DEVELOPMENT OF EXTRACELLULAR VESICLE ISOLATION FROM CELL CULTURE MEDIUM BY GEL FILTRATION METHOD
Project no. 262 · Status: Kapanmış · 2016
Summary
In recent years, it has been shown that extracellular vesicles (EVs) are secreted by all cell types in living organisms and play very important roles in physiological and pathological conditions. In the field of medicine and pharmacy, genomic and proteomics analyzes of these vesicles provide important knowledge about the formation and development of diseases. In addition, specific proteins and nucleic acids present in EVs secreted from cancer cells make it possible to use these EVs for the diagnosis of cancer. Due to their nano-sized vesicular structure, they are being evaluated for therapeutic delivery purpose. In order to be used in such studies, EVs should be isolated from other cellular components (free proteins and nucleic acids, appoptotic bodies, metabolic product etc.) as much as it is possible. All methods used for EV insulation have their own drawbacks, therefore, cannot provide highly pure EV isolates. Therefore, the aim of this project was to evaluate the gel filtration chromatography technique to isolate extracellular vesicles free from other cellular components from the cell culture medium. First, the medium of Calu1 cells is concentrated by diafiltration. This concentrated culture medium was loaded onto Sepharose CL-4B filled columns and eluted. In the collected fractions, protein concentration was measured, protein density was evaluated by SDS-PAGE, particle size was measured by DLS and morphology was evaluated by SEM imaging. The longer one of the tested two different-length columns was more effective in separating the Evs. According to the SEM images of the fractions obtained, it was observed that EV types were not completely separated. However, the larger vesicles eluted first and then smaller ones followed. According to these results, gel filtration chromatography is better than the ultracentrifuge method in order to obtain different EV species separately. The method can also be applied to biological fluids in order to investigate its potential to be applied in disease diagnosis and treatment.