This comprehensive analyses of CE-EV proteins using FACS, Western blot, and proteomics, along with the assessment of their relative enrichment compared to CE-cells and together with the current literature have enhanced our understanding of the interactions involved in corneal epithelial wound healing (Figure ). Efficacy of CE-EVs in healing corneal epithelium. To further evaluate the potential of CE-EVs in corneal epithelial wound healing, an in vitro wound healing assay (scratch assay) was carried out. Human corneal epithelial cells were grown on a collagen-coated surface to form a confluent monolayer and then scratched using a pipette tip to create a wound mimic. Then CE-EVs in different concentrations (20, 100, and 200 µg/mL) were added and left to incubate for 24h before studying the closure of the scratched area (Figure ). All CE-EV concentrations showed significantly higher healing compared to the negative control (Opti-MEM) (Figure ). However, the results for 20 µg/mL were more varying (66.8 ± 8.3 %) and significantly lower (p<0.05) than 100 µg/mL (78.8 ± 3.8 %). Both 100µg/mL and 200 µg/mL (72.3 ± 4.7 %) showed significantly higher (p<0.001) healing compared to the control (55.8 ± 1.9 %) but not significantly different from each other. The data indicate that higher concentrations of CE-EVs enhance healing up to a certain threshold, beyond which no additional benefits are observed. This finding highlights the potential for scale-up and GMP production for future clinical applications, as the optimal effective concentration can be achieved relatively easily and is suitable for integration with a delivery matrix. Sustained and on-demand delivery of CE-EVs using hydrogel matrix. EV-delivery to the eye presents several significant challenges. Topical delivery methods often face rapid clearance due to blinking and tear production whereas systemic delivery approaches are unsuitable due to cornea being immune-privileged, making localized delivery methods more crucial. Our earlier developed crosslinked collagen hydrogel showed promise in corneal perforation due to its injectable and adhesive nature. Furthermore, due to the shape-retaining nature of this hydrogel and the cytocompatibility of the crosslinking reaction, it would be possible to use such a hydrogel for in situ molding of corneal defects. Additionally, since these hydrogels can be stored in buffers without swelling and allow the proliferation of corneal epithelial cells, they are potentially useful as corneal implants during Deep Anterior Lamellar Keratoplasty (DALK). Sustained delivery of EVs during corneal perforation, in situ molding of corneal defects, and keratoplasty using corneal implants would thus be highly desirable. However, a prerequisite would be the wide range of delivery time scales; a perforation would require a relatively fast to moderate release rate as the hydrogel was supposed to act only as a temporary scaffold lasting over a few weeks, an in situ molding of stromal defects would require a release time over a few months as stromal remodeling is slow, and complete stromal regeneration after keratoplasty with hydrogel stromal substitute could take even longer. Although, it is impossible to mimic the exact in vivo scenario in vitro, and nearly impossible to achieve all of these release time-scales through one hydrogel, it would be highly valuable if the release time-scales could be as broad as from days to years. For this purpose, we further evaluated the release of encapsulated CE-EVs from our previously reported collagen hydrogel as well as the distribution and mobility of these EVs inside the hydrogel matrix.