A simplified and efficient method for isolating small extracellular vesicles for comparative and comprehensive translational research.

Small extracellular vesicles (sEVs) can provide information about the pathophysiology of the cells; therefore, sEVs have attracted considerable interest as possible diagnostic biomarkers. A key challenge lies in the necessity for simple and cost-effective sEV isolation methods to achieve high purity and yield suitable for research and clinical applications. We are introducing a comprehensive study on isolating sEVs using a novel cocktail strategy that integrates chemical precipitation and ultrafiltration with a two-step filtering process to ensure a highly pure and homogeneous population and further compared with PEG-based precipitation, ultra-centrifugation, and size-exclusion-chromatography columns. The isolated sEVs from each protocol are quantified for size and yield using nanoparticle tracking analysis, morphologically characterized through transmission electron microscopy, and validated by quantifying the expression profiles of sEV surface biomarkers. Furthermore, the study explores the applicability of our method for downstream multi-omics analyses. The results highlight the efficacy of the proposed protocol, demonstrating the ease and efficiency of isolating sEVs from different biofluids with minimal laboratory requirements and confirming the compatibility with multi-omics analyses. These findings position our method as particularly valuable for translational research, offering a promising avenue for advancing the study and application of sEVs in diagnostic and therapeutic research.