AFM-Based High-Throughput Nanomechanical Screening of Single Extracellular Vesicles

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  • Andrea Ridolfi, Università Degli Studi di Firenze, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Consiglio Nazionale delle Ricerche
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  • Marco Brucale, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Consiglio Nazionale delle Ricerche
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  • Costanza Montis, Università Degli Studi di Firenze, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase
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  • Lucrezia Caselli, Università Degli Studi di Firenze
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  • Lucia Paolini, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Università degli Studi di Brescia
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  • Anne Borup
  • Anders T Boysen
  • Francesca Loria, HansaBiomed Life Sciences
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  • Martijn J C van Herwijnen, Utrecht University
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  • Marije Kleinjan, Utrecht University
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  • Peter Nejsum
  • Natasa Zarovni, HansaBiomed Life Sciences
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  • Marca H M Wauben, Utrecht University
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  • Debora Berti, Università Degli Studi di Firenze, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase
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  • Paolo Bergese, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Università degli Studi di Brescia
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  • Francesco Valle, Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Consiglio Nazionale delle Ricerche

The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role.

Original languageEnglish
JournalAnalytical Chemistry
Volume92
Issue15
Pages (from-to)10274-10282
Number of pages9
ISSN0003-2700
DOIs
Publication statusPublished - Aug 2020

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