Ex vivo corneal wound healing model using an innovative bioreactor

Abstract

Purpose : Experimental models of corneal epithelial healing are critically important to study cellular mechanisms and new therapeutics. Available model generally use animal corneas and do not enable long-term storage, creation of an appropriate epithelial environment and preservation of the endothelium that influence wound healing. We designed a bioreactor (BR) for human ex-vivo corneal storage that restores a pressure equivalent to the intraocular pressure in the endothelial chamber while allowing continuous renewing of media endothelial chambers. The epithelial chamber is alternatively exposed to air and to a specific epithelial medium. Aim: to study epithelial wound healing in the BR compared to a standard ex-vivo model of human corneas.

Methods : Human corneas not suitable for graft were used. Corneas were stored either in a BR or in petri dish (immersed or airlifted with epithelial culture medium). A calibrated epithelial debridement was done with a crescent knife. Histological structure and immunohistological staining (K3-K12, Laminin-5, type IV collagen) were compared to conventionally stored corneas after 1 week. Healing rate was monitored with fluorescein staining, digital pictures, and image analysis with ImageJ. Experiments were done in triplicate.

Results : Faster epithelial healing was observed for cornea stored in bioreactor. In bioreactor corneal epithelium was differentiated (K3-K12 immunostaining) and multi-layered, whereas a 1-3 layer thick epithelium was observed in Petri Dish. Basement membrane was also restored (continuous laminin-5 and type IV collagen immunostaining) in BR whereas it was fragmented in Petri dish.

Conclusions : This innovative bioreactor could be a new ex-vivo assay to study corneal wound healing and to assess efficacy or toxicity of new therapeutics.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.