Abstract
Extracellular vesicle (EV) isolation from conditioned cell culture medium has been a challenging topic. It is particularly difficult to obtain pure and intact EVs at a large scale. The commonly used methods such as differential centrifugation, ultracentrifugation, size exclusion chromatography, polyethylene glycol (PEG) precipitation, filtration, and affinity-based purification each have their advantages and limitations. Here, we present a tangential-flow filtration (TFF) based, multi-step purification protocol that combines filtration, PEG precipitation, and Capto Core 700 multimodal chromatography (MMC) to isolate EVs at high purity from large volumes of cell culture conditioned medium. Inserting the TFF step before PEG precipitation removes proteins, which may aggregate in subsequent steps and co-purify with EVs.
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
Similar content being viewed by others
References
Kalluri R, LeBleu VS (2020) The biology, function, and biomedical applications of exosomes. Science 367(6478):eaau6977
Théry C, Witwer KW, Aikawa E et al (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7(1):1535750
Théry C, Amigorena S, Raposo G et al (2006. Chapter 3) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 3:22
Lobb RJ, Becker M, Wen SW et al (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 4:27031
McNamara RP, Caro-Vegas CP, Costantini LM et al (2018) Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles. J Extracell Vesicles 7(1):1541396
Konoshenko MY, Lekchnov EA, Vlassov AV et al (2018) Isolation of extracellular vesicles: general methodologies and latest trends. Biomed Res Int 2018:8545347
Rider MA, Hurwitz SN, Meckes DG (2016) ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles. Sci Rep 6(1):23978
Sidhom K, Obi PO, Saleem A (2020) A review of Exosomal isolation methods: is size exclusion chromatography the best option? Int J Mol Sci 21(18):6466
Burgess RR (2018) A brief practical review of size exclusion chromatography: rules of thumb, limitations, and troubleshooting. Protein Expr Purif 150:81–85
Gámez-Valero A, Monguió-Tortajada M, Carreras-Planella L et al (2016) Size-exclusion chromatography-based isolation minimally alters extracellular Vesicles’ characteristics compared to precipitating agents. Sci Rep 6(1):33641
Benedikter BJ, Bouwman FG, Vajen T et al (2017) Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. Sci Rep 7(1):15297
McNamara RP, Dittmer DP (2020) Modern techniques for the isolation of extracellular vesicles and viruses. J Neuroimmune Pharmacol 15(3):459–472
McNamara RP, Eason AB, Zhou Y et al (2022) Exosome-encased nucleic acid scaffold chemotherapeutic agents for superior anti-tumor and anti-angiogenesis activity. ACS Bio Med Chem Au, vol 2, p 140
McNamara RP, Zhou Y, Eason AB et al (2022) Imaging of surface microdomains on individual extracellular vesicles in 3-D. J Extracell Vesicles 11(3):e12191–e12191
Chambers MG, McNamara RP, Dittmer DP (2021) Direct stochastic optical reconstruction microscopy of extracellular vesicles in three dimensions. J Vis Exp 174
Acknowledgments
This work was supported by public health service grant R01-CA228172 to D.P.D.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Yuan, R., Zhou, Y., Arias, G.F., Dittmer, D.P. (2023). Extracellular Vesicle Isolation by a Tangential-Flow Filtration-Based Large-Scale Purification Method. In: Vainio, S. (eds) Cell-Secreted Vesicles. Methods in Molecular Biology, vol 2668. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3203-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3203-1_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3202-4
Online ISBN: 978-1-0716-3203-1
eBook Packages: Springer Protocols