BACKGROUND
Globally, more than 12 million people are awaiting corneal transplantation and cornea donor reduction has been observed since the outbreak of the COVID-19 pandemic, negatively influencing the availability of human corneas for research purposes as well. Therefore, the exploitation of ex vivo animal models in this field is of great value. The present study aimed at the development of a novel experimental model of porcine cornea ex vivo and lamellar tissue preparation to investigate the effects of storage conditions on corneal preservation.

METHODS
Twelve fresh porcine eye bulbs were disinfected by immersion in 10 mL of 5% povidone-iodine under orbital mixing for 5 minutes at room temperature. The corneoscleral rims were dissected, and stored in Tissue-C (Alchimia S.r.l., n=6) at 31°C and in Eusol-C (Alchimia S.r.l., n=6) at 4°C up to 14 days. The evaluation of Endothelial Cell Density (ECD) and endothelial mortality was performed using vital dye Trypan Blue staining (TB-S, Alchimia S.r.l.). Digital 1X pictures of TB-stained corneal endothelium were acquired and percentage of stained area was quantified using FIJI ImageJ software. ECD and endothelial mortality were determined at 0, 3, 7 and 14 days. Medium turbidity detected by naked eye was considered as proof of tissue contamination. Additionally, non-vital staining of the endothelium with Alizarin Red (AR) was performed and the endothelial morphology was investigated at Day 14 in both whole corneas and dissected endothelial lamellae.

RESULTS
The contamination rate of porcine corneas corresponded to <10% and 0% in Tissue-C and Eusol-C after 14 days, respectively. Porcine corneas stored in Tissue-C and Eusol-C showed <10% and <20% mortality in Tissue-C and Eusol-C respectively at the end of storage. Preliminary ECD determination (range 3700-4100 cells/mm2) at Day 0 aligned with data present in the literature (Meltendorf et al., Graefe’s Arch Clin Exp Ophthalmol, 2007). Whole cornea and dissected lamellae stained with TB and AR showed comparable endothelial morphology after incubation in Tissue-C and Eusol-C for 14 days. The lamellar tissue allowed endothelium morphology analysis at higher magnification compared to whole cornea.

CONCLUSION
The presented ex vivo porcine model allows evaluation of the performance and safety of storage conditions. Future perspectives of this method will be the extension of the porcine corneas storage up to 28 days.

Background: The endothelial cell mortality of the donor corneas is determined qualitatively by the eye bank technicians using the trypan blue staining of the endothelium and visual evaluation of the mortality zones. The present study aimed at developing a quantitative method to determine the endothelial cell mortality based on the trypan blue staining and Image Analysis Using ImageJ software.

Method: Porcine eyes were retrieved at local abattoir, transported to our labs, the cornea was dissected and endothelial cell mortality was immediately assessed. Evaluation of corneas was repeated in five different experiments each including 10 corneas. Endothelial cell mortality was determined by staining the endothelium with trypan blue (TB-S Alchimia S.r.l.) and acquiring stereomicroscopy pictures with 10x magnification. The pictures were analyzed with the Fiji ImageJ software, adopting a customized macro sequence to count blue pixels, corresponding to dead cells, in the central 8-mm diameter area. Surface irregularity of the corneal tissue was compensated during macro sequence optimization phase. Mean percentage of cell mortality was determined for each group of tissues. The normal distribution of percentages of cell mortality within groups was evaluated with Shapiro-Wilk Normality Test and differences between groups was verified with ANOVA one-way analysis of variance.

Results: Customized macro sequence of Fiji ImageJ software provided an accurate and repeatable quantification of the mortality area of corneal endothelium despite the irregular surface of the corneal tissue. The mean endothelial cell mortality was 4.50 % ± 0.94%; the low standard error indicated the repeatability of the method. The distribution of values within groups resulted to be normal according to Shapiro-Wilk Normality Test (p>0.05) and no significant differences were observed between groups based on ANOVA test (p>0.05).

Conclusion: The present study allowed the development of a semi-automatized quantitative method for the measurement of corneal endothelial cell mortality using image analysis with Fiji ImageJ customized macro sequence with optimal method sensibility and repeatability. Additional method optimization will be investigated in order to implement the endothelial cell mortality evaluation within the daily routine of the eye banks.

Purpose: To test the feasibility of a cell therapy approach to treat corneal endothelial (CE) disorders using an in vitro model of human corneal decompensation.

Methods: A CE decompensation model was established by removal of the Descemet membrane/endothelium complex from cadaveric human corneas in an air interface organ culture system (group 2) and compared with normal corneas (group 1). The posterior stroma of decompensated corneas was seeded with immortalized human corneal endothelial cells (HCEC-12) in group 3 and passage 0 primary human CE cells in group 4 corneas. Functional effects on stromal thickness were determined with histological analysis 3 to 10 days after cell therapy treatment.

Results: Removal of the Descemet membrane/endothelium complex in group 2 corneas resulted in a stromal thickness of 903 ± 86 μm at 12 hours compared with 557 ± 72 μm in group 1 corneas. Stromal thickness reduced from 1218 ± 153 μm to 458 ± 90 μm (63% ± 6%, P = 0.001) after cell transplantation in group 3 and from 1100 ± 86 μm to 489 ± 94 μm (55% ± 7%, P = 0.00004) in group 4. Posttransplantation histology demonstrated formation of a monolayer of corneal endothelium attached to the posterior stromal surface.

Conclusions: Direct transplantation of cultured human CE cells and immortalized HCEC-12 to bare posterior corneal stroma resulted in formation of anendothelial monolayer and restoration of stromal hydration to physiological thickness, demonstrating the feasibility of cell therapy in treatment of CE decompensation in a human in vitro model.

Abstract: Eye Banks are institutions charged with evaluating, storing and distributing corneal tissues for transplantation. The use of specific culture media allowed to extend the storage time of tissues in the eye bank, thus meeting the increasing demand for tissues intended for corneal transplantation. In particular, TISSUE-C allows to store donor corneas for a period of 4 weeks. In addition, the use of 0,25% trypan blue (TB-S) and 1,4% sucrose (SR-S) allowed to significantly increase the quality of tissues for transplantation. In fact, the two products allow to carefully assess the quality of the corneal endothelium, through evaluation of cell viability and morphology.