Isolation of intact extracellular vesicles from cryopreserved samples.

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 inter...

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Autores principales:	Shannon N Tessier, Lauren D Bookstaver, Cindy Angpraseuth, Cleo J Stannard, Beatriz Marques, Uyen K Ho, Alona Muzikansky, Berent Aldikacti, Eduardo Reátegui, Daniel C Rabe, Mehmet Toner, Shannon L Stott
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/37571d608dcc43d79e2f83e12ac8c324
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spelling	oai:doaj.org-article:37571d608dcc43d79e2f83e12ac8c3242021-12-02T20:05:38ZIsolation of intact extracellular vesicles from cryopreserved samples.1932-620310.1371/journal.pone.0251290https://doaj.org/article/37571d608dcc43d79e2f83e12ac8c3242021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0251290https://doaj.org/toc/1932-6203Extracellular 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.Shannon N TessierLauren D BookstaverCindy AngpraseuthCleo J StannardBeatriz MarquesUyen K HoAlona MuzikanskyBerent AldikactiEduardo ReáteguiDaniel C RabeMehmet TonerShannon L StottPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 5, p e0251290 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Shannon N Tessier Lauren D Bookstaver Cindy Angpraseuth Cleo J Stannard Beatriz Marques Uyen K Ho Alona Muzikansky Berent Aldikacti Eduardo Reátegui Daniel C Rabe Mehmet Toner Shannon L Stott Isolation of intact extracellular vesicles from cryopreserved samples.
description	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.
format	article
author	Shannon N Tessier Lauren D Bookstaver Cindy Angpraseuth Cleo J Stannard Beatriz Marques Uyen K Ho Alona Muzikansky Berent Aldikacti Eduardo Reátegui Daniel C Rabe Mehmet Toner Shannon L Stott
author_facet	Shannon N Tessier Lauren D Bookstaver Cindy Angpraseuth Cleo J Stannard Beatriz Marques Uyen K Ho Alona Muzikansky Berent Aldikacti Eduardo Reátegui Daniel C Rabe Mehmet Toner Shannon L Stott
author_sort	Shannon N Tessier
title	Isolation of intact extracellular vesicles from cryopreserved samples.
title_short	Isolation of intact extracellular vesicles from cryopreserved samples.
title_full	Isolation of intact extracellular vesicles from cryopreserved samples.
title_fullStr	Isolation of intact extracellular vesicles from cryopreserved samples.
title_full_unstemmed	Isolation of intact extracellular vesicles from cryopreserved samples.
title_sort	isolation of intact extracellular vesicles from cryopreserved samples.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/37571d608dcc43d79e2f83e12ac8c324
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_version_	1718375417090408448

Isolation of intact extracellular vesicles from cryopreserved samples.

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