Journal
JOURNAL OF EXTRACELLULAR VESICLES
Volume 10, Issue 9, Pages -Publisher
WILEY
DOI: 10.1002/jev2.12128
Keywords
EV isolation; EVs; exosomes; extracellular vesicles; microvesicles; MV; plasma; proteomics; subpopulations
Categories
Funding
- Knut and Alice Wallenberg Foundation, National Bioinformatics Infrastructure Sweden at SciLifeLab
- Swedish Medical Research Council
- Swedish Cancer Society
- Cancer Research Foundations of Radiumhemmet
- Swedish Heart-Lung Association
- Hesselman's foundation
- Cancer- and Allergy Foundation
- Karolinska Institutet's KID grant
- Karolinska Institutet Foundations
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The method for isolating EVs impacts sample type, interest in EV subtypes, sample volume, and budget, and there is no one-size-fits-all method for all studies.
Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 mu l or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.
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