Efficient enrichment of plasma-derived extracellular vesicles from small volumes of bovine blood.

There is a growing interest in small extracellular vesicles (sEVs). These nanoparticles, which range in diameter from 30 to 150 nm, are secreted by cells into their surrounding environment and transfer biological content to distant cells. However, the lack of consensus on sEV isolation, from bovine plasma limits their study. This work aimed to develop an optimized method to enrich sEVs from 4 mL of bovine blood plasma. To increase the yield of sEVs while reducing contamination from other particles and free proteins, sEVs were isolated from 38 bovine plasma samples of crossbred heifers using sequential centrifugation and filtration with size-exclusion chromatography. In accordance with the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines, the sEV preparations were characterized in terms of size, particles concentration, morphology, and sEV markers. To accurately estimate particle size and distribution, we used a combination of three methods. This approach confirmed that 76% of the particles fell within the expected range of 30-150 nm for sEVs. The preparations were pure, with an average particle-to-protein ratio of 2.4 × 108 particles/µg of protein. This is comparable to or exceeds recent observations in bovine and other mammalian species when blood plasma and serum are used. Moreover, albumin, accounted for only 1.8-6.5% of the final protein abundance, indicating a 90-98% depletion relatively to raw plasma. Microscopy confirmed the presence of cup-shaped particles characteristic of sEVs. Proteomic characterization identified 417 proteins (FDR 1%, ≥ 2 peptides), corresponding to 372 unique homologous human gene names, including the cytosolic (HSPA8, SDCBP, ACT, TUB, GAPDH) and membrane (CD9, CD81) markers of sEVs. Of these proteins, 347 (93%) are referenced in  Vesiclepedia, an international database of sEV proteome, suggesting a strong enrichment of sEVs during the purification process. This finding is supported by the identification of 172 significantly enriched Gene Ontology terms related to sEV annotation (P < 0.01, Fisher's one-tailed test with Benjamin-Hochberg correction) such as GO:0005615 (extracellular space) and GO:1903561 (extracellular vesicle). According to the MISEV guidelines and proteomic requirements, the proposed optimized sEV enrichment protocol is suitable for 4 mL of plasma. These results pave the way for future research into the role of sEVs in relation to animal health and performance.