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.