The Changes of Apoptosis and Viability in Stem Cells after Thawing with Lapse of the Time

Park, Seun Joo; Bae, Sang Young; Ryu, Kyung Ha; Whang, Il Tae; Kim, Kyung Hyo; Hong, Young Mi; Kim, Kyung Hee; Lee, Keun; Seoh, Joo Young

Abstract

PURPOSE: Thawed stem cells should be infused as early as possible because delay of infusion leads to decrease of cell viability and formation of DNA clumping. The procedure of 10% Dimethyl sulfoxide(DMSO) removal and a long distance from the thawing location to the patient are the main causes of delay of infusion that results in the loss of cell viability and apoptosis. Therefore, we investigated the changes of cell viability and apoptosis after thawing with lapse of time.
METHODS
Five samples of mobilized peripheral blood were evaluated. We measured cell viability, colony forming unit(CFU) and apoptosis at 0, 1, 2, 4, and 24 hours after thawing. The state of stem cells were divided into live, apoptotic and dead with double staining using annexin V and 7-aminoactinomycin D(7-AAD) in flow cytometry.
RESULTS
Viability of the total cells after thawing was 77.3(53.3-97.7)%. The percentage which recovered to initial CFU at 1, 2, 4 and 24 hours after thawing decreased to 63.9%, 50.2%, 45.8% and 11.6%, respectively. The proportion of apoptotic cells among CD34+ cells after thawing were increased from 0.2% at 0 hour to 16.5% at 1 hour, 21.9% at 2 hours, and then decreased to 15.0% at 4 hours, 2.7% at 24 hours because they were replaced by dead cells.
CONCLUSION
Thawed cells changed to apoptotic and had less colony forming capacity from 1 hour after thawing, and were then replaced by dead cells from 4 hours after thawing.