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Employing Flow Cytometry to Extracellular Vesicles Sample Microvolume Analysis and Quality Control.

ABSTRACT: Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. EVs-based liquid biopsies usually require EVs isolation before content analysis, which frequently increases sample volume requirements. We here present a Flow Cytometry (FC) strategy that does not require isolation or concentration of EVs prior to staining. By doing so, it enables population analysis of EVs in samples characterized by challenging small volumes, while reducing overall sample processing time. To illustrate its application, we performed longitudinal non-lethal population analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By quantifying the proportion of vesicular particles in purified and non-purified biological samples, this method also serves as a precious tool to quality control isolates of EVs purified by different protocols. Our FC strategy has an unexplored clinical potential to analyze EVs in biofluids with intrinsically limited volumes and to multiply the number of different analytes in EVs that can be studied from a single collection of biofluid.

SUBMITTER: Maia J

PROVIDER: S-EPMC7661467 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Employing Flow Cytometry to Extracellular Vesicles Sample Microvolume Analysis and Quality Control.

Maia Joana J Batista Silvia S Couto Nuno N Gregório Ana C AC Bodo Cristian C Elzanowska Julia J Strano Moraes Maria Carolina MC Costa-Silva Bruno B

Frontiers in cell and developmental biology 20201030

Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. EVs-based liquid biopsies usually require EVs isolation before content analysis, which frequently increases sample volume requirements. We here present a Flow Cytometry (FC) strategy that does not require isolation or concentratio ...[more]

PMID: 33195266

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Project description:The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform their use as biomarkers for disease.

Project description: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.

Dataset Information

Employing Flow Cytometry to Extracellular Vesicles Sample Microvolume Analysis and Quality Control.

Publications

Employing Flow Cytometry to Extracellular Vesicles Sample Microvolume Analysis and Quality Control.

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