Improvement of small extracellular vesicle isolation from mouse model blood

<b>Aim:</b> Small extracellular vesicles (sEVs) are membrane-bound nanoparticles secreted by virtually all cell types that have emerged as promising sources of protein biomarkers for a wide range of diseases, including central nervous system disorders. Blood sampling is the most informative and non-invasive biomarker source. Notably, mouse models represent essential systems for studying <i>in vivo</i> disease mechanisms and testing therapeutic strategies. Therefore, in this study, we investigated the suitability of two different isolation methods for sEV recovery starting from non-terminal mouse blood sampling, with the aim of identifying the most effective protocol for downstream biomarker discovery. <b>Methods:</b> We performed and compared size exclusion chromatography (SEC) and ultracentrifugation followed by iodixanol density gradient (UC-IDG). Additionally, we optimized extracellular vesicle (EV) isolation from small-volume samples of both serum and plasma, since these represent the most used sources for <i>in vivo</i> preclinical biomarker research. Both methods were evaluated in terms of yield, purity, and EV protein content by nanoparticle tracking analysis, electron microscopy, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) proteomics. <b>Results:</b> SEC showed a higher number of isolated vesicles and EV-associated markers, while reporting a reduced percentage of blood-abundant co-isolated proteins, compared to UC-IDG. The use of plasma as a starting material resulted in a cleaner background, showing fewer protein aggregates. The obtained results emphasize the advantage of SEC in enhancing vesicle yield and purity levels. <b>Conclusion:</b> This work contributes to sEV-derived biomarker research in mouse models by confirming plasma, rather than serum, as the most reliable source of EVs and providing evidence that SEC is more suitable than UC-IDG for EV isolation.