Low concentration of polyethylene glycol facilitates separation of extracellular vesicles from bronchoalveolar lavage fluid

Extracellular vesicles (EVs) in bodily fluids play an essential role in cell-cell cross talk and potentially serve as novel biomarkers in "liquid biopsy." It is crucial to have a consistent, efficient, and reliable method to separate EVs from bodily fluids. Currently, there is no universally accepted, "best" method to separate EVs. Besides differential ultracentrifugation (UC), polyethylene glycol (PEG) is among the commonly used methods for EV separation from bodily fluids. However, the optimal concentration of PEG to be used remains inadequately addressed. We initially observed that the concentration of PEG has a significant impact on the amount of separated EVs and EV-cargos, which are recovered from bronchoalveolar lavage fluid (BALF). To determine the optimal PEG concentration to be used in EV separation from BALF, we first separated the BALF and serum from wild-type C57BL/6 mice. Next, various concentrations of PEG (5%, 10%, and 15% PEG), a commercial kit, and UC were used to obtain EVs from BALF and serum. EVs were characterized, and EV-cargo protein, RNA, and miRNA levels were determined. We found that high concentration of PEG (10% and 15%) altered various EV parameters that are frequently used in EV studies, including EV yield, purity, and morphology. Using miR-15a, miR-142, and miR-223 as examples, we found that 10% and 15% PEG robustly reduced the detected levels of EV-cargo miRNAs compared with those in the EVs separated using UC or 5% PEG. Collectively, low concentration of PEG facilitates the optimal BALF EV separation.