Feasibility of cell preservation as a form of cell suspension for a cell-based therapy in a rabbit corneal endothelial dysfunction model

Abstract

Purpose : We recently initiated a clinical trial of cultured corneal endothelial cell (CEC) injection for treating corneal endothelial dysfunction in Japan. In this trial, cultured human CECs are harvested in a cell processing center and immediately injected into patients. In this study, we evaluated whether or not cultured CECs can be preserved for 24 hours as a form of cell suspension and can regenerate corneal endothelium in a rabbit corneal endothelial dysfunction model.

Methods : Corneal endothelium was intensively scraped off in 12 rabbit eyes to create a corneal endothelial dysfunction model. Cultured rabbit CECs were recovered from the culture plate by trypsin digestion, suspended in medium, and preserved at 4°C for 24 hours. A 5.0×10⁵ amount of CECs was then injected with rho-associated protein kinase inhibitor into the anterior chamber of the corneal endothelial dysfunction model (Preservation Group). As a control, CECs recovered from the culture plate were immediately injected (Control Group). All eyes were then evaluated by slit-lamp microscopy, Pentacam^® HR, and tonometer for 2 weeks. At 2-weeks post treatment, the phenotype of the reconstructed corneal endothelium was evaluated by immunohistochemical analysis of ZO-1, N-cadherin, and Na⁺/K⁺-ATPase.

Results : Corneal clarity was recovered in all eyes, and no severe adverse effect such as intraocular pressure elevation or graft rejection was observed. After 14 days, central corneal thickness recovered in a similar fashion (Control Group: 369.0±2.5μm; Preservation Group: 369.3±12.4μm). In accordance with central corneal thickness, the regenerated corneal endothelium cell density was nearly the same in both groups (Control Group: 2781.5±52.2 cells/mm²; Preservation Group: 2755.3±262.0 cells/mm²). In both groups, immunohistochemical analysis showed that the regenerated corneal endothelium was a monolayer of polygonal cells expressing ZO-1, N-cadherin, and Na⁺/K⁺-ATPase.

Conclusions : Our findings demonstrate that cultured rabbit CECs can regenerate CECs with high cell density and functional phenotype in a rabbit corneal endothelial dysfunction model even after 24-hours preservation at 4°C, and provide beneficial information for the transplantation of cultured CECs; e.g. final product protocol and how long cells can be preserved as a cell suspension pre transplantation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.