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.