As cell culture media typically contains serum, which includes serum-derived particles that affect EV purity, it is necessary to use serum-free, chemically defined, or EV-depleted medium for collection.
Once cells in the recommended number of culture flasks (20 flasks containing 35 mL of medium each) reach approximately 90% confluency (corresponding to a total culture volume of ~700 mL), wash the cells twice with phosphate‑buffered saline (PBS). The cells are then cultured under serum-free conditions for 24-48 hours, depending on cell viability to allow EV accumulation.
After collecting the conditioned medium, immediately centrifuge it at 800 × g for 30 minutes at 4 °C to remove cells and cell debris.
We recommend shipping the supernatant on the same day as media collection, with morning delivery before 10:30 am preferred.
