Severe corneal damage leads to complete vision loss, thereby affecting life quality and impinging heavily on the healthcare system. Current clinical approaches to manage corneal wounds suffer from severe drawbacks, thus requiring the development of alternative strategies. Of late, mesenchymal stromal/stem cell (MSC)-derived extracellular vesicles (EVs) have become a promising tool in the ophthalmic field. In the present study, we topically delivered bone-marrow-derived MSC-EVs (BMSC-EVs), embedded in methylcellulose, in a murine model of alkali-burn-induced corneal damage in order to evaluate their role in corneal repair through histological and molecular analyses, with the support of magnetic resonance imaging. Our data show that BMSC-EVs, used for the first time in this specific formulation on the damaged cornea, modulate cell death, inflammation and angiogenetic programs in the injured tissue, thus leading to a faster recovery of corneal damage. These results were confirmed on cadaveric donor-derived human corneal epithelial cells in vitro. Thus, BMSC-EVs modulate corneal repair dynamics and are promising as a new cell-free approach for intervening on burn wounds, especially in the avascularized region of the eye.

We investigated which specific correlation exists between the endothelial cell density (ECD) of corneal endothelial cell (CEC) cultures and the features of the donor corneas from which they originate. CEC cultures were prepared from one donor cornea or by pooling together cells of more corneas from elderly donors with ECDs lower or higher than 2000 cells/mm². The ECDs of such primary cultures were evaluated and showed that that ECDs > 2000 cells/mm² can be obtained only when CECs are isolated from (1) corneas of young donors; (2) at least two elderly donor corneas (if ECD > 2000 cells/mm²), or three elderly donor corneas (if ECD < 2000 cells/mm²). Secondary cultures are all characterized by ECDs < 2000 cells/mm². Our study highlights the difficulties in obtaining cultures with ECDs > 2000 cells/mm². Even if achievable with corneas from young donors, this becomes a challenging task when corneas from elderly donors are used (i.e., the overall majority of those collected by eye banks) and particularly when corneas from elderly donors with ECD < 2000 cells/mm² are used. Pooling more of two corneas to obtain suitable CECs could technically overcome the problem. The above issues should be tackled appropriately before moving into clinical studies.

Background/Aims To set up the in vitro conditions for renewal of the conjunctival epithelium using healthy fragments of conjunctival tissue glued over an amniotic membrane. Methods We evaluated the capability of conjunctival tissue fragments to generate conjunctival cell outgrowth after seeding them onto amniotic membrane and culture plates; we then assessed conjunctival molecular marker expression by immunofluorescence. We also evaluated the efficiency of glueing the fragments over the amniotic membrane to determine the best setting and the feasibility of shipping preloaded amniotic membranes. Results Epithelial outgrowth was detected in 65%–80% of conjunctival fragments starting 48–72 hours after glueing, without major differences between type of membrane preparation and fragment size. Within 6–13 days, a full epithelium covered the surface of the amniotic membrane. Specific marker expression (conjunctival epithelium, Muc1, K19, K13; stemness, p63; tight junctions, ZO-1) was detected. Results of the shipping test showed that only 31% of the fragments were still glued over the epithelial side of the membrane within 24 hours compared to more than 90% of fragments stayed attached in the remaining conditions. Conclusion The in vitro regeneration of conjunctival epithelium following outgrowth from conjunctival tissue fragments glued over an amniotic membrane may offer a viable strategy to renew the epithelium in vivo once applied over the ocular surface at the recipient site.