Abstract
An effective isolation protocol for outgrowth endothelial cells (OEC) resulting in higher cell numbers and a reduced expansion time would facilitate the therapeutical application. In this study a standard protocol based on the isolation of mononuclear cells from adult peripheral blood was modified by adding a passaging step 7 days after the isolation. OEC colonies gained by both protocols were evaluated after 28 days and resulted in different frequencies of OEC colonies depending on the donor and culture protocol. Accordingly, we defined two groups, namely, high colony-forming cultures (HCC) and low colony-forming cultures (LCC) for further analysis. LCC revealed no increase in OEC colonies by the modified protocol, whereas in HCC the frequency of OEC colonies was significantly improved by the passaging step. Quantitative real-time polymerase chain reaction, flow cytometry, and immunofluorescence for endothelial markers indicated an enrichment of OEC by protocol modification in HCC. In addition, HCC revealed higher expression of CD34 and CD133 compared to LCC and resulted in higher numbers of OEC gained per donor, which was further improved by the modified protocol. We conclude that the modified protocol supports the selection of OEC from adult peripheral blood with a high clonogenic potential and results in a better efficacy in OEC isolation.