ECFC recovery from human steady-state PB in an animal serum-free humanized system. (A,B) Peripheral blood (PB) from healthy volunteers was density gradient-separated (+) to enrich for mononuclear cells () or immediately diluted (□) and seeded in EGM/10% pHPL in 75-cm² cell culture flasks. Culture surfaces were coated () with collagen only when indicated (+). PB from patients with stable cardiovascular disease was also immediately diluted and seeded in EGM/10% pHPL (▨). (A) The initial appearance of visible colonies was determined by daily culture observation. (B) Colony number was counted at the end of the primary 7- to 19-day culture period. Results are shown as mean plus or minus SEM of 6 independent experiments. * indicates statistically significant difference, P < .05. (C-H) Representative early colonies (day 8) and parts of large expanded colonies (day 13) from healthy volunteers are depicted with 40× initial magnification corresponding to different recovery strategies as indicated. (A composite picture of 1 representative large ECFC colony is shown in Figure S1D.) Images were captured with a DS-Fi1 camera on a Nikon (Lijnden, Netherlands) Diaphot 300 inverted microscope (original magnification 4×/0.13 NA objective) with the NIS-Elements D3.0 image acquisition software (Nikon). (J) Population doublings () and expanded cell number () determined after large-scale expansion of ECFCs from 6 healthy volunteers (healthy controls) and 3 CVD patients are shown. (K) Cumulative population doublings (mean ± SD) as obtained during large-scale expansion of ECFCs from 6 healthy volunteers after large-scale expansion are shown. Large-scale expansion-derived cells bear a history of mean 21 population doublings before initiating long-term culture at cell seeding densities of 10 (◆), 100 (■), 1000 (▲), and 10 000 cells/cm² (x). Cells were reseeded during long-term culture at indicated time points according to their initial seeding density.