Optimized exosome isolation protocol for cell culture supernatant and human plasma

By R. Lobb, Melanie Becker, Shu Wen Wen, Christina S. F. Wong, A. Wiegmans, Antoine Leimgruber, Andreas Möller. Journal of Extracellular Vesicles, 2015

1,562 citations recorded by the source corpus.

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 analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.

Subfields: Biomedical Engineering, Cancer Research, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a89f9b283c21b1c1c77f4eefccf180009b99ccbd

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Citations in the corpus

High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources (2016, methodology): “Available purification methods include separation based on size [differential ultracentrifugation (14) and ultrafiltration (15)], density [OptiPrep (16) and sucrose (17)], floatation velocity (18), immunoaffinity (19) and polyethylene glycol (PEG)-based precipitation (15).”
Isolation of Extracellular Vesicles: General Methodologies and Latest Trends (2018, methodology): “In addition, THP is capable of binding calcium ions [147].”
Precision Cancer Medicine: Challenges and Opportunities (2019, background): “After the first cancer genomes were reported in 2008 [111, 112], tens of thousands of tumors have been comprehensively analyzed at the whole-genome or whole-exome scale, either by individual research groups or by large consortiums such as TCGA (The Cancer Genome Atlas) and ICGC (International Cancer”
Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics (2020, background): “, gradient ultracentrifugation) can address this problem [55, 56].”
A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option? (2020, methodology): “This technique relies on the use of membranes with specified pore diameters to isolate particles of a pre-determined size range [19,63,64].”
Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods (2015, result): “This finding contradicts recent results which show that commercially available 10 mL ExoSpin or qEV columns separate exosomes from soluble contaminants with high efficiency [19, 24].”