Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials

Endothelial progenitor cells (EPC) are markers of endothelial injury and may serve as a surrogate marker for vascular repair in interventional clinical trials. Objectives of this study were to modify a method of isolation of peripheral blood mononuclear cells (PBMC) and enumeration of EPC and mature endothelial cells (EC) from peripheral blood and to evaluate influence of cryopreservation on viability of PBMC and on numbers of EPC and EC.

EPC and EC were analyzed in healthy volunteers in freshly isolated PBMC collected in CPT (cell preparation tubes) and in PBMC cryopreserved with: 1) Gibco Recovery^™ Cell Culture Freezing Medium, 2) custom freezing medium. Viability of PBMC was tested using DAPI. EPC were gated for CD45^- CD34⁺CD133^+/-VEGFR2^+/- and EC were gated for CD45^-CD146⁺CD34^+/-VEGFR2^+/-.

Cryopreservation for 7 days at -80°C decreased viable PBMC from 94 ± 0.5% (fresh) to 84 ± 4% (the custom medium) and to 69 ± 8% (Gibco medium), while cryopreservation at -65°C decreased viability to 60 ± 6% (p<0.001, the custom medium) and 49 ± 5% (p<0.001, Gibco medium). In fresh samples early EPC (CD45^- CD34⁺CD133⁺VEGFR2⁺) were enumerated as 0.2 ± 0.06%, late EPC(CD45^-CD146⁺CD34⁺VEGFR²⁺) as 0.6 ± 0.1% and mature EC (CD45^-CD146⁺CD34^-VEGFR2⁺) as 0.8 ± 0.3%of live PBMC. Cryopreservation with Gibco and the custom freezing medium at -80°C for 7 days decreased numbers EPC and EC, however, this decrease was not statistically significant.

Our data indicate that cryopreservation at -80°C for 7 days decreases, although not significantly, viability of PBMC and numbers of subsets of EC and EPC. This method may provide an optimized approach to isolation and short-term cryopreservation of subsets of EPC and of mature EC suitable for multicenter trials.