Al.chi.mi.a. Srl, Ponte San Nicolò, Italy.
Fondazione Banca degli Occhi del Veneto Onlus, Venezia, Italy.
BMJ Open Ophthalmol. 2022 Nov;7(Suppl 2):A8. doi: 10.1136/bmjophth-2022-EEBA.18.
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.
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.
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.
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.
在全球范围内，有超过 1200 万人在等待角膜移植，自 COVID-19 大流行爆发以来，角膜供体减少的情况已经出现，这对研究用人类角膜的供应产生了负面影响。因此，在该领域开发离体动物模型具有重要价值。本研究旨在建立一种新型的离体猪角膜模型和板层组织制备方法，以研究储存条件对角膜保存的影响。
将 12 个新鲜的猪眼球在室温下用 10ml 5%聚维酮碘浸泡 5 分钟进行消毒。然后将角膜巩膜边缘切开，分别在 31°C 的 Tissue-C（Alchimia S.r.l.，n=6）和 4°C 的 Eusol-C（Alchimia S.r.l.，n=6）中储存，储存时间长达 14 天。使用活体染料台盼蓝染色（TB-S，Alchimia S.r.l.）来评估内皮细胞密度（ECD）和内皮细胞死亡率。用 FIJI ImageJ 软件对 TB 染色的角膜内皮进行数字 1X 拍摄，并对染色区域的百分比进行量化。在 0、3、7 和 14 天时分别测定 ECD 和内皮细胞死亡率。用肉眼检测到的中等浑浊度被认为是组织污染的证据。此外，还对内皮进行了茜素红（AR）非活体染色，并在第 14 天对整个角膜和分离的内皮板层进行了内皮形态学研究。
在 Tissue-C 和 Eusol-C 中，储存 14 天后猪角膜的污染率分别为<10%和 0%。在 Tissue-C 和 Eusol-C 中，储存结束时，猪角膜的死亡率分别<10%和<20%。第 0 天初步测定的 ECD 值（范围为 3700-4100 个细胞/mm2）与文献中的数据一致（Meltendorf 等人，Graefe's Arch Clin Exp Ophthalmol，2007）。在 Tissue-C 和 Eusol-C 中孵育 14 天后，用 TB 和 AR 染色的整个角膜和分离的板层均显示出相似的内皮形态。与整个角膜相比，板层组织可以在更高的放大倍数下分析内皮形态。
本研究提出的离体猪模型可以评估储存条件的性能和安全性。该方法的未来展望是将猪角膜的储存时间延长至 28 天。
