In the absence of the maternal tract, pre-implantation bovine embryos cultured in group are able to promote their own development in vitro by releasing autocrine embryotropins. Recently we have identified extracellular vesicles (EV) among these embryotropins as one of the communication mechanisms among embryos. Extracellular vesicles are nano-sized (25-250nm), with a lipid bilayer, and are functionally active, since they contain proteins, lipids, and nucleic acids, including RNA and miRNA. However, one of the major challenges in isolating EV is an inadequate volume of medium conditioned by bovine embryo. As it requires larger volumes of conditioned medium to isolate EV, our study mainly focused on isolating high yields of functional EV from a minimal volume. There are 3 known isolation methods for EV: differential ultracentrifugation (DU), OptiPrep™ density gradient ultracentrifugation (ODGU), and size-exclusion chromatography (SEC). We have used these 3 protocols to determine the method that yielded the highest number of EV. We used routine in vitro maturation and fertilization methods, but for in vitro culture presumed zygotes were cultured until 8 days post-insemination (dpi) in medium (synthetic oviducal fluid supplemented with insulin, transferrin, selenium, and bovine serum albumin) that was ultracentrifuged to remove any possible contaminating EV. In vitro embryo culture took place in groups of 25 presumed zygotes in 50-mL drops, covered with mineral oil and incubated at 38°C in 5% CO2, 5% O2, and 90% N2. On 8 dpi, medium conditioned by bovine embryo was collected and pooled until 3mL. For each isolation method, 1mL of conditioned medium was used, and next, EV isolated from each isolation method were analysed with nanoparticle tracking, electron microscopy, and Western blot (CD9, Flotillin 1, and AGO 2). We observed higher concentrations (1.03×109 particles mL−1) of EV were isolated from the SEC compared with the other 2 methods (301.5×108 particles mL−1 and 64.5×108 particles mL−1 for DU and ODGU, respectively; P<0.05), whereas smaller size EV (20-50nm) were lost during the ultracentrifugation methods. Besides, it takes only 2h of time to perform size-exclusion chromatography for isolating EV, whereas it takes more than 1 day to perform ultracentrifugation methods. Therefore, we propose to use SEC for further downstream processing and sequencing of miRNA in isolated EV. We are currently focusing on optimizing an EV isolation protocol to extract EV from very low volumes of conditioned medium (less than 500 µL).
Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples