Simple innervated 3D corneal constructs for in vitro testing

Abstract

Purpose : The purpose of this study was to develop a 3D corneal construct appropriate for in vitro studies of corneal disease conditions and therapies. The construct was designed to be similar to a human cornea, in part by use of human components.

Methods : Human corneal constructs were created with chemically cross-linked extracellular matrix (ECM) analog comprising mainly collagen with layer-by-layer concept. The whole construct was developed in a cell culture insert. The corneal cell layers were generated from immortalized human corneal cell lines. Endothelial cells were first seeded on the insert membrane and then ECM containing stromal cells was added on the single cell endothelial layer. Neural cells derived from hybrid neuroblastoma cells were grafted inside the ECM. A stratified epithelial cell layer was developed on the top of the ECM. A simplified culture medium composition was selected for the culture of the construct with the different cell types. Assays for cytotoxicity, cell proliferation, histology, and protein expression were used to characterize the constructs.

Results : We identified a single media that can be used to culture multiple corneal cell types used in the construct. Proliferation studies of the individual cell types confirmed similar cell growth with the modified media when compared with the cell-specific culture media. In vitro differentiation of neuroblastoma cells to phenotypically healthy appearing neurons validated use of these cells as the neural source. Live/dead staining of the construct confirmed the viability of all cell types within the construct. Histological evaluation of the construct showed a stratified epithelium formation on the top of the construct with five to seven layers of epithelial cells. Western blot analysis of the construct confirmed appropriate cell type-specific protein expression by each cell type.

Conclusions : The corneal construct effectively mimicked a living human cornea, while maintaining individual cell phenotypes and anatomically correct cell loci. The corneal construct may be useful in assessment of specific corneal conditions and in the testing of novel therapeutics for corneal diseases, potentially reducing the necessity for animals in corneal research.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.