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

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 categor...

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Autores principales: Frigerio, Roberto, Musicò, Angelo, Brucale, Marco, Ridolfi, Andrea, Galbiati, Silvia, Vago, Riccardo, Bergamaschi, Greta, Ferretti, Anna Maria, Chiari, Marcella, Valle, Francesco, Gori, Alessandro, Cretich, Marina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001372/
https://www.ncbi.nlm.nih.gov/pubmed/33806297
http://dx.doi.org/10.3390/cells10030544
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author Frigerio, Roberto
Musicò, Angelo
Brucale, Marco
Ridolfi, Andrea
Galbiati, Silvia
Vago, Riccardo
Bergamaschi, Greta
Ferretti, Anna Maria
Chiari, Marcella
Valle, Francesco
Gori, Alessandro
Cretich, Marina
author_facet Frigerio, Roberto
Musicò, Angelo
Brucale, Marco
Ridolfi, Andrea
Galbiati, Silvia
Vago, Riccardo
Bergamaschi, Greta
Ferretti, Anna Maria
Chiari, Marcella
Valle, Francesco
Gori, Alessandro
Cretich, Marina
author_sort Frigerio, Roberto
collection PubMed
description 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.
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spelling pubmed-80013722021-03-28 Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis Frigerio, Roberto Musicò, Angelo Brucale, Marco Ridolfi, Andrea Galbiati, Silvia Vago, Riccardo Bergamaschi, Greta Ferretti, Anna Maria Chiari, Marcella Valle, Francesco Gori, Alessandro Cretich, Marina Cells Technical Note 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. MDPI 2021-03-04 /pmc/articles/PMC8001372/ /pubmed/33806297 http://dx.doi.org/10.3390/cells10030544 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ).
spellingShingle Technical Note
Frigerio, Roberto
Musicò, Angelo
Brucale, Marco
Ridolfi, Andrea
Galbiati, Silvia
Vago, Riccardo
Bergamaschi, Greta
Ferretti, Anna Maria
Chiari, Marcella
Valle, Francesco
Gori, Alessandro
Cretich, Marina
Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis
title Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis
title_full Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis
title_fullStr Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis
title_full_unstemmed Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis
title_short Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis
title_sort extracellular vesicles analysis in the covid-19 era: insights on serum inactivation protocols towards downstream isolation and analysis
topic Technical Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001372/
https://www.ncbi.nlm.nih.gov/pubmed/33806297
http://dx.doi.org/10.3390/cells10030544
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