Improvement of small extracellular vesicle isolation from mouse model blood.

Aim: 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 in vivo 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.

Methods

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 in vivo 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.

Results

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.

Conclusion

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.