation rate (OEC-CFR) under various conditions on the basis of the following 4 points: 1, cell density; 2, pre-selection of CD45^(-) cells; 3, culture medium; and 4, influence of cryopreservation. The OEC-CFR from CD45^(-) cells was 0.250 colony/5 × 10⁷ ,cells and was dependent on the initial cell density, while the OEC-CFR from total mononuclear cells was 0.347 colony/5 × 10⁷ cells. This result suggested that pre-selection of CD45^(-) cells was not necessary to obtain OECs. Supplementation of the culture medium with microvascular endothelial growth medium (EGM-2-MV) caused an increase in the OEC-CFR. Furthermore, we obtained at least 1 colony from each sample of total mononuclear cells after cryopreservation, although the OEC-CFR was lower than that from fresh cells. Flow cytometric analysis and immunocytochemistry showed that isolated OECs expressed CD31, CD34, CD73, CD146, CD144 and CD309. By confocal microscopy, we confirmed the formation of tubes by OECs on Matrigel. In this study, we proposed an optimized method for the isolation of OECs. This method could be useful for the clinical application of OECs.