In vitro isolation and cultivation of endothelial progenitor cells from human umbilical cord blood

Objective To establish a stable method for isolation and cultivation of endothelial progenitor cells (EPCs) from human umbilical cord blood. Methods Mononuclear cells (MNCs) were isolated from human umbilical cord blood by density gradient centrifugation, then they were seeded in 6-well plates pre-coated with fibronectin (Fn) and cultured in EGM-2. EPCs were identified by morphology, cell surface markers, ability to uptake Ac-LDL and bind UEA-1 and in vitro tube formation characteristic. Results The morphology of freshly isolated MNCs was small and spherically shaped. A small amount of adherent cells with circular, oval and spindle shapes appeared after 4 days culture. Colonies formed after 8 days culture. The cells exhibited typical cobblestone morphology after 14 days culture. Some cells formed network structures and linear cord-like structures. EPCs could uptake Ac-LDL, bind UEA-1, express cell surface markers, CD34, CD133 and VEGFR-2, and possess in vitro tube formation ability. Conclusions EPCs can be successfully isolated by density gradient centrifugation from human umbilical cord blood and cultivated for further research. Key words: Human umbilical cord blood; Mononuclear cells; Endothelial progenitor cells; Cell culture