Zsofia Onodi1, Csilla Pelyhe1, Gabor Brenner1, Kittel Agnes2, Csilla Terezia Nagy1, Mateja Mancek-Keber3, Ferdinandy Peter1, Zoltan Giricz1
1 Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest
2 Institute of Experimental Medicine, Hungarian Academy of Sciencies, Budapest
3 Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana
Background: Blood-derived extracellular vesicles (EVs) are extensively investigated both as biomarkers and therapeutics. However, efficient isolation of EVs from a limited amount of sample is a great challenge. Thus, the aim of this study was to identify a method to isolate the majority of EVs from blood plasma, while eliminating impurities such as lipoprotein particles and soluble proteins.
Methods: Rat blood samples underwent low-speed centrifugations to remove cells, debris and large particles. Density gradient ultracentrifugation (DGUC) was performed by layering 50, 30 and 10% iodixanol solutions on top of which sample was loaded and centrifuged at 120,000×g for 24 hours. Ten fractions were collected. Fractions with highest EV content were further purified by bind-elute chromatography. Efficiency and purity were assessed by Western blot. Morphology and size distribution of particles were examined by dynamic light scattering (DLS) and electron microscopy (EM).
Results: Highest band intensities of EV markers Alix and Tsg101 were detected at a density of 1.13-1.17g/mL. Presence of EVs was confirmed by EM and DLS, showing particles with a mean diameter of 38±2 nm. By DGUC, approximately 90% of lipoprotein- and 80% of albumin contamination was separated from EV-containing fractions. However, 60% of the total fibrinogen content was present in EV-rich fractions, indicating the need for further purification. After loading EV-rich fractions on HiScreen Capto Core 700 column, albumin was not detectable, while the majority of EV markers was observed in 2mL eluate, but fibrinogen was still present in EV-rich eluate.
Conclusion: DGUC with iodixanol shows higher efficiency than currently described methods for the isolation of blood-derived exosomes. It separates EVs from the majority of vesicle-like lipoproteins, and reduces the amount of soluble proteins. Further purification of EV-rich DGUC fractions by bind-elute chromatography column yields EVs without contamination by non-EV plasma components, however, fibrinogen still needs to be removed.
Doctoral School: Pharmaceutical Sciences
Program: Experimental and Clinical Pharmacology
Supervisor: Peter Ferdinandy
E-mail address: email@example.com