The culture media was collected for isolation and purification of EVs using ultrafiltration coupled with size-exclusion chromatography (UF-SEC), as this combination of techniques ensures the efficient enrichment of small-sized EVs suitable for compositional and functional studies.42 (link),43 (link) First, the media was centrifuged for 10 min at 300xg to remove dead cells, for 10 min at 2500xg to remove cell debris and apoptotic bodies, and then filtered through a 0.2 μm membrane to remove large vesicles and aggregates. The media was concentrated using 100 kDa centrifugal ultrafiltration unit Amicon Ultra (UFC9100, Millipore/Sigma Aldrich) according to the instructions of the manufacturer. For EV enrichment, 1 ml of concentrated cell culture media from approximately six T150 flasks was subjected to SEC on a 30 cm length and 1.5 cm diameter Sephacryl S-200 HR (Sigma, GE17-0584-01) column at 4°C using sterile phosphate-buffered saline (PBS) as mobile phase. The absorbance of the collected fractions (approximately 30 fractions of 1 ml each) was measured at 280 nm to determine the EV-containing fractions, which were then further concentrated to a smaller volume, characterized, and used for subsequent studies.