Optimized exosome isolation protocol for cell culture supernatant and human plasma

作者：Richard J. Lobb, Melanie Becker, Shu Wen, Christina S.F. Wong, Adrian P. Wiegmans, Antoine Leimgruber, Andreas Möller · 发表于：Journal of Extracellular Vesicles · 年份：2015 · DOI：10.3402/jev.v4.27031 · 被引用次数：1752 · 研究领域：Extracellular vesicles in disease、MicroRNA in disease regulation、Nanopore and Nanochannel Transport Studies

Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative ...