A simple, economical, and high-yield method for polyethylene glycol-based extraction of follicular and serum-derived extracellular vesicles

Objectives:The optimization of polyethylene glycol (PEG)-based extracellular vesicles (EVs) extraction from human follicular fluid (FF) and serum was investigated, and their functional analysis was confirmed. The PEG-based EV results were compared to the ExoQuick (ExoQ)-based EV.Materials and Methods:FF-EVs and serum-EVs were extracted by using different concentrations of PEG (8000). Nanoparticle tracking analysis was used to count the particles, and electron microscopy of EVs was performed for visualization. Exosomes were confirmed by the western blot analysis with exosome-specific markers. RNA and microRNA were extracted from exosomes and quantitative polymerase chain reaction analysis was performed. Fallopian tube epithelial (FTE) cells were used for the EV uptake experiment and an anchorage-independent growth test to confirm that extracted EVs harbor transformation activity.Results:The PEG 8% enriched method produced the highest yield and the lowest carry-over protein. Salt containing PEG 8% produced a higher yield than nonsalted PEG 8%. Overnight enrichment increased four times and 18 times for PEG 8% and ExoQ-based EV extraction from FF. For serum EV, the same overnight enrichment moderately increased yield for both PEG 8% and ExoQ methods. Less carry-over protein resulted in more EV-promoted transformation activity.Conclusion:This study overcomes the time-consuming, expensive, laborious, and complicated machine-dependent EV extraction methods. The study highlights that longer incubation time is needed for EV extraction from FF. PEG 8000-based EV extraction provided a higher yield and less carry-over protein than ExoQ-based EV extraction.