Bioengineered multilayered human corneas from discarded human corneal tissue

Corneal transplantation has become a common procedure to improve visual acuity by replacing the opaque or distorted host tissue with clear healthy donor corneal tissue. However, globally its wide spread clinical utility is limited due to a lack of supply of high quality corneas. Bioengineered neo-corneas using discarded human corneas to isolate corneal endothelial and epithelial cells, as well as corneal stroma as a scaffolding material, could help address this shortage. The objective of this study was to fabricate multilayered corneal equivalents that could be suitable for full thickness cornea transplantation. To achieve this goal human corneal endothelial cells (hCEC) and human limbal epithelial cells (hLEC) were isolated from discarded human corneas and expanded in vitro, maintaining their phenotype for at least 3 passages. We used our previously described process of human cornea decellularization to create corneal scaffolds that preserve the native extracellular matrix of the corneal stroma. The corneal scaffolds were seeded with hCEC and hLEC, using a special apparatus that enabled seeding both sides of the scaffold. The cell-seeded corneal constructs supported hCEC and hLEC growth and multi-cellular organization for 2 weeks in vitro. Immunohistochemical analysis showed expression of typical hCEC and hLEC markers on their corresponding sides. Importantly, the cell-seeded corneal constructs were more transparent than non-seeded corneal scaffolds. Taken together, this study demonstrates the feasibility of creating multilayered cornea equivalents, exclusively from human donor-derived materials. These constructs may be suitable for corneal transplantation, and as a short-term application, may serve for ophthalmological drug testing purposes.