Assembly and Application of a Three-Dimensional Human Corneal Tissue Model.

The cornea provides a functional barrier separating the outside environment from the intraocular environment, thereby protecting posterior segments of the eye from infection and damage. Pathological changes that compromise the structure or integrity of the cornea may occur as a result of injury or disease and can lead to debilitating effects on visual acuity. Over 10 million people worldwide are visually impaired or blind due to corneal opacity. Thus, physiologically relevant in vitro approaches to predict corneal toxicity of chemicals or effective treatments for disease prior to ocular exposure, as well as to study the corneal effects of systemic, chronic conditions, such as diabetes, are needed to reduce use of animal testing and accelerate therapeutic development. We have previously bioengineered an innervated corneal tissue model using silk protein scaffolds to recapitulate the structural and mechanical elements of the anterior cornea and to model the functional aspects of corneal sensation with the inclusion of epithelial, stromal, and neural components. The purpose of this unit is to provide a step-by-step guide for preparation, assembly, and application of this three-dimensional corneal tissue system to enable the study of corneal tissue biology. © 2019 by John Wiley & Sons, Inc.

Modeling Diabetic Corneal Neuropathy in a 3D In Vitro Cornea System.

Diabetes mellitus is a disease caused by innate or acquired insulin deficiency, resulting in altered glucose metabolism and high blood glucose levels. Chronic hyperglycemia is linked to development of several ocular pathologies affecting the anterior segment, including diabetic corneal neuropathy and keratopathy, neovascular glaucoma, edema, and cataracts leading to significant visual defects. Due to increasing disease prevalence, related medical care costs, and visual impairment resulting from diabetes, a need has arisen to devise alternative systems to study molecular mechanisms involved in disease onset and progression. In our current study, we applied a novel 3D in vitro model of the human cornea comprising of epithelial, stromal, and neuronal components cultured in silk scaffolds to study the pathological effects of hyperglycemia on development of diabetic corneal neuropathy. Specifically, exposure to sustained levels of high glucose, ranging from 35 mM to 45 mM, were applied to determine concentration-dependent effects on nerve morphology, length and density of axons, and expression of metabolic enzymes involved in glucose metabolism. By comparing these metrics to in vivo studies, we have developed a functional 3D in vitro model for diabetic corneal neuropathy as a means to investigate corneal pathophysiology resulting from prolonged exposure to hyperglycemia.