| dc.description.abstract | Exosomes represent a distinct class of extracellular vesicles of endocytic origin secreted by multiple cell types, including tumour cells. An increased release of exosomes, which appears to be a rich source of biomarkers, has been reported from tumour cells. However, current strategies concerning the isolation (and characterization) of exosomes from fluids differ significantly and no consensus method is established. The goal of this work was to evaluate two different exosome isolation methods with two different breast cancer cell lines in culture: differential ultracentrifugation and commercial isolation kit. Evaluation was done using a bicinchoninic acid assay (protein concentration), transmission electron microscopy (morphology), dynamic light scattering (hydrodynamic size), western blotting (targeted protein exosome markers) and nano liquid chromatography tandem mass spectrometry (comprehensive protein identification). The characterization techniques confirmed the isolation of exosomes with both isolation kit and ultracentrifugation. However, the isolated samples did contain contaminations, and there was a clear difference in the protein amount, particle size and populations identified with the two isolation methods. In addition, the majority of the characterization techniques provided poor repeatability, reproducibility and/or demanded extensive optimization. The evaluation showed that the exosome isolation procedures used in this thesis appear to be far from mature. Additionally, the majority of the characterization techniques used in this study provided challenges. | eng |