Advances in extracellular vesicle research: tools and techniques

Extracellular vesicles (EVs) are lipid bilayer vesicles produced endogenously by all cell types and they contain cytosolic, extracellular, and membrane signaling molecules. Due to the physiological and pathological significance of EVs, they have emerged as potential biomarkers of diseases and as vectors for delivering therapeutic molecules. For accurate functional and clinical analysis of EVs, reliable methods for isolation, labelling, imaging and characterization are required. In this review, we discuss the progress in research on methods for isolating and analyzing EVs over the past few years. While different reviews have focused on different aspects of EV research, a detailed, comparative report spanning isolation, imaging, labelling and characterization is lacking. Our review will provide a comprehensive analysis of currently available techniques for EV research by discussing traditional and emerging methods of isolation including ultracentrifugation, ultrafiltration, chromatographic-based methods, precipitation-based methods and microfluidics approaches with their advantages and disadvantages. Furthermore, for tracking and visualization of EVs, various labeling strategies, including fluorescent labeling, nanoparticle labeling, radiolabeling and genetic labeling are summarized. In addition to imaging and characterization of EVs, various state-of-the-art technologies, including electron microscopy, fluorescence microscopy, flow cytometry, mass spectrometry and other molecular profiling techniques have been discussed. This review aims to help researchers select appropriate methods for isolating and analyzing EVs, in both fundamental and clinical research, by comparing the performance, scalability and reproducibility of methods through application-specific suitability. Future research should focus on improving detection methods, developing novel analytical platforms and integrating artificial intelligence to improve the specificity of EV characterization methods.