Culture of Rabbit Corneal Epithelial Cells on Fibrin Gel for Autologous Grafting

Abstract

Abstract: : Purpose: Patients who suffer from limbal stem cells deficiency are unable to regenerate a normal corneal epithelium. Using a rabbit model, we studied epithelial regeneration initiated by autologous limbal cells cultured on fibrin gel. We have set up a culture of rabbit corneal epithelial cells (RCEC) from a small limbal biopsy in order to study corneal regeneration following grafting of cultured corneal cells. Methods: We first developed a method for RCEC growth and culture. RCEC cultures were established from freshly harvested small limbal biopsies. Epithelium was separated from stroma after dispase digestion and cells were put in culture with a murine 3T3 feeder layer. At the first passage, RCEC were cultivated on a fibrin gel matrix. When RCEC reached confluence, they were either grafted in vivo on the denuded rabbit autologous cornea or processed for macroscopic and histologic analyses. Grafted rabbit corneas were observed at different times after transplantation by slit lamp microscopy and then harvested for immunofluorescence analysis. Results: From a small limbal biopsy, we were able to produce enough RCEC to prepare several grafts and to perform cell analysis. Only two weeks were necessary to produce a confluent RCEC on fibrin gel ready to grafting. Ten days after grafting, an improvement was observed for rabbit corneas that received a fibrin gel with RCEC compared with non-grafted corneas. Histologic analysis of grafted corneas shows a 5 layer corneal-like epithelium with cuboidal basal cells. K3 staining of RCEC allows to confirm the corneal origin of these cells. Work is in progress to transplant these cultured substitute on a long-term basis. Conclusions: Our results demonstrate that an autologous graft can be prepared from a small limbal biopsy by culturing RCEC on a physiologically degradable matrix. Our results indicate that grafting cultured limbal cells allows healing of a total stem cell deficiency.

Keywords: cornea: epithelium • transplantation • animal model