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

## TL;DR

This paper studies how to best preserve red-blood-cell-derived extracellular vesicles during freezing, finding that a specific solution (PBS-HAT) works better than common methods.

## Contribution

The study introduces PBS-HAT as a novel and effective cryopreservation method for red-blood-cell-derived extracellular vesicles.

## Key findings

- Phosphate-buffered saline (PBS) causes significant losses in vesicle number and compositional changes.
- The PBS-HAT solution preserves the biochemical and biophysical integrity of REVs more effectively.
- Higher initial REV concentration correlates with better particle recovery after freezing.

## Abstract

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 × 1011 particles/mL.
Our findings demonstrate that PBS-HAT offers an effective strategy
for the short- to midterm cryopreservation of REVs above the initial
concentration of 1012 particles/mL.