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Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum.

ABSTRACT: Ultracentrifugation remains the gold standard for isolation of small extracellular vesicles (sEV), particularly for cancer applications. The objective of this study was to determine if a widely used ultracentrifugation protocol for isolation of serum sEV could be modified to reduce the number of ultracentrifugation cycles and increase efficiency, while maintaining equal or better sample purity and yield. Serum was obtained from two healthy subjects. sEVs were isolated from 1 mL aliquots using three different ultracentrifugation protocols. Co-isolation of RNA carrier protein was assessed by performing Western blots for ApoA-I, ApoB, and Ago2. Small RNA-sequencing was performed on the sEV isolates, and differential detection of small ncRNA was compared across isolation protocols. Reduction from three- to two-ultracentrifuge cycles with no sucrose cushion resulted in a much higher sEV yield but also had the highest levels of lipoprotein and Ago2 contamination. However, the two-ultracentrifugation cycle protocol that incorporated a 30% sucrose cushion into the first cycle resulted in slightly higher sEV yields with lower levels of protein contamination compared to the lengthier three-ultracentrifugation cycle approach, therefore presenting a more efficient alternative approach for isolation of serum sEVs. It was also notable that there were some differences in sEV ncRNA cargo according to protocol, although it was less than expected given the differences in co-isolated RNA carrier proteins. Our results suggest that use of the modified serum sEV isolation protocol with two ultracentrifugation cycles and incorporating a 30% sucrose cushion offers a more efficient approach in terms of efficiency and purity.

SUBMITTER: Langevin SM

PROVIDER: S-EPMC6380284 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Project description:Background: Extracellular vesicles (EVs) (isolated from blood plasma) are currently being extensively researched, both as biomarkers and for their therapeutic possibilities. One challenging aspect to this research is the efficient isolation of high-purity EVs from blood plasma in quantities sufficient for in vivo experiments. In accordance with this challenge, the aim of this study was to develop an isolation method in which to separate the majority of EVs from major impurities such as lipoprotein particles and the abundant plasma proteins albumin and fibrinogen. Methods: Samples of rat blood were centrifuged to remove cells, platelets, large EVs and protein aggregates without prior filtration. Density gradient ultracentrifugation was performed by loading plasma sample onto 50, 30, and 10% iodixanol layers and then centrifuged at 120,000 ×g for 24 h. Ten fractions (F1-10) were collected from top to bottom. Fractions with the highest EV content were further purified by ultracentrifugation, size exclusion, or bind-elute chromatography. Efficiency and purity were assessed by Western blots. Morphology and size distribution of particles were examined by dynamic light scattering and electron microscopy (EM). Results: The highest band intensities of EV markers Alix, Tsg101 and CD81 were detected by Western blot in F6 of small-scale DGUC (61.5 ± 10.4%; 48.1 ± 5.8%; 41.9 ± 3.8%, respectively) at a density of 1.128-1.174 g/mL, where the presence of vesicles with a mean diameter of 38 ± 2 nm was confirmed by EM and DLS. Only 1.4 ± 0.5% of LDL and chylomicron marker, 3.0 ± 1.3% of HDL marker, and 9.9 ± 0.4% of albumin remained in the EV-rich F6. However, 32.8 ± 1.5% of the total fibrinogen beta was found in this fraction. Second-step purification by UC or SEC did not improve EV separation, while after BEC on HiScreen Capto Core 700 albumin and lipoprotein contamination were below detection limit in EV-rich fractions. However, BEC decreased efficiency of EV isolation, and fibrinogen was still present in EV-rich fractions. Conclusion: This is the first demonstration that DGUC is able to markedly reduce the lipoprotein content of EV isolates while it separates EVs with high efficiency. Moreover, isolation of lipoprotein- and albumin-free EVs from blood plasma can be achieved by DGUC followed by BEC, however, on the expense of reduced EV yield.

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Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum.

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