University of Louisville
The Recipe for Freeze-Drying: Red Blood Cell Edition
Grade Level at Time of Presentation
Junior
Major
Biology
Minor
Public Health
2nd Grade Level at Time of Presentation
Junior
2nd Student Major
Chemistry
2nd Student Minor
History and Biology
KY House District #
76; 31
KY Senate District #
28; 20
Faculty Advisor/ Mentor
Michael Menze, PhD
Department
Dept. of Biology
Abstract
Transfusions of red blood cells (RBCs) are vital to many medical procedures, including surgery. Medical procedures must be delayed or rescheduled when blood donations decrease, like during the COVID-19 pandemic. The main reason for shortages of RBC units is their short shelf-life and inability to develop strategic reserves. RBC units have a maximum FDA-approved shelf-life of 42 days and require constant cooling at 1 – 6 °C. Many animals, such as tardigrades and brine shrimp, have evolved mechanisms to survive severe drying. These organisms can be dried to less than 5 percent of body water and remain dormant in a state approaching suspended animation for years until appropriate conditions for life arise. Researchers have shown that these animals intracellularly accumulate high concentrations of the non-toxic sugar trehalose. It has been hypothesized that trehalose provides stability to cellular structures in the dry state. Therefore, our project implemented trehalose to protect RBCs during freeze-drying. We studied three parameters during freeze-drying to identify conditions that yield the highest quality RBCs after rehydration. Before freeze-drying, trehalose loading procedures were performed, and intracellular trehalose concentrations were measured to assess its effect on RBC recovery after rehydration. Additionally, various chemical additions to the freeze-drying buffer were employed to evaluate the protection provided by multiple compounds in a range of concentrations. Finally, after freeze-drying, different proton concentrations in the rehydration buffers were assessed to determine if the pH plays a role in RBC recovery after rehydration. The obtained data show that RBC loss (hemolysis) could be significantly decreased by optimizing the freeze-drying parameters, trehalose loading techniques, the freeze-drying buffer utilized, and the rehydration buffer used for resuspension. A thorough understanding of how animals survive extreme drying (desiccation) will bring us closer to meeting the extensive daily demand for RBC units in the United States.
The Recipe for Freeze-Drying: Red Blood Cell Edition
Transfusions of red blood cells (RBCs) are vital to many medical procedures, including surgery. Medical procedures must be delayed or rescheduled when blood donations decrease, like during the COVID-19 pandemic. The main reason for shortages of RBC units is their short shelf-life and inability to develop strategic reserves. RBC units have a maximum FDA-approved shelf-life of 42 days and require constant cooling at 1 – 6 °C. Many animals, such as tardigrades and brine shrimp, have evolved mechanisms to survive severe drying. These organisms can be dried to less than 5 percent of body water and remain dormant in a state approaching suspended animation for years until appropriate conditions for life arise. Researchers have shown that these animals intracellularly accumulate high concentrations of the non-toxic sugar trehalose. It has been hypothesized that trehalose provides stability to cellular structures in the dry state. Therefore, our project implemented trehalose to protect RBCs during freeze-drying. We studied three parameters during freeze-drying to identify conditions that yield the highest quality RBCs after rehydration. Before freeze-drying, trehalose loading procedures were performed, and intracellular trehalose concentrations were measured to assess its effect on RBC recovery after rehydration. Additionally, various chemical additions to the freeze-drying buffer were employed to evaluate the protection provided by multiple compounds in a range of concentrations. Finally, after freeze-drying, different proton concentrations in the rehydration buffers were assessed to determine if the pH plays a role in RBC recovery after rehydration. The obtained data show that RBC loss (hemolysis) could be significantly decreased by optimizing the freeze-drying parameters, trehalose loading techniques, the freeze-drying buffer utilized, and the rehydration buffer used for resuspension. A thorough understanding of how animals survive extreme drying (desiccation) will bring us closer to meeting the extensive daily demand for RBC units in the United States.