Isolation of intact extracellular vesicles from cryopreserved samples

Shannon N. Tessier, Lauren D. Bookstaver, Cindy Angpraseuth, Cleo J. Stannard, Beatriz Marques, Uyen K. Ho, Alona Muzikansky, Berent Aldikacti, Eduardo Reategui, Daniel C. Rabe, Mehmet Toner, Shannon L. Stott

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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.

Original languageEnglish (US)
Article numbere0251290
JournalPloS one
Issue number5 May
StatePublished - May 2021

Bibliographical note

Publisher Copyright:
© 2021 Tessier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

NSF EEC 1941543

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.


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