A ‘Living’ Biological Bandage for Management of Ocular Injuries

Abstract

Purpose: A significant portion (22.1%) of corneal blindness is caused by chemical injuries, for which conventional therapy could result in complications such as glaucoma and post-transplantation immunological rejection. In addition, several of these management strategies are costly. Cell sheet engineering via an Aqueous-Two-Phase-System (ATPS) is an emerging technique which may be adopted for construction of an epithelial bandage for healing of corneal epithelial wounds. In this study, we investigate the properties of the corneal epithelial cell sheet, as well as measure the efficacy of the cell sheet therapy using an in vitro chemical injury model and an ex vivo model. Setting: The study will be taken place in vitro and ex vivo. Methods: Primary human corneal epithelial cell line was used for the experiments. A liquid-liquid interface was created between a Poly(ethylene glycol)(PEG)-rich layer and a Dextran(DEX)-rich layer, within which the corneal epithelial cell sheet was constructed. In vitro tests were carried out to investigate the properties of the cell sheet, including cytotoxicity assays for ATPS components. An in-vitro transwell air-liquid interface chemical injury model was constructed for measurement of time to complete re-epithelialisation. The ex vivo model was used for testing of integration and proliferation of cell sheet on the wound. Results: The optimised cell number density and time for incubation within ATPS was found to be 10,000 cells per mm2 and 3 hours respectively. MTS assay revealed insignificant cytotoxic effects of PEG and DEX on seeded HCECs at 3 hours incubation time. Once the cell sheet was able to attach to culture plates and proliferate, a significant difference in cell density was found between cells directly seeded from the cell sheet, and cells proliferating out from the cell sheet. Measurement of percentage defect area remaining at time points 1, 5, and 8 days post-transplantation illustrated a statistically significant reduction in wound healing time compared to control group without transplantation performed. Ex vivo model demonstrated adherence of cell sheet on exposed stromal surface. Conclusions: The ATPS cell sheet engineering method may be potentially advantageous when compared to current management strategies, in that it is cheaper and more efficient to produce. Future directions point towards the construction of an ex-vivo porcine cornea model for testing the efficacy of transplantation of cell sheet on induced chemical injuries.