Epithelial wound healing in a model of porcine corneas stored in an innovative bioreactor

Abstract

PurposeExperimental models of corneal epithelial healing are critically important to study cellular mechanisms and new therapeutics. We designed a bioreactor (BR) for exvivo corneal storage. It restores a pressure equivalent to the intraocular pressure in the endothelial chamber while allowing continuous renewing of media in both epithelial and endothelial chambers. It comprised 2 transparent windows for direct observation of epi and endo layers. Initially designed for human corneas, we adapted a prototype for porcine corneas to develop a new experimental platform. Aim: to study epithelial wound healing in the BR compared to a standard ex‐vivo model of porcine corneas.MethodsPorcine eyeballs were obtained from a local slaughterhouse within 4 hours after death. Excised corneas were stored either in a BR or in a Petri dish with culture medium and agar for 4 weeks. A 5 mm epithelial debridmentulcer was performed. Exposition of the epithelium to various conditions was assessed in parallel: air‐lifting, immersion in different media, alternating media and air. Experiments were done in triplicate. Healing rate was monitored with fluorescein staining, digital pictures, and image analysis with ImageJ. After complete healing, corneas were processed for histology and immunolabelling with K3‐K12, laminin‐5, 5‐ethynyl‐2′‐deoxyuridine (EdU), ABCB5, and PAX6.ResultsFaster epithelial healing was observed for cornea stored in bioreactor. In bioreactor epithelial layer was mature (K3‐K12 immunostaining) and multi‐layered. Basement membrane was also restored (laminin‐5 immunostaining). Repeated epithelial debridement successfully healed indicating adequate survival of progenitorsConclusionsThis innovative porcine bioreactor could be a new ex‐vivo assay to study corneal wound healing and to assess efficacy or toxicity of new therapeutics. Grant: UJM, ANSM.