Bioprinting of human pluripotent stem cell derived corneal endothelial cells with hydrazone crosslinked hyaluronic acid bioink

Abstract

Background: Human corneal endothelial cells lack regenerative capacity through cell division in vivo. Thus, in the case of trauma or dystrophy, the only treatment modality currently available is corneal tissue or primary corneal endothelial cell transplantation from cadaveric donor with high global shortage. Our ultimate goal is to use the state-of-the-art 3D-bioprint technology for automated production of human partial and full-thickness corneal tissues using human stem cells and functional bioinks. Here, we explored the possibility to bioprint corneal endothelium using human pluripotent stem cell derived corneal endothelial cells and hydrazone crosslinked hyaluronic acid bioink. Methods: Corneal endothelial cells differentiated from human pluripotent stem cells were bioprinted using optimized hydrazone crosslinked hyaluronic acid based bioink. Before bioprinting, the biocompatibility of the bioink with cells was first analyzed with transplantation on ex vivo denuded porcine corneas and on denuded human Descemet membrane. Then bioprinting was proceeded and the viability of human pluripotent stem cell derived corneal endothelial cells after bioprinting was verified with live/dead stainings. Histological and immunofluorescence stainings with ZO1, Na+/K+-ATPase and CD166 were used to confirm corneal endothelial cell phenotype in all experiments and STEM121 marker was used to identify human cells from the ex vivo porcine corneas. Results: The bioink modified for human pluripotent stem cell derived corneal endothelial cells successfully supported the viability and printability of the cells. After 10 days of ex vivo transplantations, STEM121 positive cells were verified on the Descemet membrane of porcine cornea showing the biocompatibility of the bioink. Furthermore, biocompatibility was confirmed on denuded human Descemet membrane showing corneal endothelial like characteristics. Seven days after bioprinting, the corneal endothelial like cells were viable and showed polygonal morphology with expression and native-like localization of ZO-1, Na+/K+-ATPase and CD166. Nevertheless, mesenchymal-like cells in some parts of the cultures were evident and those cells spread underneath the corneal endothelial-like cell layer. Conclusions: Our results demonstrate that human pluripotent stem cell derived corneal endothelial cells can be bioprinted in covalently crosslinked hyaluronic acid bioink. This approach has potential as a corneal endothelium transplant and furthermore, can be used in the mission of bioprinting the full-thickness human cornea.