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Isolation of intact extracellular vesicles from cryopreserved samples
Shannon N Tessier1,2, Lauren D Bookstaver1, Cindy Angpraseuth1
1Department of Surgery, Center for Engineering in Medicine and BioMEMS Resource Center Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States of America.
Plos One
|May 13, 2021
Summary
Cryopreservation of extracellular vesicles (EVs) at -80°C significantly reduces EV particle count and RNA yield. However, RNA quality remains high, and cryoprotectants like DMSO can mitigate these losses.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Extracellular vesicles (EVs) are crucial biomarkers for diagnostics due to their protected molecular cargo.
- Cryopreservation of EV samples can introduce stressors, potentially affecting EV integrity and yield.
Purpose of the Study:
- To systematically evaluate the impact of short-term cryopreservation (-80°C, up to 12 days) on plasma and media-derived EVs.
- To assess changes in EV particle count, RNA yield, and RNA quality after cryopreservation.
Main Methods:
- Comparison of fresh control samples with cryopreserved samples (plasma and media-derived EVs).
- Analysis of EV particle count, RNA yield, and RNA quality (RNA traces).
- Evaluation of cryoprotective agents (DMSO) and controlled cooling/warming rates.
Main Results:
- Cryopreservation led to a significant decrease in plasma-derived EV concentration (to 23% of fresh samples).
- RNA yield decreased significantly in cryopreserved plasma-derived EVs (47-52% of fresh) and media-derived EVs (58% of fresh).
- No significant decrease in RNA quality was observed, and cryoprotectants improved RNA yield.
Conclusions:
- Short-term cryopreservation without cryoprotectants causes substantial loss of EVs and RNA yield.
- High-quality RNA can still be obtained from cryopreserved samples, but yield is compromised.
- Cryoprotective agents and controlled freezing/thawing are essential for preserving EV integrity and maximizing RNA yield.
