Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles

Extracellular vesicles (EVs) are heterogeneous lipid containers with a complex molecular cargo comprising several
populations with unique roles in biological processes. These vesicles are closely associated with specific physiological
features, which makes them invaluable in the detection and monitoring of various diseases. EVs play a key role in
pathophysiological processes by actively triggering genetic or metabolic responses. However, the heterogeneity of their
structure and composition hinders their application in medical diagnosis and therapies. This diversity makes it difficult to
establish their exact physiological roles, and the functions and composition of different EV (sub)populations. Ensemble
averaging approaches currently employed for EV characterization, such as western blotting or ‘omics’ technologies, tend
to obscure rather than reveal these heterogeneities. Recent developments in single-vesicle analysis have made it possible
to overcome these limitations and have facilitated the development of practical clinical applications. In this review, we
discuss the benefits and challenges inherent to the current methods for the analysis of single vesicles and review the
contribution of these approaches to the understanding of EV biology. We describe the contributions of these recent
technological advances to the characterization and phenotyping of EVs, examination of the role of EVs in cell-to-cell
communication pathways and the identification and validation of EVs as disease biomarkers. Finally, we discuss the
potential of innovative single-vesicle imaging and analysis methodologies using microfluidic devices, which promise to
deliver rapid and effective basic and practical applications for minimally invasive prognosis systems.