The impact of storage on extracellular vesicles: A systematic study

Abstract

Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While −80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of −80°C storage and freeze‐thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent‐tagged EVs populations (GFP and mCherry), mixed before preservation. We found that −80°C storage reduces EVs concentration and sample purity in a time‐dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size‐charge plots. None of the tested conditions prevented the observed effects. Freeze‐thaw cycles lead to an EVs reduction after the first cycle and to a cycle‐dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double‐positive EVs (GFP+‐mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non‐archival, samples in majority of cases.

Identification of storage conditions stabilizing extracellular vesicles preparations

“…There are likely other storage-related factors influencing EV stability since we observe general downtrends over time at all temperatures and in most buffers. Previous studies have also attributed loss of particles to aggregation (Bosch et al, 2016) or fusion (Gelibter et al, 2022); however, we observed no buffer-related consistent increase of particle diameters. In addition to observed quantitative differences, we included assays aiming to evaluate EV intactness and function and compared genetically engineered EVs in previously established uptake and cytokine binding assays as well as stability of intravesicular fluorescent proteins in different buffers, over time.…”

“…A few studies in the last years have investigated the impact of storage temperature and time (Cheng et al., 2019 ; Lőrincz et al., 2014 ; Park et al., 2018 ), focused on the role of plastic tubes used for isolation or storage (Evtushenko et al., 2021 ; Resnik et al., 2020 ), or explored the use of cryoprotective additives such as DMSO (Tegegn et al., 2016 ) or trehalose (Bosch et al., 2016 ). More recent comprehensive studies investigated EV stability in context of lyophilisation (Trenkenschuh et al., 2022 ), storage‐related particle loss through vesicle fusion (Gelibter et al., 2022 ), and the short‐term influence of storage conditions and concentration methods on EV recovery and function (van de Wakker et al., 2021 ). Generally, reported findings have sometimes been conflicting, likely related to a focus on storage procedures for specific biofluid samples, to specific storage strategies in terms of EV isolation procedures, or to limited downstream read‐outs only covering partial aspects of EVs such as small RNA quantification or bulk protein content in several studies (Jeyaram & Jay, 2017 ; Kusuma et al., 2018 ; Qin et al., 2020 ; Welch et al., 2017 ; Yuan et al., 2021 ).…”

Prostate-Specific Membrane Antigen (PSMA)-Positive Extracellular Vesicles in Urine—A Potential Liquid Biopsy Strategy for Prostate Cancer Diagnosis?

“…Gelibter et al found a significant increase in the particle size while the particle concentration was reduced when isolated blood plasma EVs were stored at −80 °C for 6 months. On the contrary, when blood plasma was stored under the same conditions and the EVs were isolated after thawing, the changes were less severe, indicating a possible protective effect by the biofluid [ 22 ]. In addition, it was shown that the EVs in urine were found to be relatively resistant (up to 18 h at 37 °C) toward endogenous proteolytic activity [ 23 ].…”

Impact of Storage Conditions on EV Integrity/Surface Markers and Cargos

“…However, storage at −80 °C for 6 months of plasma before EV isolation and of isolated EVs was associated with a decrease in particle concentration and an increase in protein content and particle size. The freeze–thaw process also lowered the yield, increased particle size, and triggered membrane breakdown and re-micellization [ 72 ]. Meanwhile, Jin et al, 2016 reported that serum-derived exosomes stored at RT showed significant changes in CD63, TSG101, and DNA concentrations after 24 h. In addition, the increased stability of isolated exosomes stored at 4 °C for seven days, without effects on CD63, TSG101, or nucleic acid concentrations, was observed [ 65 ].…”

“…Non-penetrating cryoprotectants (e.g., sucrose, mannose, and trehalose) can form hydrogen bonds with water, reducing the damage to EVs [ 94 ]. According to Gelibter et al, 2022, after six months of storage at −80 °C, there was no significant reduction in EV concentrations when EVs were stored with or without various cryoprotectants such as trehalose 25 mM, DMSO 6 and 10%, glycerol 30%, protease inhibitors, and sodium azide at 4 °C or after lyophilized with trehalose [ 72 ]. However, different cell-derived EVs showed other features after storage.…”