*2.4. EV Isolation*

EVs from primary mHSC or human LX-2 hHSC were enriched from their respective serum-free conditioned media by sequential centrifugation of the supernatant obtained after 300× *g* for 15 min, 2000× *g* for 20 min, 10,000× *g* for 30 min and ultracentrifugation at 100,000× *g* for 70 min at 4 ◦C (T-70 i fixed-angle rotor; Beckman Coulter, Brea, CA, US, USA). The pellet from the latter step was dispersed in phosphate-buffered saline (PBS) and underwent a repeat round of ultracentrifugation. The use of differential ultracentrifugation is consistent with current recommendations for EV enrichment and is the technique most commonly used by researchers for primary EV separation and concentration [23]. The resulting pellet was dispersed in PBS and the constituent EVs were characterized as described below.
