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

Livshits MA, Livshts MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM - Sci Rep (2015)

Bottom Line: 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.Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for "fixed-angle" rotors.Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32, Vavilova str., Moscow, 119991, Russia.

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.

No MeSH data available.


Related in: MedlinePlus

Schematic sketches of the two types of rotors: (a) “swinging bucket” and (b) “fixed angle”.
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f1: Schematic sketches of the two types of rotors: (a) “swinging bucket” and (b) “fixed angle”.

Mentions: Two types of rotors are commonly used in microvesicles research3035: “swinging bucket” (SW) rotors (Fig 1A) and “fixed-angle” (FA) rotors (Fig. 1B). These rotors are fundamentally different in their geometry and consequently in their sedimentation properties. The major differences between these rotors that are important for the theoretical description of sedimentation include the following. For FA-rotors, the maximal path length for sedimenting particles usually is small in comparison with the rotation radius, allowing an approximation of the constant migration rate and simplifying the description. However, the second difference is that the horizontal cross-sections of a circular FA-tube are elliptical and the path lengths of different particles differ depending on the distance of the trajectory from the long axis of the ellipse. Peripheral particles may sediment faster because of the shorter path length.


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

Livshits MA, Livshts MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM - Sci Rep (2015)

Schematic sketches of the two types of rotors: (a) “swinging bucket” and (b) “fixed angle”.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663484&req=5

f1: Schematic sketches of the two types of rotors: (a) “swinging bucket” and (b) “fixed angle”.
Mentions: Two types of rotors are commonly used in microvesicles research3035: “swinging bucket” (SW) rotors (Fig 1A) and “fixed-angle” (FA) rotors (Fig. 1B). These rotors are fundamentally different in their geometry and consequently in their sedimentation properties. The major differences between these rotors that are important for the theoretical description of sedimentation include the following. For FA-rotors, the maximal path length for sedimenting particles usually is small in comparison with the rotation radius, allowing an approximation of the constant migration rate and simplifying the description. However, the second difference is that the horizontal cross-sections of a circular FA-tube are elliptical and the path lengths of different particles differ depending on the distance of the trajectory from the long axis of the ellipse. Peripheral particles may sediment faster because of the shorter path length.

Bottom Line: 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.Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for "fixed-angle" rotors.Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32, Vavilova str., Moscow, 119991, Russia.

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.

No MeSH data available.


Related in: MedlinePlus