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Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.

ABSTRACT: 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.

SUBMITTER: Livshits MA

PROVIDER: S-EPMC4663484 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.

Livshits Mikhail A MA Khomyakova Elena E Evtushenko Evgeniy G EG Lazarev Vassili N VN Kulemin Nikolay A NA Semina Svetlana E SE Generozov Edward V EV Govorun Vadim M VM

Scientific reports 20151130

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 ac ...[more]

PMID: 26616523

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Project description:BackgroundExtracellular vesicles (EVs) is a group of heterogeneous cell-derived membrane structures, which is composed of a large number of exosomes released by cells, microbubbles (MVs) and apoptotic bodies. The formation of exocrine body is a process of fine regulation, which includes four stages: initiation, endocytosis, polycystic body formation and exocrine body secretion. Ultracentrifugation is currently the gold standard for external body separation; it includes a series of centrifugation steps at a rotation speed of 100,000 rpm or more to purify exocrine bodies from protein contaminants. Thus far, some in vitro separation methods, such as ultracentrifugation, polymer-based exosome separation kits and immune affinity-based isolation using antibodies against exosome surface proteins, have been used for tumor exosome isolation. It is not very clear which method is more suitable for the separation of serum exosomes from lung cancer patients.MethodsTwo methods for the extraction of exosomes from serum samples of lung cancer patients, namely, ultra-high speed centrifugation (Ultra-Exo) and precipitation (Prekit-Exo), were analyzed and compared. The biological morphologies of the extracted exosomes were studied by negative staining matter with transmission electron microscopy and cryo-electron microscopy. The particle size and the distribution were detected using nanoparticle tracking analysis (NTA).ResultsBio-transmission electron microscopy revealed that the morphologies of exosomes extracted by ultracentrifugation were superior to exosomes extracted with the Prekit-Exo kit. Ultracentrifugation was able to extract more exosomes compared to the Prekit-Exo kit. NTA showed that the exosomes obtained by ultra-high speed centrifugation had a smaller particle size compared to exosomes obtained by precipitation (30.4±26.8 vs. 150.3±6.8 nm, respectively). It is possible that the precipitant used in the precipitation kit was extracted with the exosomes, thereby causing the particle size to increase. Notably, the particle size of the exosomes extracted by the precipitation kit method showed a relatively narrow range in size. This could be due to the coating effect of the precipitation reagent, reducing the difference in the particle size of the exosomes.ConclusionsExosomes collected from the serum of lung cancer patients using the two extraction methods differed in morphology and numbers, with the ultracentrifugation method being superior to the precipitation method.

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Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.

Publications

Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.

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