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Technical Note

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

1
Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, Italy
2
Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy
3
Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI), Via della Lastruccia 3, Sesto Fiorentino, 50019 Firenze, Italy
4
IRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
These authors contributed equally to this work.
Academic Editors: Alexander E. Kalyuzhny, Mahmoud Huleihel and Mauro Pistello
Cells 2021, 10(3), 544; https://doi.org/10.3390/cells10030544
Received: 2 February 2021 / Revised: 24 February 2021 / Accepted: 1 March 2021 / Published: 4 March 2021
(This article belongs to the Section Organelle Function)
Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators’ safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis. View Full-Text
Keywords: extracellular vesicles; COVID-19; SARS-CoV-2; virus inactivation; biosafety; pre-analytical protocols; human serum; serum specimen extracellular vesicles; COVID-19; SARS-CoV-2; virus inactivation; biosafety; pre-analytical protocols; human serum; serum specimen
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MDPI and ACS Style

Frigerio, R.; Musicò, A.; Brucale, M.; Ridolfi, A.; Galbiati, S.; Vago, R.; Bergamaschi, G.; Ferretti, A.M.; Chiari, M.; Valle, F.; Gori, A.; Cretich, M. Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis. Cells 2021, 10, 544. https://doi.org/10.3390/cells10030544

AMA Style

Frigerio R, Musicò A, Brucale M, Ridolfi A, Galbiati S, Vago R, Bergamaschi G, Ferretti AM, Chiari M, Valle F, Gori A, Cretich M. Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis. Cells. 2021; 10(3):544. https://doi.org/10.3390/cells10030544

Chicago/Turabian Style

Frigerio, Roberto, Angelo Musicò, Marco Brucale, Andrea Ridolfi, Silvia Galbiati, Riccardo Vago, Greta Bergamaschi, Anna Maria Ferretti, Marcella Chiari, Francesco Valle, Alessandro Gori, and Marina Cretich. 2021. "Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis" Cells 10, no. 3: 544. https://doi.org/10.3390/cells10030544

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