EV isolation, purification and characterization
The lens capsules were bathed in a freshly prepared digestion solution containing 0.2% DNase I (Roche, Germany, Cat No.11284932001) and 2mg/mL collagenase D (Roche, Germany, Cat No.11088866001) with RPMI 1640 cell medium (Gibco, USA) was applied for the release of Ti-EVs. The mixture was incubated at 37°C for 1 hour with constant agitation to ensure thorough dissociation. After digestion, the suspensions underwent a two-step centrifugation: initially at 1,000 ×g for 10 minutes to remove intact cells and large particles, followed by a second centrifugation at 10,000 ×g for 20 minutes to eliminate apoptotic bodies and cellular debris. The supernatants were then purified using an ExoDisc filtration device (LabSpinnerTM, Ulsan, South Korea) to isolate EVs, which were subsequently resuspended in PBS for further analysis. The size distribution and particle concentration of the purified EVs were assessed using NanoFlow CytoMetry (NanoFCM, Xiamen, China). Western blotting was performed to confirm the presence of key EV markers, including CD9, CD81 and CD63. In addition, transmission electron microscopy (TEM) imaging was used to visualize the morphology of the isolated EVs.