(B-59) Characterization of ultrasound-mediated sonoporation and freezing rates on production of freeze-dried red blood cells

Introduction:: Hemorrhage is the leading cause of preventable death worldwide, and blood transfusions with red blood cells (RBCs) play a critical role in saving lives in emergencies. However, the unavailability of blood products in certain areas or during high demand can pose a significant challenge. Currently, RBCs are stored in refrigerators at 2-6 °C with a limited shelf life of up to 42 days. Glycerol has been used to freeze RBCs for long-term storage to address this issue. However, glycerol must be removed from thawed RBCs before transfusion, and this process currently takes up to two hours which can be problematic in emergency situations. One potential solution to this problem could be freeze-dried RBCs, which have a longer shelf life and can be stored at ambient temperatures. To produce freeze-dried RBCs, we are developing a novel approach for the intracellular delivery of trehalose, a naturally occurring cell-protectant sugar that enables some organisms to survive long-term dehydration. Our technique utilizes ultrasound waves to drive microbubble oscillation for transient permeabilization of cell membranes (“sonoporation”) to rapidly deliver trehalose intracellularly and confer protection during freeze-drying and rehydration. In this study, we investigated the effect of ultrasound-mediated sonoporation and other parameters, such as freezing rate and hematocrit, on the quality and viability of freeze-dried RBCs. We also investigated the oxygen-binding dynamics of freeze-dried RBCs. This study provides important insights into processing methods for optimizing the production of freeze-dried red blood cells that could enable transfusions in austere environments

Materials and Methods:: Porcine RBCs were collected from an abattoir. RBCs were stored under refrigeration for up to four weeks and then resuspended immediately before ultrasound treatment in a saline solution containing 300 mM trehalose. Phospholipid-coated perfluorobutane microbubbles were added to RBC solutions to induce sonoporation and load trehalose or a fluorescent marker (calcein). Solutions were pumped through a 3D-printed flow chamber with a peristaltic pump at 1.5 mL/min for ultrasound treatment. B-mode ultrasound pulses (2.5 MHz center frequency) were transmitted into the flow chamber using an ATL P4-1 transducer on a Verasonics Vantage ultrasound system. Trehalose-loaded RBC samples were frozen at -1 °C/min using CoolCell containers or frozen at approximately -300 °C/min using liquid nitrogen before thawing in a 37 °C water bath or freeze-drying for three days. Freeze-dried samples were stored at ambient temperature and rehydrated with saline solutions containing 20% dextran immediately before

analysis. The number of intact RBCs was enumerated using a hemocytometer. Oxygen-binding isotherms of rehydrated RBC solutions were recorded using a Loligo Systems blood oxygen binding system at a constant temperature of 30 °C, and the gas mixture temperature was kept between 30.5-31.5 °C.


Results, Conclusions, and Discussions:: The results of this study showed that rapid freezing with liquid nitrogen generally maintained more intact RBCs compared to slow freezing. When employing the slow freezing technique at an RBC concentration of 5 million/mL, sonoporation treatment yielded a significant increase in the recovery of intact cells (p< 0.001, n=12), but this effect was not observed at an RBC concentration of 5 billion/mL. The opposite trend was observed for freeze-dried samples, with significantly higher recovery of intact RBCs after slow freezing than rapid freezing (p=0.04, n=8). The oxygen-binding capacity of freeze-dried RBCs was also measured after rehydration, and the partial pressure to achieve 50% oxygen saturation (p50) was 10.5±2.0 pO2 mmHg (n=4) and unchanged from control RBCs which were stored with refrigeration (9.7±1.6 pO2 mmHg, n=6, p=0.5), demonstrating that the freeze-dried samples maintained their oxygen-binding ability.

In this study, we identified essential parameters to optimize the production of freeze-dried RBCs through ultrasound-mediated sonoporation. These results indicate that the freezing process can significantly impact cell viability and integrity after freeze-drying and rehydration. Rapid freezing generally resulted in better recovery of intact RBCs after thawing than slow freezing. However, the opposite trend was observed for freeze-dried RBCs, which indicates that the optimal freezing rates are different for freezing and thawing compared to freeze-drying and rehydration.

This study demonstrated that optimizing freezing rates can help preserve the integrity and viability of RBCs during processing. Moreover, ultrasound-mediated sonoporation was found to further improve recovery of intact cells during slow freezing only at a low RBC concentration of 5 million/mL, but sonoporation did not provide added benefit at a higher RBC concentration of 5 billion/mL. In summary, the freeze-drying process shows potential for producing RBCs that have a longer shelf life and can be easily transported and stored at room temperature while maintaining normal oxygen-binding dynamics, making them a potentially viable solution for emergencies in austere environments.


Acknowledgements (Optional): : This project was supported by funding from the U.S. Defense Medical Research and Development Program (award #W81XWH-20-1-0866). Co-authors JAK and MAM disclose ownership in DesiCorp, Inc.

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