Systematic Evaluation of Isolation Techniques and Freeze-Thaw Effects on Plasma Extracellular Vesicle Heterogeneity and Subpopulation Profiling.

Extracellular vesicles (EVs) are increasingly recognized as promising disease biomarkers and therapeutic carriers. However, standardizing blood-derived EV isolation remains challenging due to the heterogeneity of EV populations and variability among isolation techniques. In this study, we systematically evaluated three distinct EV isolation methods, including asymmetrical flow field-flow fractionation (AF4), size-exclusion chromatography (SEC) and automated centrifugal microfluidic disc system combined with functionalized membranes (Exo-CMDS), to compare their efficiency in isolating EVs from both freshly frozen and freeze-thawed plasma samples. We utilized an integrative approach combining Proximity-dependent Barcoding Assay (PBA) for single-EV surface protein profiling, Liquid Chromatography-Mass Spectrometry (LC-MS/MS) for bulk proteomic analysis, along with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) to assess EV yield, morphology, surface protein expression and subpopulation diversity. Our results revealed significant differences in three EV isolation methods. AF4 is particularly enriched for EV subpopulations expressing high levels of classical tetraspanins (e.g., CD81, CD9 and CD151), and single-pass membrane proteins (e.g., ITGA4 and ITAGB1). Exo-CMDS demonstrated the highest reproducibility across samples, isolating specific EV subpopulations enriched in markers like CD5. SEC provided the highest yield but co-isolated significant amounts of non-vesicular particles, including lipoproteins. The findings contribute valuable insights toward standardized and reliable EV isolation practices for research and clinical applications.