Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation

Three widely used viability assessments were compared: (1) membrane integrity of nucleated cells using trypan blue (TB) exclusion and a fluorometric membrane integrity assay (SYTO 13 and propidium iodide), (2) enumeration of viable CD34+ cells, and (3) clonogenic assay (granulocyte-macrophage colony-forming units, CFU-GM). Post thaw peripheral hematopoietic progenitor cells (HPC) were incubated at 0, 22, and 37°C for 20-min intervals before assessment. The recovery of viable nucleated cells assessed by TB and SYTO/PI decreased significantly with time at incubation temperatures of 22 and 37°C (P<0.05), and correlated with the concentration of mononuclear cells (MNC) (r=0.936, P<0.05). The decrease in recovery of viable nucleated cells was slower when thawed cells were incubated at 0°C compared with 22°C or 37°C. The recovery, measured by absolute viable CD34+ or CFU-GM, was not affected by 2 h post thaw incubation (P>0.05) at 0, 22, and 37°C (P>0.05). There were no significant differences in the measured recovery of viable CD34+ cells and CFU-GM at all incubation times (P>0.05) and temperatures (P>0.05). Both CFU-GM and absolute CD34+ cells can be used as post thaw viability assays for HPC cryopreserved for transplantation.