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.
Researchers at the CIC bioGUNE 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. The researchers 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, they 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.
Schematic overview of the main SVA techniques
Data visualization and single-vesicle interpretation using each SVA methodology are depicted. In the center of the figure, a (tumorigenic) cell releasing EVs is shown. a–i, The techniques can be divided into two groups: label-free (a–e) and label-based (f–i) methodologies. The methods used here are cryo-electron microscopy (a), AFM (b), NTA (c), RTM (d), SP-IRIS (e), hrFC (f), ddPCR (g), SRM (h) and fluorescence microscopy (i; TIRF image of synaptic vesicles is depicted).