Cryopreservation of Red-Blood-Cell-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are crucial in many physiological and pathological processes, and therefore, they are increasingly studied for their potential as diagnostic biomarkers, therapeutic agents, and drug carriers. Red-blood-cell-derived EVs (REVs) have gained particular interest due to their beneficial properties for drug delivery and their unique biophysical and molecular characteristics, which make them a practical model system for EV research. While EV cryopreservation methods have advanced in the past decade, REVs remain relatively understudied, with their hemoglobin-rich composition presenting unique storage challenges. To address this gap, we investigated how the vesicle concentration and buffer composition affected REV preservation to identify the best storage conditions. We evaluated changes in protein and lipid contents, hemoglobin retention, particle recovery, size distribution, and optical properties using a variety of analytical methods. Our results show that phosphate-buffered saline (PBS), despite its common use, leads to significant losses in vesicle number and major compositional changes. While human serum albumin (HSA) alone showed no protective effect, the combined action of HEPES, HSA, and trehalose (PBS-HAT) preserved the biochemical and biophysical integrity of REVs much more effectively. Furthermore, we found that the initial REV concentration at the time of freezing significantly affected particle recovery but not the composition of the EVs. Notably, a loss in particle concentration (30%) was already evident at an initial concentration of 1.2 × 10