Sun, Fancheng2023-12-142023-12-142023-12-12http://hdl.handle.net/10012/20167Fuchs endothelial corneal dystrophy (FECD) is a corneal endothelium (CE) and Descemet’s membrane (DM) dysfunction symptom, which is the most common corneal endothelial disease. Despite the long history of clinical assessments regarding FECD, the only clinical treatment available currently is corneal transplantation. Therefore, a study to further explore the complex pathology of the FECD is required to develop novel therapies. Clinically, FECD is a condition indicated by the gradual loss of corneal endothelial cells (CEnCs) and the development of abnormal accumulations of extracellular matrix (ECM) on Descemet's membrane, known as guttae. By utilizing a patient-derived immortalized FECD corneal endothelial cell line FECD-SV-54F-73 and an in vitro synthetic guttata (s-guttata) model with varying dimensions, an in vitro model was constructed aiming to recapitulate the in vivo abnormal cell-ECM interactions in FECD. With this model, we hypothesize that the FECD cells seeded on the s- guttata patterns exhibit higher cytoskeleton contractility and early apoptosis rate. In addition, we speculate that the healthy CEnC line B4G12 on the synthetic guttata would undergo the endothelial to mesenchymal transition (EndMT) process, which is also observed in FECD pathology. The FECD 54F CEnCs had a significant increase in early apoptosis and cytoskeletal response in 20x20x20 μm (diameter-spacing-height) s-guttata samples. Furthermore, these apoptotic cells were localized around the s-guttata with smaller diameters and higher densities. However, after seven days, no significant upregulation of EndMT-related gene expressions (ZEB1 and SNAI1) was detected in the cells seeded on the 20x20x20 μm sample compared to the unpatterned control. In addition, by employing a curved contact lens, we also aim to explore the cell migration mechanism of the CEnCs and hypothesize that the wound healing rate of the FECD cells is higher than the healthy corneal endothelial cell line B4G12. Interestingly, we found that while the FECD 54F (2.35% of total wounded area per hour) cells migrated faster than the B4G12 (1.41% per hour) cells on the concave side of the gelatin methacrylate + (GelMA+) contact lens, B4G12 had a significantly higher migration speed on flat PDMS (3.02% of per hour) and TCPS (2.74% per hour) substrate surfaces when compared with FECD 54F (2.9% per hour on PDMS, 1.73% per hour on TCPS). However, further studies are required for the curved samples as there were not sufficient sample replicates for statistical analysis. A lithography process was also developed to explore whether the effect of micro-topography can affect cell behavior on curved surfaces. While the sub-micron patterns on the silicon wafer were successfully created via maskless photolithography, the subsequent soft lithography process that required the transfer of the designs from a flat substrate onto the curved surface was delicate and required more improvements for subsequent cell experiments on a non-planar curved surface. In general, by applying various instruments and analytical approaches, this study aims to contribute further to comprehending FECD pathophysiology and improve the current stages of FECD clinical treatment approaches.enFuchs Endothelial Corneal Dystrophy (FECD)corneal endotheliumapoptosiscytoskeletonendothelial to mesenchymal transition (EndMT)maskless photolithographysoft lithographyhydrogeltopographygelatin methacrylateMaster Thesis