Tunable resistive pulse sensing (TRPS) was used in a systematic study aimed at assessing the impact of -80°C storage and freeze-thaw cycles on the properties of extracellular vesicles (EVs).
Three sets of experiments were completed to:
- Compare EVs from fresh purified platelet-free plasma (pPFP), pPFP-EVs stored post qEV-isolation in cryogenic vials (8 different conditions at -80°C post qEV isolation), and EVs stored as pPFP prior to qEV isolation (8 different conditions at -80°C).
- Compare fresh pPFP-EVs with EVs from aliquots subject to slow freeze-thaw cycles and snap freeze-thaw cycles.
- Analyse possible fusion phenomena occurring during storage and freeze-thaw cycles, using EVs isolated from murine microglial cell lines. Cells were genetically modified to express either green fluorescent protein or mCherry as membrane proteins, with flow cytometry used to detect ‘double positive’ events that were interpreted as possible fusion events, i.e., EV membrane disruption and lipid re-micellisation – although EV aggregation could not be ruled out.
TRPS helped corroborate and provide context to the results of the study, by providing insights into the size, concentration, and zeta potential of isolated EVs. EV characterisation by TRPS revealed a reduction in EV concentration following freeze-thaw cycles, and an increase in particle size associated with freeze-thaw cycles. A shift towards larger particle size was also observed in samples stored as isolated EVs, compared to samples stored as plasma.
Zeta potential was measured to gauge particle surface charge and EV colloidal stability. As TRPS measures EVs on a single-particle basis – rather than providing a bulk, averaged measurement – zeta potential data of single particles can be expressed as a size vs charge plot. Gelibter et al. (2022) used this approach to compare the effect of different storage conditions over time; when plasma-derived samples were stored as isolated EVs, single-particle analysis revealed a shift towards positive zeta potential values.
In contrast, this change was not observed when EVs were recovered from frozen plasma samples, suggesting a potential protective role of plasma in this context. Importantly, these changes may not be easily detectable with ensemble measures. Altogether, along with highlighting the need for functional studies and further systematic approaches to studying storage effects, the authors suggested working with fresh samples where possible, and when storage is necessary, store the biological matrix instead of the isolated sample of EVs.
