Technical note: Efficient enrichment of plasma-derived extracellular vesicles from small volumes of bovine blood

Lay Summary This study presents a reliable method to extract small Extracellular Vesicles (sEVs) from 3-4 mL of bovine blood plasma. The sEVs are tiny particles (30-150 nm of diameter) released by cells, involved in their communication, body development, health and maintenance of homeostasis. Moreover, sEVs are known to carry different biomolecules that may serve as biomarkers of metabolic or health status, and may be related to different phenotypic expressions. However, isolating sEVs in plasma is difficult due to the presence of particles similar in size and density, such as lipoproteins. Moreover, the bovine species exhibit a specific lipoprotein profile, which results in necessary methodological adjustments to purify sEVs. To isolates sEVs, 3 steps were combined: ultra-centrifugation, size-exclusion chromatography and ultra-filtration. The sEVs size and shape were assessed combining imaging and EV-dedicated measurement techniques. A sufficient quantity of proteins was collected to enable investigation of sEVs proteome. This resulted in the identification of 417 proteins, 93% of which being known to be associated with sEVs in Vesiclepedia database. Overall, this method enables the isolation of purified plasma-derived sEVs, the investigation of their proteome, opens the possibility to study their biological functions, and finally opens new perspectives for biomarker discovery in livestock.