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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| Format: | Article |
| Language: | English |
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MDPI AG
2021-03-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/10/3/544 |
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| author | Roberto Frigerio Angelo Musicò Marco Brucale Andrea Ridolfi Silvia Galbiati Riccardo Vago Greta Bergamaschi Anna Maria Ferretti Marcella Chiari Francesco Valle Alessandro Gori Marina Cretich |
| author_facet | Roberto Frigerio Angelo Musicò Marco Brucale Andrea Ridolfi Silvia Galbiati Riccardo Vago Greta Bergamaschi Anna Maria Ferretti Marcella Chiari Francesco Valle Alessandro Gori Marina Cretich |
| author_sort | Roberto Frigerio |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-09T05:40:32Z |
| format | Article |
| id | doaj.art-c08b863e006946d9b72828ab9ca61bfc |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-09T05:40:32Z |
| publishDate | 2021-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-c08b863e006946d9b72828ab9ca61bfc2023-12-03T12:25:53ZengMDPI AGCells2073-44092021-03-0110354410.3390/cells10030544Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and AnalysisRoberto Frigerio0Angelo Musicò1Marco Brucale2Andrea Ridolfi3Silvia Galbiati4Riccardo Vago5Greta Bergamaschi6Anna Maria Ferretti7Marcella Chiari8Francesco Valle9Alessandro Gori10Marina Cretich11Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, ItalyIstituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, ItalyIstituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, ItalyIstituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, ItalyIRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, ItalyIRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, ItalyIstituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, ItalyIstituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, ItalyIstituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, ItalyIstituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, ItalyIstituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, ItalyIstituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, ItalySince 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.https://www.mdpi.com/2073-4409/10/3/544extracellular vesiclesCOVID-19SARS-CoV-2virus inactivationbiosafetypre-analytical protocols |
| spellingShingle | Roberto Frigerio Angelo Musicò Marco Brucale Andrea Ridolfi Silvia Galbiati Riccardo Vago Greta Bergamaschi Anna Maria Ferretti Marcella Chiari Francesco Valle Alessandro Gori Marina Cretich Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis Cells extracellular vesicles COVID-19 SARS-CoV-2 virus inactivation biosafety pre-analytical protocols |
| 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 | extracellular vesicles COVID-19 SARS-CoV-2 virus inactivation biosafety pre-analytical protocols |
| url | https://www.mdpi.com/2073-4409/10/3/544 |
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