Evaluation of corneal endothelial cell therapy using an in vitro human corneal model
thesisposted on 2023-08-30, 15:02 authored by Kostadin D. Rolev
Aim: To establish an in vitro human corneal decompensation model and to use it for the evaluation of a cell-therapy approach for treating corneal endothelial (CE) disorders and to test the expression profile of positive regulatory domain proteins (PRDMs) as potential markers for corneal endothelial cells (CECs). Materials and Methods: Human cadaveric corneas were obtained from Bristol and Manchester Eye Banks, UK. A CE decompensation model was established by removal of the Descemet’s membrane (DM)/Endothelium complex from donor corneas and placing them in air-interface organ culture. The corneal thickness was used as a surrogate measure of CE function and was measured using Optical Coherence Tomography (OCT). Decompensated corneas were subjected to cultured endothelial cell therapy using immortalized HCEC -12 cells (group 1), primary human corneal endothelial cells (hCECs) at 0 passage (group 2) and hCECs at passage 2 (group 3) with defined seeding cell density. The effect on stromal de-swelling in cell therapy treated corneas was assessed 3, 7 and 10 days post-transplantation followed by histological evaluation. In addition, expression of PRDM genes in the corneal endothelium was undertaken using reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and immunohistochemistry. Results: Organ culture of human cadaveric corneas in air-interface following the selective removal of the DM/Endothelium complex resulted in stromal thickness of 903.6 ± 86.51 μm, whereas normal corneas maintained a physiological thickness of 557.51 ± 72.64 μm. When transplanted directly onto the posterior corneal stroma the human CECs were able to attach and achieved physiological corneal thickness of 458.91 ± 90.07 μm, 489.65 ± 94.62 μm and 613.7 ± 94.62 μm for cell therapy groups -1, -2 and -3 respectively. The study identified PRDMs 1, 2, 4, 5 and 10 in the human CE and revealed a differential expression between normal CE and cultured hCECs. Conclusion: Removal of the DM/Endothelium complex from cadaveric human corneas held in air interface organ culture resulted in corneal endothelial decompensation. Direct transplantation of cultured primary hCECs to bare posterior corneal stroma devoid of DM resulted in the formation of an endothelial monolayer and restoration of stromal hydration to physiological thickness, substantiating the role of cell therapy to treat corneal endothelial disorders. The identification of PRDM proteins in the human corneal endothelium paves the way for future studies to understand their role in hCEC proliferation control.
InstitutionAnglia Ruskin University
- Accepted version