Regenerating corneal endothelial functionality by engineering cell construct with aqueous two-phase system (ATPS)

Abstract

Corneal endothelial transplantation is one of the major surgeries for treating corneal endothelium related corneal dystrophy. Due to the shortage of corneal donor graft, alternatives of full-cornea transplantation need to be developed. In this thesis, we focus on developing scaffold-free cell construct based on aqueous two-phase system (ATPS). With the aid of reducing osmolality difference between the two immiscible phases and centrifugation assisted assembly, the cell construct formed could be applicable in clinical application.

In chapter 2, we investigate different parameters affecting the cell construct formation. Through the process of ATPS equilibration, we can reduce the osmolality difference between the two phases. This reduction of osmolality difference could result in a higher success rate of cell construct formation. By applying centrifugal force at the cell construct formation process, the thickness of the cell construct would be more uniform. The cell construct formed would also be less likely to be fragmented. We also investigate the cell construct formation phenomenon is only applicable on PEG-DEX-based ATPS.

In chapter 3, we demonstrate the in vitro proliferation, protein expression and ex vivo injection, culture of the corneal endothelial cell construct. Porcine corneal endothelial cell is chosen as the cell type in this study. We show that the porcine corneal endothelial cell harvested is physiologically representative, based on the functionality assay examining the pump and barrier function of the cell construct. The cell construct can also grow and proliferate in vitro with the increase of the cell construct area. Immunocytochemistry is applied to illustrate the physiological markers of the corneal endothelial cell after in vitro culture. After a month of in vitro culture, we find that the cell construct can express ZO-1 and Na+/K+ ATPase, two of the physiological markers of the corneal endothelial cell. It shows its capability to reconstruct the corneal functionality after transplantation. We also demonstrate the feasibility to inject the corneal endothelial cell construct in an ex vivo porcine model using a commercially available intraocular injector. Therefore, the cell construct formed is applicable at clinical context.