Abstract
Purpose :
Corneal engineering is a potential tool to address the donor shortage. The main challenges in this matter are the expression of functional molecular markers and cellular morphology, which are mediated by the use of a proper scaffold. In this study, we analyzed the expression and location of ZO-1 and Na/K ATPase of an engineered corneal endothelium (ECE).
Methods :
Corneoscleral rings from corneas previously used for PK were obtained from the local Eye Bank and Instituto Oftalmológico Fernández-Vega, according to the Declaration of Helsinki. Rings were placed in maintenance medium Optisol/Eusol-C at 4°C. Tissues were processed within two days. The remaining CE of seven corneoscleral rings was dissected. Isolated CE was placed overnight at 37°C in a culture plate with culture media (OptiMEM-I, 8% FBS, 20 ng/ml of nerve growth factor, 5 ng/ml of epidermal growth factor, 200 mg/l of calcium chloride, 0.3 mM ascorbic acid, 0.04% chondroitin sulfate, and 1% antibiotics). Then, CE rings were digested with collagenase Type I. Obtained hCECs were cultured on 48 well plates until 90% confluence. ECE were made out of hCECs adhered to the collagen scaffold we produced: a 1:1 passage was made to transfer CECs onto an 8 mm diameter collagen scaffold until confluence. Then, constructs were fixed and co-immunocytochemistry detection of ZO-1 and Na/K ATPase markers was carried out. Uncultured human corneal endothelium rings were used as controls. Photodocumentation and 3D analysis were made using wide-field epifluorescence microscopy.
Results :
CE from 7 PK-remaining corneoscleral rings produced 4 ECE. Co-expression of Na/K ATPase and, ZO-1 was observed in all ECE and in control CE. The normal complex 3D morphology of CE with the basal podocyte-like configuration and flattened-hexagonal-perimeter apical side is appreciated at the ECE. The basolateral location of Na/K ATPase is seen as a wavy-frilly skirt around the basal perimeter and, ZO-1 as the typical zig-zag distribution between cells of the apical side.
Conclusions :
The use of PK-residual CE opens the possibility of producing two CE from a single organ donor cornea: these options must be further explored in order to optimize. Our collagen-based scaffold serves as hCECs carrier. Moreover, our ECE presents hexagonallity and molecular expression of ZO-1 and ATPase, making it suitable for potential use in translational research.
This is a 2020 ARVO Annual Meeting abstract.