The term “extracellular vesicles” (EVs) refers to lipid bilayer vesicles endogenously produced by all cell types and contain cytosolic, extracellular, and membrane signaling molecules. RecentlyEVs have been established as potential biomarkers of several diseases and their characterization is crucial for the early diagnosis and treatment; therefore, EVs have become a rapidly developing research topic over the past decade. The separation of EVs is significant for research and clinical applications of exosomes. Regardless of advancements in analytical techniques and procedures used for isolation and analysis, the standardization and characterization of EVs remains challenging. The state of art technologies like flow cytometry and mass spectrometry has significantly improved the understanding of single EVs analysis, but certain issues like high technical complexity and low throughput are limiting their applications. Other techniques including electron microscopy, Raman spectroscopy and single EV analysis have significant contribution in EV analysis. Future research must be centralized on improvement of detection methods, development of novel analytical platforms and the integration of artificial intelligence to improve the specificity of EV characterization methods. In current review, we discussed the progress in research on EV isolation, separation, and characterization methods over the past few years. Further, the limitations and advantages of these techniques have also been summarized.
