Optimized exosome isolation protocol for cell culture supernatant and human plasma

Journal of extracellular vesicles - Tập 4 Số 1 - 2015
Richard J. Lobb1,2, Melanie Becker3, Shu Wen3, Christina S.F. Wong3, Adrian P. Wiegmans3, Annette Leimgruber4,5, Andreas Möller1,2
1School of Medicine, University of Queensland, Brisbane, Qld, Australia
2Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia, School of Medicine, University of Queensland, Brisbane, QLD, Australia
3Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia
4Department of Medical Imaging, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; #R##N#Service of Nuclear Medicine and Molecular Imaging, Department of Medical Imaging, University Hospital of Lausanne, Switzerland
5Service of Nuclear Medicine and Molecular Imaging Department of Medical Imaging University Hospital of Lausanne Switzerland

Tóm tắt

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.

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Thông tin xuất bản

Journal of extracellular vesicles

Tập 4 Số 1

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