Ex vivo models of corneal epithelial regeneration in bioreactor: Respective roles of limbal, conjunctival and corneal epithelia

Abstract

AbstractPurpose: The corneas preserved in bioreactor (BR) had been shown to have not only a better endothelial viability, but also a more differentiated and stratified epithelium than corneas preserved in organoculture. Purpose: By using BR, we would analyse the respective contribution of corneal (C), limbal (L), and conjunctival (Conj) epithelia in corneal epithelial regeneration.Methods: Five pairs of corneas from body donation to Science were used with a death‐to‐collection time < 20 h. A 3‐ to 5‐mm‐wide conjunctival flange was kept intact. Five patterns were set up by fully mechanical removal of 1, 2, or 3 epithelia: C‐L + Conj+, C‐L‐Conj+, C‐L + Conj‐, C + L‐Conj‐, C‐L‐Conj‐ (control) n = 2 for each pattern. The limbus was destroyed by scraping and thermocoagulation. Corneas were then kept in BR (21 mmHg, 2.5 μl/min of Corneamax Eurobio, 31°C) for 3 weeks to allow epithelial regeneration. The epithelium was then analysed using immunofluorescence (IF) on flat mounted cornea by targeting CK12 (corneal epithelium) and CK15 (limbal epithelium). Cell nuclei were counterstained with DAPI. Corneal transparency was quantified using a transparometer.Results: No epithelium was reconstituted in the controls. In the other 4 models including the C‐L‐Conj+ group, the cornea was transparent and covered by a pluristratified corneal epithelium, characterized by CK12 expression.Conclusions: In this BR model, conjunctival epithelial cells allowed the regeneration of a typical corneal epithelium in model C‐L‐Conj+. The corneal epithelium can also migrate to the limbus and conjunctiva. We hypothesize that all 3 ocular surface epithelia (including the conj) contain stem cells or progenitors capable of migrating throughout the cornea and restoring the corneal epithelium independently of each other. The main difference between our ex vivo model and in vivo situation is absence of neovascularization. This suggests that the main cause of limbic insufficiency is due to the loss of the anti‐angiogenic barrier rather than the loss of limbic stem cells.