Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry

Abstract

Extracellular vesicles (EVs) are responsible for a multitude of physiological functions, including immunomodulation. A heterogenous mixture of small EV (sEV) subsets, including putative exosomes, is derived when commonly used “exosome” isolation techniques are employed. Subset diversity relates in part to their different intracellular origins, and can be associated with distinct functional properties. Recent progress in the EV field has enabled the categorization of such subsets based on their surface composition. For the first time, we combine such emerging subset-specific markers with advanced imaging flow cytometry (iFCM) to perform high-throughput, multiparametric, vesicle-by-vesicle characterization, and functional assessment of specific small EV subsets, and exosomes in particular. The approach allows researchers to address three important applications. First, it is known that different isolation techniques result in the divergent recovery of particular vesicle subsets. Taking three commonly used “exosome” isolation techniques as test cases (ultracentrifugation, size-exclusion chromatography, and polymer-based precipitation), the capacity for convenient and accurate isolate compositional analysis by iFCM is demonstrated. The approach was able to corroborate and to quantify the known skewing of subtype recovery among different isolation approaches. Second, exosomes are a particularly widely studied EV subset. Applying exosome-specific markers to samples collected from an optimal clinical transplantation model, we verify the capacity for iFCM to detect exosomes in circulation, to establish their tissue of origin, and to provide insights as to their functional immunological potential. Finally, we describe a technique for establishing whether the transfer of a molecule of interest to a target cell is exosomally mediated. In so doing, we highlight the approach’s utility in assessing the functional impact of circulating exosomes and in identifying their targets. In conclusion, we set out a new methodological approach by which small extracellular vesicle subsets, exosomes in particular, can be conveniently and comprehensively investigated, thereby offering novel phenotypic and functional insights.

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

“…Standardized FC has a high potential for the detection of EVs in body fluids and, when combined with specific antibodies, concurrently allows EV immunophenotypic characterization [42,44,84,131,151,152] ( Figure 2). Imaging flow cytometers combining conventional FC with fluorescence provide new, highly sensitive tools for EV studies [153,154].…”

Functions of Extracellular Vesicles in Immunity and Virulence

“…pauca 9a5c (Santiago et al, 2016). Alternatively, EVs can be isolated using immunoaffinity capture and advanced imaging flow cytometry (Li et al, 2017;Mastoridis et al, 2018). However, the latter two approaches have not been described for the isolation of EVs from plants and plantinteracting microbes (Tables 1 and 2), particularly because they depend on suitable EV biomarkers.…”

Population Analysis of Extracellular Vesicles in Microvolumes of Biofluids

“…Our FC strategy relies on the staining of vesicular particles with CFSE ( Fig.1a), as previously described in the literature. 17,19,27 A subset of the experiments showed here was performed with conditioned medium, in which cells were grown in medium containing EVs-depleted FBS. To control for the presence of serum-derived vesicles in our samples, serum-free medium and medium containing 10 % of EVs-depleted FBS were stained with CFSE and analyzed using our standard settings, with flow cytometry data being acquired for similar periods of time.…”