Comparative analysis of discrete exosome fractions obtained by differential centrifugation.
BACKGROUND
Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited.


METHODS
We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin.


RESULTS
NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics.


CONCLUSIONS
Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines.
Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
Dennis K
DK
Jeppesen
The Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Aarhus, Denmark; Department of Molecular Biology and Genetics, University of Aarhus, Aarhus, Denmark.
Michael L
ML
Hvam
Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
Bjarke
B
Primdahl-Bengtson
The Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Aarhus, Denmark; Department of Molecular Biology and Genetics, University of Aarhus, Aarhus, Denmark.
Anders T
AT
Boysen
The Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Aarhus, Denmark; Department of Molecular Biology and Genetics, University of Aarhus, Aarhus, Denmark.
Bradley
B
Whitehead
Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
Lars
L
Dyrskjøt
Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
Torben F
TF
Orntoft
The Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Aarhus, Denmark; Department of Molecular Biology and Genetics, University of Aarhus, Aarhus, Denmark.
Kenneth A
KA
Howard
Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark; marie.stampe@clin.au.dk.
Marie S
MS
Ostenfeld
differential centrifugation; exosomes; extracellular vesicles; k factor; microvesicles; nanoparticle tracking analysis