Isolation and characterization of extracellular vesicles (EVs) from renal carcinoma cells

• Main Author: Giri, K. (Khem)
• Format: Dissertation
• Language: English
• Published: University of Oulu 2017
• Subjects: MDP in Protein Science and Biotechnology
• Online Access: http://urn.fi/URN:NBN:fi:oulu-201708192751; http://nbn-resolving.de/urn:nbn:fi:oulu-201708192751

Extracellular vesicles (EVs) are small nano-sized particles released constantly by cells into body fluids like blood, saliva, urine, plasma and milk. Depending in the size, EVs are divided into exosomes (30–100 nm), microvesicles (100–1000 nm) and apoptotic bodies (50–5000 nm). During this work, I extracted exosomes from kidney cancer cells using two different centrifugation methods (sequential centrifugation and sucrose gradient ultracentrifugation) under two different conditions (hypoxia and normoxia). The characterization was done by electron microscopy, western blot and nanoparticle tracking analysis (NTA). The effect of exosomes in normal kidney cells was studied in vitro by treating metanephric mesenchyme (MM) cells with exosomes for cell proliferation and motility. The exosomes were injected in chicken embryos in vivo together with renal carcinoma (Renca YFP) cells to see if they have some role in tumor growth. The protein and RNA contents of the exosomes were also analyzed. The cells release more vesicles when they are exposed to hypoxic conditions. Electron microscopy and western blot showed the presence of exosomes expressing CD63 and CD81 markers. Cell proliferation and motility was found to enhance when cells were treated with exosomes. Chicken embryos showed formation of bigger Renca YFP tumor after treatment with exosomes. Different proteins and miRNAs were detected in exosomes which may play active roles in biological processes. In summary, I successfully purified exosomes from kidney cancer cells and characterized them. We concluded that these vesicles play important roles in cell activity and have ability to enhance the tumor growth. The presence of proteins and miRNAs suggest the potential role in cellular communication.