Sonoporation enables high-throughput loading of trehalose into red blood cells

Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Storing RBCs in a frozen or dried state is an appealing solution to address storage limitations, but many promising cryoprotectants, including the non-reducing sugar trehalose, are impermeant to mammalian cell membranes and cannot be utilized effectively using currently available compound-loading methods. We found that transient pore formation induced by ultrasound and microbubbles (sonoporation) offers an effective means of loading trehalose into RBCs to facilitate long-term storage in a frozen or desiccated state. The protective potential of trehalose loading was demonstrated by freezing processed RBCs at -1 °C/min to -80 °C, then either storing the cells at -80 °C or lyophilizing them. RBCs were either thawed or rehydrated after 42 days of storage and evaluated for membrane integrity and esterase activity to estimate recovery and cell viability. The intracellular concentration of trehalose reached 40 mM after sonoporation and over 95% of treated RBCs were recovered after loading. Loading of trehalose was sufficient to maintain RBC morphology and esterase activity in most cells during freezing (>90% RBC recovery) and to a lower degree after lyophilization and rehydration (>20% recovery). Combining sonoporation with an integrated fluidics device allowed for rapid loading of up to 70 mM trehalose into RBCs. These results demonstrate the potential of sonoporation-mediated trehalose loading to increase recovery of viable RBCs, which could lead to effective methods for long-term stabilization of RBCs.