Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol
By Mikhail A. Livshts, E. Khomyakova, E. Evtushenko, V. N. Lazarev, N. Kulemin, S. Semina, E. Generozov, V. Govorun. Scientific Reports, 2015
616 citations recorded by the source corpus.
Exosomes, small (40–100 nm) extracellular membranous vesicles, attract enormous research interest because they are carriers of disease markers and a prospective delivery system for therapeutic agents. Differential centrifugation, the prevalent method of exosome isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors. Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for “fixed-angle” rotors. For both types of rotors – “swinging bucket” and “fixed-angle” – we express the theoretically expected proportion of pelleted vesicles of a given size and the “cut-off” size of completely sedimented vesicles as dependent on the centrifugation force and duration and the sedimentation path-lengths. The proper centrifugation conditions can be selected using relatively simple theoretical estimates of the “cut-off” sizes of vesicles. Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements. Measured by the nanoparticle tracking analysis (NTA) technique, the concentration and size distribution of the vesicles after centrifugation agree with those theoretically expected. To simplify this “cut-off”-size-based adjustment of centrifugation protocol for any rotor, we developed a web-calculator.
Subfields: Cancer Research, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/2d7272c9e3beae793f3a67ae459e0dc745047536
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Citations in the corpus
- Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics (2020, positive): “Due to ease of use, little technical expertise requirement, and compatibility with large volume preparation without complicated sample pretreatment, differential ultracentrifugation has been widely employed over the past 30 years to isolate exosomes from various sources such as cell culture medium, ”
- Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis (2019, background): “As is the case with all centrifugation methods, the separation of exosomes and other EVs from the extracellular matrix depends on density, size, and shape, with larger and more dense particles sedimenting out first [112].”
- Exosome: A Review of Its Classification, Isolation Techniques, Storage, Diagnostic and Targeted Therapy Applications (2020, neutral): “48,50 This method does not need to label exosomes, which can avoid cross-contamination, but it is not conducive to downstream analysis due to its time consumption, high cost, structural damage, aggregation into blocks, and lipoprotein co-separation.”
- A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option? (2020, methodology): “dUC has been reported to potentially damage exosomes and can alter the exosome proteome, lipome, and/or genome [50]; UF can lead to the deformation and break-up of larger vesicles due to the pressure and contact with filter membranes [19]; PEG can co-precipitate non-EV components and alter exosomal ”