Autologous transplantation of rabbit limbal epithelia cultured onfibrin gels for ocular surface reconstruction

[摘要] Purpose: Regeneration of the corneal epithelium could be severelyimpaired in patients suffering from limbal stem cell deficiency. Thepurpose of this study was to evaluate the restoration of the cornealepithelium by grafting onto denuded corneas autologous limbal cellscultured on fibrin gels. The rabbit model was chosen to allow themicroscopic evaluation over time after grafting.Methods: Rabbit limbal epithelial cells (RLECs) were isolated andcultured from small limbal biopsies (3 mm2). The epithelium wasseparated from stroma after dispase digestion and put in culture onlethally irradiated fibroblasts used as a feeder layer. At the firstpassage, RLECs were cultured on a fibrin gel matrix. At confluence, thecultured epithelia were grafted in vivo on denuded autologous rabbitcorneas. At different postoperative times, grafted and control (withoutgraft or grafted with fibrin gels only) rabbit corneas were compared invivo with a slit lamp microscope, and in situ by histological andimmunohistological microscopy of harvested biopsies.Results: A small limbal biopsy was sufficient to generate enoughRLECs to prepare several grafts and to perform cell analysis. Only twoweeks were required to produce a cultured epithelium suitable forautologous transplantation. One month after grafting, a normal cornealphenotype was observed on the ocular surface of grafted rabbits incontrast to the control rabbits (ungrafted or grafted with fibrin gelonly) where histological signs of conjunctivalization were found. Theabsence of goblet cells and negative staining for keratin 4 confirmedthat the cultured cells persisted and that the epithelium regeneratedafter grafting was not from conjunctival origin.Conclusions: Our results demonstrate that an autologous epitheliumcultured on a physiologically biodegradable matrix can be prepared froma small biopsy and grafted on denuded cornea. The autologous graftallows epithelial regeneration from cultured cells and promotes cornealhealing of unilateral total stem cell deficiency.

[学科分类] 生物化学/生物物理