EV Separation: Release of Intact Extracellular Vesicles Immunocaptured on Magnetic Particles

Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell–cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supern...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2021-04, Vol.93 (13), p.5476-5483
Main Authors: Brambilla, Dario, Sola, Laura, Ferretti, Anna Maria, Chiodi, Elisa, Zarovni, Natasa, Fortunato, Diogo, Criscuoli, Mattia, Dolo, Vincenza, Giusti, Ilaria, Murdica, Valentina, Kluszczyńska, Katarzyna, Czernek, Liliana, Düchler, Markus, Vago, Riccardo, Chiari, Marcella
Format: Article
Language:English
Subjects:
Analytical chemistry
Antibodies
Cell culture
Cell interactions
Chemistry
Culture media
Deoxyribonuclease
Deoxyribonucleic acid
DNA
Drug delivery
Endonuclease
Exosomes
Extracellular vesicles
Imaging techniques
Immobilization
Nanoparticles
Physical characteristics
Physical properties
Separation
Transmission electron microscopy
Vesicles
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Summary:Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell–cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation of a single class of EVs, such as exosomes, is complex because blood and cell culture media contain many nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high-purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles usually requires harsh conditions that hinder their use in certain types of downstream analysis. A novel capture and release approach for small extracellular vesicles (sEVs) is presented based on DNA-directed immobilization of antiCD63 antibody. The flexible DNA linker increases the capture efficiency and allows for releasing EVs by exploiting the endonuclease activity of DNAse I. This separation protocol works under mild conditions, enabling the release of vesicles suitable for analysis by imaging techniques. In this study, sEVs recovered from plasma were characterized by established techniques for EV analysis, including nanoparticle tracking and transmission electron microscopy.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.0c05194