In-Cell Labeling Coupled to Direct Analysis of Extracellular Vesicles in the Conditioned Medium to Study Extracellular Vesicles Secretion with Minimum Sample Processing and Particle Loss.

Anissa Viveiros,John Monyror,Luis Carlos Morales,Vaibhavi Kadam,Desmond Pink,Aja M Rieger,Elena Posse De Chaves,Simonetta Sipione

doi:10.3390/cells11030351

Anissa Viveiros, John Monyror + Show 6 more

Open Access

https://doi.org/10.3390/cells11030351

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Abstract

Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The largest proportion of studies on EVs is based on the analysis and characterization of EVs secreted in the cell culture medium. These studies remain challenging due to the small size of the EV particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV labeling for single particle tracking and/or separation techniques. To address these problems, we characterized and validated a method for in-cell EV labeling with fluorescent lipids coupled with direct analysis of lipid-labeled EVs in the conditioned medium by imaging flow cytometry (IFC). This approach significantly reduces sample processing and loss compared to established methods for EV separation and labeling in vitro, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by size-exclusion chromatography and ultracentrifugation. Our optimized protocol for in-cell EV labeling and analysis of the conditioned medium reduces EV sample processing and loss, and is well-suited for cell biology studies that focus on modulation of EV secretion by cells in culture.

Highlights

Extracellular vesicles (EVs) are cell-derived particles delimited by a membrane
In this study, (i) we present a method to label EVs that consists of labeling parental cells with lipophilic cationic indocarbocyanine dyes prior to EV collection and analysis by imaging flow cytometry (IFC); (ii) we demonstrate that the cleared conditioned medium represents the preferable sample for analysis of EV
Secretion, with minimum processing and loss compared to EV preparations that rely on ultracentrifugation or size exclusion chromatography; and (iii) we provide evidence that, at least in some cases, EV analysis performed in the cleared conditioned medium allows detection of changes in EV secretion that cannot be distinguished if the samples undergo ultracentrifugation

Summary

Introduction

Extracellular vesicles (EVs) are cell-derived particles delimited by a membrane. They are shed by most cells and play pivotal roles in intercellular communication and signaling both in health and disease conditions [1–4]. There is a growing appreciation of the utility of EVs as therapeutics [7–10], gene editing tools [11], drug carriers [12], and in other clinical applications [13,14]. Owing to this multitude of physiological functions and therapeutic applications, the interest in EVs has grown exponentially in the past few years and recent technological advances have considerably expanded the tools available for EV studies. The small and heterogeneous size (30–1000 nm) of EVs and the absence of universal EV markers are still challenges that significantly hamper studies on the biology of EVs, with many aspects related to biogenesis, secretion, and biology still remaining unclear [15,16]

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