Objective   To investigate a stable method for the isolation

cultivation

identification of endothelial progenitor cells (EPCs) derived from human umbilical cord blood (HUCB) and the best concentration for the growth of EPCs.    Methods   EPCs were separated from HUCB by density gradient centrifugation

then the mononuclear cells at a density of 1×10

6

/ml

1.5×10

6

/ml

2×10

6

/ml

2.5×10

6

/ml

3×10

6

/ml

3.5×10

6

/ml

or 4×10

6

/ml were inoculated on culture plate coated with human fibronectin to observe cell adherence and colony. EPCs were characterized as adherent cells with double positive to DiI-acLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were documented by demonstrating the expression of cell surface makers with flow cytometry and immunofluorescence.    Results   The cell adherence and cell colony of every concentration group was different

and the best was at the density of 2.5×10

6

/ml. EPCs were characterized as adherent cell with double positive to DiI-acLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. The expression of CD34

VEGFR2 and AC133 with flow cytometry were (1.67±0.35)%

(12.63±9.70)%

and (66.81±6.63)% respectively. The results of EPCs migration and adhesion were (33.7±1.6) and (29.2±1.2) separately.    Conclusion   Combined Ficoll’density gradient centrifugation with adherence screening

we obtain optimal purity and survival rate of HUCB-derived EPCs. The optimal cell density for differentiation and proliferation is 2.5×10

6

/ml inoculation concentration.