Isolation of intact extracellular vesicles from cryopreserved samples

Abstract

Extracellular vesicles (EVs) have emerged as promising candidates in biomarker discovery and diagnostics. Protected by the lipid bilayer, the molecular content of EVs in diverse biofluids are protected from RNases and proteases in the surrounding environment that may rapidly degrade targets of interests. Nonetheless, cryopreservation of EV-containing samples to -80°C may expose the lipid bilayer to physical and biological stressors which may result in cryoinjury and contribute to changes in EV yield, function, or molecular cargo. In the present work, we systematically evaluate the effect of cryopreservation at -80°C for a relatively short duration of storage (up to 12 days) on plasma- and media-derived EV particle count and/or RNA yield/quality, as compared to paired fresh controls. On average, we found that the plasma-derived EV concentration of stored samples decreased to 23% of fresh samples. Further, this significant decrease in EV particle count was matched with a corresponding significant decrease in RNA yield whereby plasma-derived stored samples contained only 47–52% of the total RNA from fresh samples, depending on the extraction method used. Similarly, media-derived EVs showed a statistically significant decrease in RNA yield whereby stored samples were 58% of the total RNA from fresh samples. In contrast, we did not obtain clear evidence of decreased RNA quality through analysis of RNA traces. These results suggest that samples stored for up to 12 days can indeed produce high-quality RNA; however, we note that when directly comparing fresh versus cryopreserved samples without cryoprotective agents there are significant losses in total RNA. Finally, we demonstrate that the addition of the commonly used cryoprotectant agent, DMSO, alongside greater control of the rate of cooling/warming, can rescue EVs from damaging ice formation and improve RNA yield.

Application of Cell-Derived Extracellular Vesicles and Engineered Nanovesicles for Hair Growth: From Mechanisms to Therapeutics

“…This condition of storage showed loss of EVs, changes in the lipid integrity of EVs morphology, loss of cargoes (such as RNA and protein), number of EVs were decreased on the other hand size range increased. The −80°C storage have other limitations such as high cost and transportation ( Tessier et al, 2021 ; Yuan et al, 2021 ). However, the most favorable condition for storage of isolated EVs is −80°C.…”

Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases

“…Preliminary evidence was also provided to show how EV HB‐Chip can be functionalized with ACE2 receptor for SARS‐Cov2 virus or cell‐specific Ab cocktail (CD3, CD4 or CD8 T‐cells) to capture virus or cell‐specific EVs from the plasma of COVID‐19 patients, and how these EVs can be used for RNAseq to potentially identify patients suitable for immunotherapy or diagnosis of COVID‐19 based on detection of viral RNA in EVs or plasma. Although the microfluidic systems seem to allow high purity isolation of EVs from small volume plasma samples, several areas for further research and development were identified by the expert panel, such as the need to combine with sophisticated hardware and software, select optimum Ab cocktails, address limitations associated with the usage of whole blood and investigate how haemolysis, activation of blood cells, coagulation during blood or plasma storage may affect the efficiency of EV isolation (Tessier et al., 2021 ; Wong et al., 2017 ).…”

“…EVs are currently being tested for their potential clinical application based on their biological origin, lower immunogenicity, versatility in engineering the membrane or cargo and their potential for tissue‐specific targeting (Anselmo & Mitragotri, 2021 ; Herrmann et al., 2021 ; Kalluri & LeBleu, 2020 ; Mentkowski et al., 2018 ; Murphy et al., 2019 ). However, limited knowledge of the heterogeneity of EV populations or their corresponding cargos, challenges in minimizing off‐target effects and improving tissue‐specific targeting, short half‐life and bioactivity in the circulation, selection of the dose (EVs vs. therapeutic cargo), dosage strategy (size of dose vs. frequency), route of administration (intravenous or intratracheal or intramyocardial), poorly understood pharmacokinetics or pharmacodynamics in vivo, unknowns related to the scale‐up of manufacturing for the pharmaceutical grade EVs, challenges in ensuring batch to batch reproducibility and loss of EV function during cryopreservation (Tessier et al., 2021 ) pose challenges in the translation of EVs for the therapy of HLBS diseases. Besides functional optimization of engineered EVs, the process would also need to match the milestones for cell‐derived products set by regulatory agencies, such as ensuring control, standardization and reproducibility of EV sources (parent cells used for EV production), and the methods used for EV production, including appropriate product test methods, to ensure the reproducibility of the therapeutic effects of engineered EVs.…”

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

“…Another study reported that EV concentration decreased significantly in plasma stored at −80 °C for 10–12 days compared to freshly isolated plasma samples. However, no significant difference was observed in the average size of EVs between the samples [ 69 ]. Platelets were frozen and cryopreserved for 24 h at −80 °C in 5–6% dimethyl sulfoxide (DMSO).…”