Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive biomarkers are needed to aid diagnosis and treatment. Extracellular vesicles (EVs), membranous vesicles secreted by all cells, are a promising potential source for cancer biomarkers, but new methods are required that are both sensitive and specific for cancer identification.
Researchers at Johns Hopkins University School of Medicine have developed an EV isolation protocol optimized for kidney tumor and normal kidney tissue that yields a high vesicle concentration, confirmed by nanoparticle tracking analysis (NanoSight) and by nanoscale flow cytometry (NanoFCM). Using Western blot, the researchers confirmed presence of EV markers CD81, CD63, flotillin-1, and absence of cellular debris, calnexin. Transmission electron microscopy images demonstrate intact membranous EVs. This new method improves existing protocols with additional steps to reduce contaminants in the EV product. Characterization of our isolation product confirms successful isolation of EVs with minimal contamination. The particle yields of this protocol are consistent and high as assessed by both standard and novel methods. This optimized protocol will contribute to biomarker discovery and biological studies of EVs in renal cancer.
Workflow of EV isolation protocol starting from kidney tissue
1 Sample preparation: normal kidney or kidney tumor is collected in PBS and cut into 2–3 mm fragments. Tissue pieces are collected by centrifugation and the PBS removed by aspiration. 2 Incubation: the tissue pieces are incubated in DMEM with Collagenase D (20 mg/mL) and DNase I (40 U/mL) for 30 min. The tissue is removed from the conditioned media by a 70 µm strainer. 3 Differential centrifugation: non-EVs are precipitated by sequential centrifugation and discarded after each step, retaining the supernatant for subsequent centrifugation steps (three times). 4 Filtration: the supernatant is syringe-filtered with a 0.8 µm filter followed by a 0.45 µm filter. 5 Ultracentrifugation: EVs are isolated from supernatant by ultracentrifugation for 2 h. The supernatant is now aspirated. The EV pellet is retained, washed with PBS, and then precipitated again by 2 h of ultracentrifugation. PBS phosphate-buffered saline, DMEM Dulbecco’s modified Eagle’s medium.