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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

The size distribution of the HT29 exosome population changing under a test centrifugation (30 min, 10000 g) in a Beckman 60 Ti rotor: (red)–NTA distribution of pre-purified exosomes before the test centrifugation; (blue)–measured NTA distribution observed after the centrifugation; (green)–theoretically expected distributions for the assumed vesicle densities: (solid) 1.15 g/cm3, (dashed) 1.08 g/cm3, (dotted) 1.19 g/cm3.
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f3: The size distribution of the HT29 exosome population changing under a test centrifugation (30 min, 10000 g) in a Beckman 60 Ti rotor: (red)–NTA distribution of pre-purified exosomes before the test centrifugation; (blue)–measured NTA distribution observed after the centrifugation; (green)–theoretically expected distributions for the assumed vesicle densities: (solid) 1.15 g/cm3, (dashed) 1.08 g/cm3, (dotted) 1.19 g/cm3.

Mentions: We compare the results of the test centrifugation with those theoretically expected. A FA-type rotor (Beckman Type 60 Ti), being of special interest in the test because of the novelty of the theoretical description, was considered (Fig. 3). NTA PSD of the supernatant after 30 min centrifugation at 10000 g was compared with the theoretically expected distribution, calculated as superposition of the initial NTA PSD of pre-purified exosomes and the theoretically predicted proportion of vesicles of each size, which is to remain in supernatant after the testing centrifugation 1-Pell(d)FA. The calculation is performed with the assumption of the generally accepted mean vesicle density value (1.15 g/cm3) and for lower (1.08 g/cm3) and higher (1.19 g/cm3) values.


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)

The size distribution of the HT29 exosome population changing under a test centrifugation (30 min, 10000 g) in a Beckman 60 Ti rotor: (red)–NTA distribution of pre-purified exosomes before the test centrifugation; (blue)–measured NTA distribution observed after the centrifugation; (green)–theoretically expected distributions for the assumed vesicle densities: (solid) 1.15 g/cm3, (dashed) 1.08 g/cm3, (dotted) 1.19 g/cm3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The size distribution of the HT29 exosome population changing under a test centrifugation (30 min, 10000 g) in a Beckman 60 Ti rotor: (red)–NTA distribution of pre-purified exosomes before the test centrifugation; (blue)–measured NTA distribution observed after the centrifugation; (green)–theoretically expected distributions for the assumed vesicle densities: (solid) 1.15 g/cm3, (dashed) 1.08 g/cm3, (dotted) 1.19 g/cm3.
Mentions: We compare the results of the test centrifugation with those theoretically expected. A FA-type rotor (Beckman Type 60 Ti), being of special interest in the test because of the novelty of the theoretical description, was considered (Fig. 3). NTA PSD of the supernatant after 30 min centrifugation at 10000 g was compared with the theoretically expected distribution, calculated as superposition of the initial NTA PSD of pre-purified exosomes and the theoretically predicted proportion of vesicles of each size, which is to remain in supernatant after the testing centrifugation 1-Pell(d)FA. The calculation is performed with the assumption of the generally accepted mean vesicle density value (1.15 g/cm3) and for lower (1.08 g/cm3) and higher (1.19 g/cm3) values.

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