Fabrication of Tissue-Engineered Ex Vivo Cornea Model to Evaluate Anti-Inflammatory Response of Ophthalmic Medication

Abstract

The eye irritation test established in the early 1930s by John H Draize is the conventional method to evaluate the toxicity of test agents to eyes and skin. For the test, the rabbits were fixed their body and eyelid with the chamber to restrict movement, and then the toxicants were treated to eyes to score toxicity. The Draize test can observe the physiological change (e. g. swelling, loss of transparency, redness, and bleeding) of animals but, the ethical problems remain because they suffer pain and stress in this process. In addition, the test has a huge limitation on the over-prediction of the physiological response of humans. Because the rabbit has thin cornea thickness and a lower volume of a tear than humans so, they can’t enough wash out the test solution. Additionally, the rabbit’s third eyelid which nictitating membrane can prevent the removal of the drug. For these reasons, various test methods were developed to replace the animal test. Thus, this study about the fabrication of the ex vivo cornea model to evaluate anti-inflammatory response and toxicity of ophthalmic medication was performed.

In part I, The corneal stroma has a specific structure that approximately 200 lamellae containing collagen fibers and is an important role to control light transmission. This study aims to mimic the morphology of stroma with silk fibroin film that has high biocompatibility, mechanical property, and ease to transform. To fabricate a multilayer cornea model (mCM), the corneal cells isolated from the rabbit eye were seeded on non-patterned/patterned silk film (n-p/ pSF) and then were stacked to combine each lamellar. The benzalkonium chloride that preservation solution of the ophthalmic drug was utilized for evaluating the toxicity of the mCM. The permeability is an important barrier function of the cornea to supply nutrient and remove bi-product for cell culture. Furthermore the permeability assay of silk film in different concentrations was conducted with Franz diffusion cell. The lower percentage of silk film shows high permeability but, was hard to handle in wet conditions. Thus, 3% of silk film was utilized for the next experiments. In cytoskeleton observation, the phase contrast, fluorescent, and scanning electron microscopic analyses were conducted. The group of cultured cells on pSF showed uniaxial attachment and migration and the group of n-pSF appeared multiaxial cytoskeletal alignment. As a histological result of the test solution treated cornea model, the cells and extracellular matrix (ECM), a three-dimensional network produced by cells, were dissociated and eliminated from layers. The study is about the mimicking of the multilayer structure and evaluation of cell morphological change. The mCM has low reproducibility, difficult to fabricate, and can't expect cell-to-cell interaction because of the long distance between the layers. For this reason, part II was conducted to overcome limitations.

In part II, this study aims to fabricate a thick cornea model with abundant ECM for overcoming low reproducibility and cell distribution to enhance cell signal. In particular, the ascorbic acid commonly known as vitamin C promotes the synthesis of collagen that a major protein of ECM. Thus, the corneal keratocytes (CKs) were cultured with trans-well in a special medium containing ascorbic acid for stroma fabrication. In the case of the epithelium, the corneal epithelial cells (CECs) were seeded to the top of the stromal layer and exposed to the air-liquid interface to induce cell differentiation like corneal tissue. After the fabrication of the ex vivo cornea model, the anti-inflammatory response was tested with ciprofloxacin (CIP) that bacteria antibiotics used in ophthalmology. To verify collagen synthesis in the special culture medium, the intact stromal sheet was produced and observed that has thickness. However, the central region of thick tissue can be dead because of insufficient nutrients supply and the trouble of wastes removal. For this reason, the live/dead assay was conducted in the ex vivo cornea model and the cells in the central part appeared good viability during cell culture. In the histological analysis with H&E stain, the thickness was about 338 µm and the stratification of CECs was confirmed. In inflammatory response, the groups were N, I, T1 (CIP 5 µg/ml), T2 (CIP 10 µg/ml), and T3 (CIP 15 µg/ml) and pro-inflammatory cytokine expression was analyzed with the western blot. Moreover expression levels were measured with the ImageJ software. In the interleukin-1β (IL-1β), the expression level was slightly decreased in only the T1 than I group and tumor necrosis factor-α (TNF-α) expression level was decreased over half in the T1 than I group. The fabrication of an ex vivo model that is a thick stromal layer combined with a differentiated epithelial layer was successful. In a further study, will plan to research the model targeted drug and effective dose. Part II study aims to replace animal tests and presented the possibility of a new alternative method.