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.
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The authors of this review were supported by funds from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 860303. We thank MINECO for the TenTaCles (Spanish Excellence Network in Exosomes) and the Severo Ochoa Excellence Accreditation (SEV-2016-0644). This project has received funding from the Spanish Ministry of Economy and Competitiveness MINECO (RTI2018-094969-B-I00).
The authors declare no competing interests.
Peer review information Nature Protocols thanks Paolo Bergese, Cees Otto and Frederik Johannes Verweij for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Kruglik, S. G. et al. Nanoscale 11, 1661–1679 (2019): https://doi.org/10.1039/C8NR04677H
Royo, F. et al. J. Extracell. Vesicles 8, 1575678 (2019): https://doi.org/10.1080/20013078.2019.1575678
Tatischeff, I., Larquet, E., Falcon-Perez, J. M., Turpin, P.-Y. & Kruglik, S. G. J. Extracell. Vesicles 1, 19179 (2012): https://doi.org/10.3402/jev.v1i0.19179
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Bordanaba-Florit, G., Royo, F., Kruglik, S.G. et al. Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles. Nat Protoc 16, 3163–3185 (2021). https://doi.org/10.1038/s41596-021-00551-z
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