Combining Cooling Enhancement and Trehalose Dehydration to Enable Scalable Volume Cryopreservation of RBCs with Low Concentration of Glycerol

Enhancing the cooling rate is often an alternative to increasing concentrations of cryoprotectants to inhibit harmful ice growth during freezing. Processing cell suspensions as dispersed droplets can improve the cooling rate but carries the risk of difficult collection and contamination. As the most readily available cells, human red blood cells (RBCs) are used to test the feasibility of advanced cryopreservation techniques. Herein, a feasible and straightforward method for scalable cryopreservation of RBCs is presented. This method can be easily realized by selecting 5 x 6 mm polytetrafluoroethylene (PTFE) tubes with better cooling capacity and applying the trehalose dehydration technique. Finally, the highest freeze-thaw recovery rate in the available literature with trehalose alone (93.87% with 0.4 m Tre) and freezing the volume of RBCs from 2 to 20 mL with a higher freeze-thaw recovery of 95-96% after adding 5% glycerol (0.4 m Tre + 5% Gly) are achieved. A two-step washing method is also evaluated to remove the cryoprotectants, allowing the process could be completed within 20 min after thawing. Furthermore, the functional expression of postwash RBCs after cryopreservation is examined and all results show that this scalable volume cryopreservation method can be extended to current clinical RBC cryopreservation.