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Impact of Cell Source on Human Cornea Reconstructed by Tissue Engineering

¹From the Laboratoire d’Organogénèse Expérimentale, Centre de Recherche (FRSQ) du CHA; Departments of ²Oto-Rhino-Laryngology and Ophthalmology, ³Surgery, and ⁵Anatomy and Physiology, Laval University, Quebec, Canada; the ⁶Unité de Recherche en neurosciences, Centre de Recherche du CHUQ, Pavillon CHUL, Quebec, Canada; and ⁴School of Optometry, Research Unit in Ophthalmology, Montreal University, Quebec, Canada.

PURPOSE. To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro.

METHODS. Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined.

RESULTS. The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis.

CONCLUSIONS. The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis.