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 degrees 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 degrees 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 degrees C compared with 22 degrees C or 37 degrees 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 degrees 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.
Canadian Blood Services, Edmonton Centre 8249-114 Street, Edmonton, Alberta, Canada T6G 2R8. hongyou.yang@bloodservices.ca
H
H
Yang
J P
JP
Acker
M
M
Cabuhat
L E
LE
McGann
D018952:Antigens, CD34; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D036102:Peripheral Blood Stem Cell Transplantation / Q000379:methods*; D013696:Temperature; D013995:Time
D018952:Antigens, CD34