Bioengineering an Endothelialized Neo-Cornea

Abstract

Methods: : Human corneal endothelial cells (hCECs) were collected from scleral rims of cadaveric corneas by digestion in collagenase II for 1 hour at 37° C. The cells were cultured in EGM-2 (10% FBS) for 28 days and characterized by morphology and immunoexpression of Connexin-43, VE-Cadherin, ZO-1, Na⁺/K⁺ATPase, and CD31. Corneal scaffolds were prepared using a microtome to cut whole donor corneas into 125 micron-thick slices. These slices were decellularized in 2% Triton X100, 0.1% NH₄OH in DI H₂0 for 3 days at 4^o C. hCECs (100-3,000 cells/mm²) were seeded on decellularized scaffolds. The resulting corneal constructs (scaffold plus cells) were placed in EGM-2 (10%FBS) media for seven days and then processed for scanning electoron microscope (SEM) analysis and immunostaining with H&E, DAPI, and ZO-1, Connexin-43, and Na⁺/K⁺ATPase.

Results: : . hCECs were isolated from multiple donor corneas (cadaveric age 14-75 years). The isolated cells expanded well in culture and formed confluent monolayers with a flat hexagonal phenotype. The hCECs stained positively for Connexin-43, VE-Cadherin, ZO-1 and Na⁺/K⁺ATPase, but not CD31. The decellularization process removed cells from the entire thickness of the corneal scaffolds. SEM analysis indicated that hCECs formed a monolayer when seeded on corneal scaffolds. The seeded cells expressed endothelial markers ZO-1, Connexin-43 and Na⁺/K⁺ATPase.

Conclusions: : We successfully isolated hCECs from discarded scleral rims. These cells were expanded to large populations that had both the phenotypic and functional characteristics typical of hCECs. Furthermore, when seeded on decellularized scaffolds, the hCECs retained their phenotypic and functional characteristics. These bioengineered corneal constructs populated with hCECs resemble the current donor DSEK grafts and could be used in human corneal transplantation for endothelial dysfunction.

Keywords: cornea: endothelium • transplantation • regeneration