Release studies. Corneal epithelial EVs were isolated and purified as described above. The EVs were then stained using carboxyfluorescein succinimidyl ester (CFSE). 1 µl CFSE was diluted in PBS and incubated 5 min at RT. The EVs were then added to the CFSE solution to reach a final concentration of 5µM CFSE and the mixture was incubated for 25 min at 37C. After the incubation the sample was then centrifuged at 1000 RCF in a 300kDa Vivaspin® 20 Centrifugal Concentrator using PBS to wash off excess CFSE (in total 6500 times diluted). Due to low fluorescence the sample was stained an additional time following the same procedure but using a Amicon® Ultra Centrifugal Filter 2 mL, 10 kDa MWCO and centrifuging at 2000 RCF using PBS to wash off excess CFSE (in total 730 times diluted). The stained EV solution was measured in NTA, fluorescence spectroscopy and in confocal microscope.