The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos

Abstract

Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrepTM density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrepTM density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02–1.04, 1.20–1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development.

Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality

“…In the current study, we identified that SEC (qEV IZON™ single column) resulted in some protein aggregates (possibly the lipoproteins). These findings are in agreement with our previous study [ 23 ], in which EVs were isolated from a medium conditioned by bovine embryos by means of SEC but still showed lipoprotein contamination. Similarly, Takov et al [ 27 ] demonstrated that lipoproteins were co-isolated with EVs derived from plasma when SEC is applied.…”

“…This study demonstrated that ODG UC supersedes SEC using qEV IZON™ single column in terms of EV purity and functionality preservation. It was previously demonstrated by our group [ 12 , 23 , 25 ] that ODG UC is superior in conserving EV-associated proteins and RNAs compared with other standard multistep ultracentrifugation protocols and the SEC method. In the current study, we identified that SEC (qEV IZON™ single column) resulted in some protein aggregates (possibly the lipoproteins).…”

“…To date, most of the studies performing functionality testing of EVs derived from FF have been using the “gold standard” ultracentrifugation isolation protocol [ 7 , 14 , 16 , 17 , 18 ]. However, it has been demonstrated that using high-speed ultracentrifugation can damage the EVs due to shear forces [ 12 , 23 , 24 , 25 ]. OptiPrep TM density gradient ultracentrifugation is a reliable isolation technique to purify EVs without contamination of other nanoparticles (high-density lipoproteins and ribonucleoproteins) [ 23 , 25 ].…”

“…However, it has been demonstrated that using high-speed ultracentrifugation can damage the EVs due to shear forces [ 12 , 23 , 24 , 25 ]. OptiPrep TM density gradient ultracentrifugation is a reliable isolation technique to purify EVs without contamination of other nanoparticles (high-density lipoproteins and ribonucleoproteins) [ 23 , 25 ]. Additionally, size exclusion chromatography may be a useful alternative to obtain a higher functionality of purified EVs [ 26 ].…”

Functional Relationship between Osteogenesis and Angiogenesis in Tissue Regeneration

“…EVs have been recognized as one of the intercellular communication mechanisms. EVs are lipid membrane vesicles released by cells with a strategic role that includes paracrine or autocrine biological effects in tissue metabolism [70]. They are present in biological fluids and contain several different active biomolecules, such as proteins, nucleic acids and metabolites.…”