Human Corneal Model for Ocular Irritation Assay

描述（由申请人提供）：用Draize兔眼测试替代眼刺激测试仍然是一个难以实现的目标。许多人认为有必要建立角膜全厚度（CFT）模型来完整地模拟外源化学物质与人角膜的相互作用。在第一阶段，从天然组织中分离出人角膜上皮细胞、间质细胞和内皮细胞，在单层培养中扩增，并冷冻保存。培养条件被确定为产生高度分化的2层（角膜上皮和含角化细胞的基质）和3层（上皮、基质和内皮）CFT组织。CFT组织在屏障和分化表型方面优于上皮组织。开发了一种检测和预测模型（PM），使CFT组织能够以100%的准确度区分刺激物和非刺激物。其他功能研究表明，CFT组织似乎适合评估化学损伤后的恢复时间（TTR）和损伤深度（DOI）。最后，对CFT组织生产的分析表明，CFT具有良好的经济效益。第二阶段将在第一阶段结果的基础上进一步发展CFT组织模型。最初的目标将集中在为商业生产准备CFT组织模型-细胞库存将扩大，以确保稳定的细胞供应和质量控制参数将开发。将扩大使用CFT测试的材料数据库，将最终确定刺激性/非刺激性分类的PM，并将证明该分析的实验室间可转移性。最后，CFT组织将用于TTR和DOI测量，并将开发替代Draize测试的分析和预测模型。由于这些增强的功能，CFT将为毒理学家和科学家提供一种工具，即使不能完全消除，也将大大减少动物试验来确定眼部刺激的需要。

DESCRIPTION (provided by applicant): Replacement of the Draize rabbit eye test for ocular irritancy testing remains an elusive goal. Many believe a corneal full thickness (CFT) model is necessary to completely model the interaction of exogenous chemicals with human cornea. During Phase 1, human corneal epithelial, stromal, and endothelial cells were isolated from native tissue, expanded in monolayer culture, and cryopreserved. Culture conditions were identified to produce highly differentiated 2-layer (corneal epithelium and keratocyte-containing stroma) and 3- layer (epithelium, stroma, and endothelium) CFT tissues. The CFT tissues exhibited improvements over epithelial tissues in terms of barrier and differentiated phenotype. An assay and prediction model (PM) was developed which allowed the CFT tissue to distinguish between irritants and non-irritants with 100% accuracy. Other functional studies showed that the CFT tissue appears suitable to evaluate time to recovery (TTR) and depth of injury (DOI) following chemical injury. Finally, an analysis of CFT tissue production showed that the economics of CFT are favorable. Phase II will expand upon the Phase 1 result to further develop the CFT tissue model. Initial goals will focus on preparing the CFT tissue model for commercial production - cell stocks will be expanded to insure a stable cell supply and quality control parameters will be developed. The database of materials tested using the CFT will be expanded, the PM for irritant/non-irritant classification will be finalized, and inter-laboratory transferability of the assay will be demonstrated. Finally, the CFT tissues will be used for TTR and DOI measurements and an assay and prediction model for replacement of the Draize test will be developed. Due to these enhanced capabilities, the CFT will provide toxicologists and scientists with a tool that will dramatically decrease, if not entirely eliminate, the need for animal testing to determine ocular irritancy. PUBLIC HEALTH RELEVANCE: Safety assessment of all new chemicals and products requires evaluation of potential ocular irritancy in case of intentional or unintentional exposure to the eye. Current animal-based test methods are suboptimal because of animal welfare concerns and in vitro methods lack the complexity required to completely model in vivo responses. We will develop a corneal full thickness tissue and in vitro test methods that will dramatically reduce or eliminate the need to use animals to assess ocular irritancy of chemicals and products.