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.