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AFM-Based High-Throughput Nanomechanical Screening of Single Extracellular Vesicles
journal contribution
posted on 2020-07-13, 20:29 authored by Andrea Ridolfi, Marco Brucale, Costanza Montis, Lucrezia Caselli, Lucia Paolini, Anne Borup, Anders T. Boysen, Francesca Loria, Martijn J. C. van Herwijnen, Marije Kleinjan, Peter Nejsum, Natasa Zarovni, Marca H. M. Wauben, Debora Berti, Paolo Bergese, Francesco ValleThe 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.
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AFM equipmentnanomechanical responseAFM-Based High-Throughput Nanomecha...EV samples contaminationforce microscopyprocedure yieldsEV samplesextracellular vesiclesnanosized EVsEV samplenonvesicular contaminantsdata analysisEV researchershour time scalehigh-throughput methodmeasure EVscolorectal carcinoma cell culturestiffness distributionsnanomechanical characteristicsforce spectroscopyside observationnonvesicular objectsnonvesicular aggregatesEV stiffness valuesFS techniquesSingle Extracellular Vesicles
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