Extracellular Vesicle Isolation from Cell Culture

HepG2, Hep3B, SNU387 cells were cultured in 18 Nunc EasYDish dishes (145 cm², Thermo Fisher Scientific) for 72 h. Then the culture medium was switched to serum-free culture medium (Thermo Fisher Scientific) to starve the cells for 24–48 h. The serum-free culture medium incubated with cells was finally collected for EV isolation. After first centrifugation at 300 × g (4 °C) for 10 min to remove cells and cell debris, the supernatant was collected and transferred to new tubes and centrifuged at 2800 × g (4 °C) for 10 min to further eliminate the remaining cellular debris and large particles. The supernatant was carefully transferred to Ultra-Clear Tubes (38.5 mL, Beckman Coulter, Inc., USA), followed by ultracentrifugation using an Optima L-100 XP Ultracentrifuge (Beckman Coulter, Inc, USA) at 100,000 × g (4 °C) for 70 min. The EV pellets at the bottom of the tubes were carefully collected and resuspended in 200 µL fresh PBS. In control experiments, the EV pellets collected in 200 µL PBS were treated with RNase^{61 (link),62 (link)} at 37 °C for 30 min first, then PBS was added to wash and recollect the EV pellets by ultracentrifugation at 100,000 × g (4 °C) for 70 min. For the artificial plasma samples, each 10 µL aliquot of EV pellets was spiked into 90 µL healthy donors’ plasma or cirrhotic patients’ plasma.