Development and testing of a stepwise thaw and dilute protocol for cryopreserved umbilical cord blood units

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.

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.

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.

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.