The OBSERV platform (Ophthalmic Bioreactor Specialized in Experimental Research & Valorisation): an innovative ex vivo model of human herpetic keratitis.

Abstract

Purpose : Our university lab BiiGC patented 2 versions of a bioreactor (BR): one for long-term eyebanking (in the process of industrialization), the other for preclinical experimentations, called OBSERV and supported by the French Agence Nationale de Sécurité du Médicament et des produits de Santé. Aim: to present an innovative ex vivo model of human herpetic keratitis (HSV-1).

Methods : By restoring intraocular pressure and medium renewal, the BR maintained the viability of human or animal corneas over a prolonged period. Its transparency allowed characterizing the tissue with existing or customized devices without compromising its sterility. Six corneas discarded after organ culture (OC) were placed in the BR for 14 days at 21mmHg and 5µL/H medium renewal. We previously showed that these 2 weeks allowed restoring a multilayered epithelium. The BR lid was opened for HSV inoculation: the epithelium was scarified and immersed for 1H in a solution of 10⁵ to 10⁶ Plaque-Forming Unit/mL HSV-1. The active storage was then continued for 3 to 6 days. Anti-HSV labeling was done on whole corneas maintained in the BR by incubating primary and secondary antibodies only with the epithelium. Comparison was made with OC cornea pairs infected in a concave support and incubated in OC without epithelial restoration step in the BR. Observations: on the whole cornea using a macroscope and, after flat-mounting, confocal microscopy. Corneas were also observed in transmission electronic microscopy (TEM).

Results : In the BR, pseudo-dendritic or geographic ulcers appeared. Immunostaining revealed infected epithelial cells and several areas with ulcers. Outside these spots, the epithelium remained multilayered. In several infected areas rounded and desquamating cells were observed. HSV-1 viral particles were observed by TEM. Two OC corneas had no epithelial cells at the beginning. In the 4 others that had only a few epithelial layers, viral lesions appeared as small “buds”. Controls without HSV remained unstained.

Conclusions : The OBSERV platform is an efficient tool to restore and maintain a multilayered epithelium on corneas discarded after OC, and allows HSV inoculation in more physiologic conditions than on a desquamated epithelium. The BR can complete or replace animal experimentation for academic or industrial research.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Corneas infected by HSV-1 in BR and in OC.