Porcine Cornea Storage Ex Vivo Model as an Alternative to Human Donor Tissues for Investigations of Endothelial Layer Preservation

Abstract

Purpose: Due to the growing shortage of human corneas for research, we developed a porcine cornea storage model with qualitative features comparable to human tissues.

Methods: We established a decontamination procedure for porcine eye bulbs to ensure corneal storage at 31°C to 35°C for up to 28 days without contamination. We compared human and porcine corneas under hypothermic (2-8°C) or culture (31-35°C) conditions for central corneal thickness (CCT), corneal transparency, endothelial morphology, endothelial cell density (ECD), and a novel method to quantify whole endothelial mortality. We also examined portions of lamellar tissues consisting of Descemet's membrane and endothelial cells under the microscope after Alizarin red staining.

Results: Our decontamination procedure reduced corneal contamination from 94% (control corneas without decontamination) to 18% after 28 days of storage at 31°C to 35°C. ECD, CCT, transparency, and morphology were significantly higher in porcine corneas than in human corneas at day 0. Nevertheless, the qualitative parameters of porcine and human corneas showed comparable trends under both investigated storage conditions for up to 14 days.

Conclusions: The presented corneal storage model provides a reliable alternative to human tissues for preliminary corneal investigations.

Translational relevance: The porcine cornea storage model can be used to investigate the efficacy and safety of new media, substances, or storage conditions. Furthermore, the method developed to assess the percentage of endothelial mortality is tissue conservative and can be used in eye banks to monitor endothelial mortality during storage of tissues intended for transplantation.

Figure 1.

Representative pictures of porcine (A) and human (B) corneal endothelium after TB staining after different days of hypothermic storage (upper panels) and the Fiji processed pictures for TB-stained area quantification (lower panels, in black and white) of corneas stored 14 days in hypothermic storage medium (porcine: n = 9; human: n = 3) and cytotoxic control group (porcine: n = 3; human n = 3) at 2°C to 8°C. Photographs were taken at days 0, 3, 7, and 14 of hypothermic storage. (C, D) Mean and standard deviation of endothelial mortality in porcine (C) and human (D) corneas stored under hypothermic storage conditions and in a cytotoxic control group at different time points. H.S.M., hypothermic storage medium; ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 2.

TB and AR staining images of fresh porcine and human corneas. (A–D) Representative 100× images of porcine (A, C) and human (B, D) corneas stained with TB and AR dyes before the beginning of the storage (day 0). Tissue staining allowed to properly assess the ECD and EC morphology of the endothelial hexagonality mosaic in both species, since cellular margins appeared sharp and clearly visible (scale bar: 200 µm). (E, F) Representative 400× images of porcine (E) and human (F) DM lamellar tissues. TB and AR dyes were used to stain nuclei and cellular membranes, respectively. This tissue preparation allowed high-magnification examination of both porcine and human endothelia (scale bar: 100 µm). (G, H) Representative images of porcine (G) and human (H) endothelia (scale bar: 200 µm) obtained with specular microscopy at day 0.

Figure 3.

TB and AR staining images of porcine and human corneas after 14 days of storage at 2°C to 8°C. (A–D) Representative 100× images of porcine (A, C) and human (B, D) corneas stained with TB and AR dyes after 14 days in hypothermic storage medium at 2°C to 8°C (scale bar: 200 µm). (E, F) Representative 400× images of porcine and human DM lamellar tissues (scale bar: 100 µm). (G–L) Graphs showing the ECD (G), EC morphology (H), corneal transparency (I), and CCT (L) in porcine (black line) and human (dotted lines) corneas at selected time points during hypothermic storage medium at 2°C to 8°C. Bars are standard deviations. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 4.

TB and AR staining images of porcine and human corneas after 14 days of storage at 31°C to 35°C. (A–D) Representative 100× images of porcine (A, C) and human (B, D) corneas stained with TB and AR dyes after 14 days in corneal culture medium at 31°C to 35°C (scale bar: 200 µm). (E, F) Representative 400× images of porcine and human DM lamellas (scale bar: 100 µm). (G, H) Graphic showing the ECD (G) and EC morphology (H) in porcine (black line) and human (dotted lines) corneas at selected time points during storage in corneal culture medium at 31°C to 35°C. Bars are standard deviations. *P < 0.05. **P < 0.01. ***P < 0.001.