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Abstract
Background
On performing umbilical cord blood (UCB) transplantation, faster engraftment may lead better clinical outcome. Because transplanted viable cell count in UCB is related to the engraftment, accurate evaluation of viability of CD34+ cells in cryopreserved UCB has clinical implication. We examined the difference in viability of cells in cryopreserved UCB according to the duration of cryopreservation and different methods.
Methods
A total of 60 UCB samples which were cryopreserved for 1 to 4 years were used in this study. Viability of cryopreserved cells were examined with trypan blue exclusion assay, DNA contents analysis, caspase-3 activation test, intracellular esterase activity and Annexin-V/PI staining.
Results
After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+ cells were viable as identified by trypan blue exclusion assay. In the CD34+ cell population, the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis. However, cspase-3 activity failed to document apoptosis. The intracellular esterase activity test also showed a cell death rate of about 10∼20% at 2, 4, and 6 hours after thawing.
Conclusion
Viable cells in UCB should be measured by several compensatory techniques rather than a single method. Discordance among Annexin-V/PI staining versus trypan blue exclusion, DNA contents analysis, and the caspase-3 activation test or intracellular esterase activity should be clarified in order to apply these techniques for actual cord blood transplantation.
References
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Fig. 1.
Mononuclear cell (MNC) viabilities of cryopreserved umbilical cord blood (UCB) samples after thawing. The mean and standard deviation value of viable cell percents from 60 umbilical cord blood (UCB) samples was 88.9±6.8 by trypan blue staining (A). The result was not affected by the duration of cryopreservation (B).
Fig. 2.
Successfully isolated CD34+ cells (purity of 83%) showed 83.9% of viability similar to MNC viability (A, B). Live cells and dead cells were detected by esterase activity and EthD-1, respectively (C).
Fig. 3.
33.1±9.9% and 16.5±9.3% of CD34+ cells expressed Annexin-V/PI (+/?) (early apoptosis) and Annexin-V/PI (+/+) cells (late apoptosis), respectively (A). However DNA contents analysis showed only 3.1±1.2% of CD34+ cells lost their 2N meaning apoptosis (B, C). Profound discordance was observed between two techniques, Annexin-V/PI staining and DNA contents analysis. 5 out of 10 units UCB selected randomly were represented.
Fig. 4.
Caspase-3 activity test for CD34+ cells from randomly selected 10 samples. UCB #3 sample showed faint caspase-3 activity compared with apop-tosis-induced tumor cell line (A). CD34+ UCB cells did not express active or cleaved form of caspase-3 regardless of Annexin-V or Propidium Iodide (PI) positivity (B). Actual activity of caspase-3 should be examined by cleaved fraction which was not detected at any sample (C). T, tumor cell line (neuroblastoma); T+A, apoptosis-induced tumor cell line.
Fig. 5.
Result of intracellular esterase activity test for CD34+ cells. Randomly selected one sample was checked its intracellular esterase activity at 0, 2, 4, 6 hours after thawing. The cells were incubated in RPMI1,640 media at CO2 incubator. There was no decrement of esterase activity which means CD34+ UCB cells do not die at least within 6 hours.
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