Comparison of methods for isolation of extracellular vesicles from bronchoalveolar lavage fluid

Aim: Extracellular vesicles (EVs) have been described and isolated from a variety of biological samples, including bronchoalveolar lavage (BAL). While EVs have been isolated from BAL by various methods, there has not been a comparative study to determine optimal EV isolation methods for this complex and unique biologically derived fluid. Methods: Membrane affinity binding (exoEasy by Qiagen), ultracentrifugation (UC), and size-exclusion chromatography (SEC) EV isolation methods were compared to determine the best utilization of each method when isolating EVs from BAL. Results: Both SEC and UC were able to isolate significant EV populations reliably, with UC yielding higher apparent yields by western blotting and Nanosight particle tracking. However, transmission electron microscopy, total protein assays, and proteomic analysis suggested this was due to free protein and protein aggregate contamination. The exoEasy kit demonstrated inconsistent yields and protein measurements that were frequently higher than unprocessed BAL samples. Furthermore, ExoEasy preps contained many unique proteins and higher lipoproteins compared to SEC and UC. This may indicate false signal as a result of the EV isolation process making analysis and downstream applications unreliable without an additional buffer exchange step. Most of the protein in BAL was found in the non-EV fractions. In contrast, virtually all the nucleic acids, both RNA and dsDNA, found in BAL were in the protected environment of EVs. Conclusion: SEC fractions containing EVs, when concentrated using 30 kDa centrifugal filters, yielded the lowest contaminating free protein and the highest nucleic acid content. This, coupled with good yield and preservation of EV structure and function for downstream use, makes it the ideal EV isolation protocol from BAL.