Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.

By Bow J. Tauro, David W. Greening, Rommel A Mathias, H. Ji, Suresh Mathivanan, A. Scott, Richard J. Simpson. Methods, 2012

1,105 citations recorded by the source corpus.

Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling.

Subfields: Biomedical Engineering, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/5c914ccb8944deaa31529c066099762960d32f90

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Citations in the corpus

ExoCarta as a resource for exosomal research (2012, methodology): “…viii) Koppen et al. used ExoCarta to identify orthologs of exosomal markers to characterize exosomes secreted by drosophila cells (42). ix) It has been reported that immunoaffinity capture yields high quality exosomes, provided antibodies exist for the exosomal membrane protein of choice (43).”
Optimized exosome isolation protocol for cell culture supernatant and human plasma (2015, methodology): “techniques have been carried out (6,13,20), but there is”
Bovine milk-derived exosomes for drug delivery (2015, background): “However, all isolation procedures unvaryingly have some disadvantages and contain proportions of other membranous vesicles as contaminants [32].”
Mechanisms of mesenchymal stem/stromal cell function (2016, neutral): “same source by different methods may differ in amount and/or content [179–181].”
Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy. (2012, methodology): “There are currently various methods to isolate specific extracellular vesicle populations; from ultracentrifugation protocols (97), to newer alternative methods such as filtration (98), immunoaffinity capture with beads (99) and microfluidics approaches (100).”
Focus on Extracellular Vesicles: Introducing the Next Small Big Thing (2016, methodology): “The most commonly used method for isolating exosomes is ultracentrifugation at 100,000–120,000ˆ g [8,21,154].”
Isolation of Extracellular Vesicles: General Methodologies and Latest Trends (2018, methodology): “This gradient was used to isolate the EV fraction with a particle size of 50–100 nm [36].”
Standardization of sample collection, isolation and analysis methods in extracellular vesicle research (2013, positive): “Immunologic techniques have the potential for high specificity (241,242), an important consideration in the characterization of unique EV populations.”