Abstract
Extracellular vesicles (EVs) are secreted nanoscale particles that transfer biomolecular cargo between cells in multicellular organisms. EVs play a variety of roles in intercellular communication and are being explored as potential vehicles for delivery of therapeutic biomolecules. However, EVs are highly heterogeneous in composition and biogenesis route, and this poses substantial challenges for understanding the role of EVs in biology and for harnessing these mechanisms for therapeutic applications, for which purifying therapeutic EVs from mixed EV populations may be necessary. Currently, technologies for isolating EV subsets are limited by overlapping physical properties among EV subsets. To meet this need, here we report an affinity chromatography-based method for enriching a specific EV subset from a heterogeneous EV starting population. By displaying an affinity tagged protein (tag-protein) on the EV surface, tagged EVs may be specifically isolated using simple affinity chromatography. Moreover, recovered EVs are enriched in the tag-protein relative to the starting population of EVs and relative to EVs purified from cell culture supernatant by standard differential centrifugation. Furthermore, chromatographically enriched EVs confer enhanced delivery of a cargo protein to recipient cells (via enhancing the amount of cargo protein per EV) relative to EVs isolated by centrifugation. Altogether, affinity chromatographic enrichment of EV subsets is a viable and facile strategy for investigating EV biology and for harnessing EVs for therapeutic applications.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gyorgy B, Hung ME, Breakefield XO, Leonard JN (2015) Therapeutic applications of extracellular vesicles: clinical promise and open questions. Annu Rev Pharmacol Toxicol 55:439–464. https://doi.org/10.1146/annurev-pharmtox-010814-124630
El-Andaloussi S, Mager I, Breakefield XO, Wood MJ (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12(5):347–357. https://doi.org/10.1038/nrd3978
Gyorgy B, Szabo TG, Pasztoi M, Pal Z, Misjak P, Aradi B, Laszlo V, Pallinger E, Pap E, Kittel A, Nagy G, Falus A, Buzas EI (2011) Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 68(16):2667–2688. https://doi.org/10.1007/s00018-011-0689-3
Smith ZJ, Lee C, Rojalin T, Carney RP, Hazari S, Knudson A, Lam K, Heikki S, Ibanez EL, Viitala T, Laaksonen T, Yliperttula M, Wachsmann-Hogiu S (2015) Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content. J Extracell Vesicles 4:28533
Willms E, Johansson HJ, Mager I, Lee Y, Blomberg KE, Sadik M, Alaarg A, Smith CI, Lehtio J, El Andaloussi S, Wood MJ, Vader P (2016) Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep 6:22519. https://doi.org/10.1038/srep22519
Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol/editorial board, Juan S. Bonifacino [et al.] Chapter 3:Unit 3.22. https://doi.org/10.1002/0471143030.cb0322s30
Mathivanan S, Lim JW, Tauro BJ, Ji H, Moritz RL, Simpson RJ (2010) Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature. Mol Cell Proteomics 9(2):197–208. https://doi.org/10.1074/mcp.M900152-MCP200
Hung ME, Leonard JN (2015) Stabilization of exosome-targeting peptides via engineered glycosylation. J Biol Chem 290(13):8166–8172. https://doi.org/10.1074/jbc.M114.621383
Witwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J, Nolte-'t Hoen EN, Piper MG, Sivaraman S, Skog J, Thery C, Wauben MH, Hochberg F (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2. https://doi.org/10.3402/jev.v2i0.20360
Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 29(4):341–345. https://doi.org/10.1038/nbt.1807
Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C (2005) Exosomal-like vesicles are present in human blood plasma. Int Immunol 17(7):879–887. https://doi.org/10.1093/intimm/dxh267
Hung ME, Leonard JN (2016) A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery. J Extracell Vesicles 5:31027
Reichelt S (2015) Introduction to macroporous cryogels. Methods Mol Biol 1286:173–181. https://doi.org/10.1007/978-1-4939-2447-9_14
Whitford W, Ludlow J, Cadwell J (2015) Continuous production of exosomes: utilizing the technical advantages of hollow-fiber bioreactor technology. Genet Eng Biotechnol News 35(16):34. https://doi.org/10.1089/gen.35.16.15
Acknowledgements
Theint Aung and Dr. Arabela Grigorescu (Northwestern University) provided assistance with NanoSight analysis, which was performed at the Northwestern University Keck Biophysics Facility. Traditional sequencing services were performed at the Northwestern University Genomics Core Facility. Andrew Younger (Northwestern University) provided assistance with plasmid construction. This work was supported by a 3M Non-tenured Faculty Award, the Lynn Sage Breast Cancer Foundation and Northwestern Memorial Foundation, and the Northwestern University Prostate Cancer Specialized Program of Research Excellence (SPORE) through NIH award P50 CA090386 (to J.N.L.). This work was also supported by the National Science Foundation’s Graduate Research Fellowship Program (NSF GRFP) award DGE-0824162 (to M.E.H.) and award DGE-1324585 (to D.M.S.). M.E.H. and J.N.L. are inventors on a US patent application covering inventions described in this manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hung, M.E., Lenzini, S.B., Stranford, D.M., Leonard, J.N. (2018). Enrichment of Extracellular Vesicle Subpopulations Via Affinity Chromatography. In: Patel, T. (eds) Extracellular RNA. Methods in Molecular Biology, vol 1740. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7652-2_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7652-2_9
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7651-5
Online ISBN: 978-1-4939-7652-2
eBook Packages: Springer Protocols