Dataset Information

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

ABSTRACT: 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.

SUBMITTER: Lobb RJ

PROVIDER: S-EPMC4507751 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Lobb Richard J RJ Becker Melanie M Wen Shu Wen SW Wong Christina S F CS Wiegmans Adrian P AP Leimgruber Antoine A Möller Andreas A

Journal of extracellular vesicles 20150717

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. ...[more]

PMID: 26194179

Similar Datasets

Project description:Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, Fc?RI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield. Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression. Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry. Results: We observed a significant increase in the yield of total human lung CD45+ immune cells and an even more pronounced increase in the yield of CD117+ mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods. Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

Dataset Information

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Publications

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets