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A Simplified Protocol for Intact Exosome Separation using Low-Pressure Size-Exclusion Chromatography

Authors: Morávek Ondřej | Vaňková Zuzana | Manzi Malena | Jirásko Robert | Kozovská Zuzana | Kupčík Rudolf | Bílková Zuzana | Holčapek Michal

Year: 2024

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	A Simplified Protocol for Intact Exosome Separation using Low-Pressure Size-Exclusion Chromatography	Exosomes, small extracellular vesicles, are formed through the inward budding of the endosomal membrane (endocytosis), leading to the accumulation of intraluminal vesicles within multivesicular bodies, which subsequently fuse with the plasma membrane, releasing exosomes into the extracellular space. They are an essential component of cellular and tissue processes, such as intercellular communication, immune response, programmed cell death, inflammation, and the transport of morphogens. Because of their versatile role, exosomes also play a decisive role in various pathological conditions, such as cancer or neurodegenerative diseases. These nano-sized vesicles carry a diverse cargo of biomolecules, including nucleic acids, proteins, lipids, and metabolites. Studies have shown that the composition of exosomes often reflects the physiological or pathological state of cells, tissues, organs, and organisms. This characteristic makes exosomes, especially oncosomes, promising structures to investigate the pathogenesis of disease and allow early and accurate disease diagnosis. Despite their potential, the widespread use of exosomes is hampered by the limited possibility of isolating exosomes quantitatively and in sufficient quality. Consequently, efforts have been made to implement a robust exosome isolation method that offers a high yield of intact exosomes without contaminating proteins, lipoproteins, or microvesicles. This study shows a specific configuration with a low-pressure size-exclusion chromatography, which disposes of several advantages, such as a high capacity, robustness, providing a pure fraction of intact exosomes with minimal contamination. What makes this technique unique? Optimized conditions enabled us to separate exosomes into fractions varying in exosome size. We established a protocol for the isolation and fractionation of exosomes. Methods such as DLS, NTA, Dot-Blot, ELISA, and MS were applied to control the separation process.