Reconstruction of a Human Cornea by the Self-Assembly Approach of Tissue Engineering Using the Three Native Cell Types

Abstract

Purpose: : The purpose of this study was to produce and to characterize a human tissue-engineered cornea using normal cell populations of the three cell types.

Methods: : Fibroblasts cultivated in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets, which were superposed to reconstruct the stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology, immunofluorescence labelling, scanning electron microscopy (SEM) and transmission electronic microscopy (TEM).

Results: : Histology of the tissue-engineered cornea was similar to the structure of native corneas; the epithelium showed 4-5 cell layers and was well differentiated. Epithelial and endothelial cells adhered to the reconstructed stroma. Histology, SEM and TEM showed that the endothelial cells formed a monolayer. Immunofluorescence labelling and TEM showed that the presence of endothelial cells on the engineered cornea improved the differentiation of epithelial cells and the formation of a basement membrane. The reconstructed endothelium expressed the function related protein NA⁺/K⁺-ATPase 1.

Conclusions: : This study demonstrates the feasibility of producing a complete tissue-engineered human cornea using fibroblasts, epithelial and endothelial cells, without exogenous biomaterial, nor viral transformation. This approach would be useful for pharmaceutical and clinical studies concerning the treatment of corneal pathologies that affect all layers of the cornea.

Keywords: cornea: endothelium • cornea: epithelium • cornea: stroma and keratocytes