It is clinically important to maintain high viability and potency of umbilical cord blood units (CBUs) for transplantation during thawing. In the absence of a standard thawing protocol, this study was designed to develop one based on the consensus practice of transplant centers and address the shortage of dextran 40 thawing solution.STUDY DESIGN AND METHODS:
Frozen CBU aliquots were thawed using dextran 40 thawing solution while manipulating temperature and volume of diluent and mode of dilution. The effects of these on CD45+ and CD34+ cell viability were measured through annexin V and SYTOX green staining. The developed protocol was then used to compare dextran 40 and PLASMA-LYTE A thawing solutions and finally tested on whole CBUs.RESULTS:
Step-by-step investigations resulted in the development of a protocol that thaws and dilutes CBUs with room temperature diluent to five times the original volume using two sequential dilutions separated by equilibration times. PLASMA-LYTE A diluent provided superior viability of CD45+ and CD34+ cells than dextran 40 and recovered more colony-forming units. However, both diluents were equally effective in maintaining stability of the thawed CBU for 4 hours. Moreover, the stem cell-enriched CD34+CD38- subpopulations appeared more resistant to cryoinjuries than their CD34+CD38+ counterpart.CONCLUSION:
The developed thawing protocol recovers viable CD45+ and CD34+ cells above the standard thresholds and maintains CBU potency. PLASMA-LYTE A for thawing solution proved to be an efficient alternative to dextran 40. Finally, greater dilution should be avoided to maintain the viability of CD45+ cells and maximize graft cell dose.