Transplantation of human corneal endothelial cells using functional biomaterials: Poly(N-isopropylacrylamide) and gelatin

Purpose: To evaluate the feasibility of human corneal endothelial cell (HCEC) transplantation by harvesting the HCEC sheet on a thermoresponsive surface, delivering it with a gelatin disc, and testing it in a rabbit model. Methods: Cultivated human adult HCECs labeled with a red fluorescent dye (PKH26) were seeded on a poly(N-isopropylacrylamide) (PNIPAAM)-grafted surface culture dish at 37°C. After reaching confluence, the HCEC monolayer was detached by reducing the incubation temperature to 20°C and immediately delivered by means of a 7-mm gelatin disc to the rabbit's cornea denuded with endothelial cells (HCEC group, n = 8). The morphology, viability, pump, and barrier functions of the harvested HCEC were evaluated. Traumatized rabbit corneas with only the gelatin disc graft (gelatin disc group, n = 4) and without any transplantation (wound group, n = 4) were the sham controls. Surgical corneas of each group underwent histological and clinical evaluations including corneal thickness, intraocular pressure (IOP), and corneal clarity at different time points during a follow-up period of 12 weeks. Results: Cell morphology, viability, densities, Na⁺/K⁺ ATPase, and zonula occluden-1 (ZO-1) of the cultivated HCEC monolayer were similar with those of native HCECs. After endothelial removal, corneas of three groups turned severely edematous and opaque. In the HCEC group, the clarity of cornea recovered within 2 weeks with a corneal thickness of 552 ± 18 μm, which was significantly less than those (>1,000 μm) of the control groups (p <0.05). Histological examinations showed that the PKH26-labeled HCECs were spread over the Descemet's membrane with tight junction formation in the HCEC group, but none in the control group. The postoperative IOP of all three groups was within normal limits. Conclusion: This study provides a novel strategy for reconstruction of corneal endothelial cells using cultured adult HCECs and functional biomaterials including PNIPAAM and gelatin.