Ex Vivo Functionality of 3D Bioprinted Corneal Endothelium Engineered with Ribonuclease 5-Overexpressing Human Corneal Endothelial Cells.

Human corneal endothelial cells (HCECs) are scarcely proliferative in vivo. The cultured HCECs engineered to overexpress ribonuclease (RNase) 5 (R5-HCECs) are prepared after transient transfection with RNase 5 plasmid vector. As candidate targets of R5-HCECs for enhancement of cellular proliferation and survival of R5-HCECs, programmed cell death protein 4 is inhibited, and cyclin D1 and cyclin E1 are activated. The cultured R5-HCECs and control HCECs on lyophilized amniotic membrane (AM) are deposited as a carrier by extrusion-based 3D bioprinting to prepare transplantable RNase 5 vector-transfected HCECs-laden AM graft (R5-Graft) and the control HCECs-laden AM graft (Ct-Graft), respectively. The ready-to-use R5-Graft shows clearer basolateral expression of Na+ -K+ ATPase pump and higher cell confluency than Ct-Graft. From 2 weeks after graft transplantation, both R5-Graft and Ct-Graft start restoring clarity of the rabbit corneas, and their central corneal edema are much less than those in the control group at 3 and 4 weeks. The ex vivo expression of corneal endothelial phenotypical markers is clear in R5-Grafs rather than in Ct-Grafts at 4 weeks. In conclusion, the fabricated corneal endothelium with cultured HCECs easily survives and functions as corneal endothelium in vivo. Furthermore, the use of the cultured HCECs engineered to overexpress RNase 5 (R5-HCECs) may be an option to obtain higher graft cellularity and to enhance the function of transplanted grafts.