StrataScreenSENS, Skin Sensitizer Detection in Luminescent 3D Human Skin Models

描述（由申请人提供）：该项目的最终目标是开发一种创新的体外试验，用于皮肤致敏剂的分类，允许在高度相关的人体皮肤三维模型中评估化学物质，更接近地模仿体内环境。过敏性接触性皮炎（ACD）是一种广泛存在的公共卫生问题，是由于人类皮肤在工作场所和家庭中暴露于某些反应性化学物质而引起的。确定哪些化学物质会诱发ACD对减少人类接触至关重要。目前还没有有效的皮肤致敏试验可以排除动物使用，但令人信服的经济和伦理原因支持开发新的体外方法，准确预测化学物质或混合物的致敏潜力，并消除对动物的需求。所提出的StrataScreenSENS 96孔分析背后的关键技术创新是一种独特的三维人体皮肤模型，该模型利用荧光素酶活性作为皮肤致敏电位的快速，简单和准确的定量分析。在人体皮肤模型的背景下，对皮肤致敏化学物质提供准确和稳健反应的体外试验将1)允许将测试化学物质应用于皮肤表面，模拟ACD中实际的正常暴露途径；2)比目前使用的二维单层培养系统更能模拟体内生理环境；3)采用96孔格式，提高筛分吞吐量；4)减少或取代皮肤致敏剂评价中动物试验的需要。StrataScreenSENS的优势直接解决了21世纪毒理学面临的挑战和限制，并与国家环境健康科学研究所的使命有关。该I期SBIR提案的目标是生成和表征用于StrataScreenSENS试验中报告皮肤模型生产的独特细胞试剂。这将通过使用Stratatech的平台技术NIKS细胞来完成，NIKS细胞是一种非致瘤性、无病原体的人类角化细胞祖细胞的一致来源。基因工程NIKS角质形成细胞和产生三维皮肤组织的能力，产生外源性蛋白质，为开发对皮肤致敏化学物质有反应的报告皮肤模型提供了独特的机会。I期目标的成功完成将证明我们的方法的可行性，并产生用于StrataScreenSENS试验的细胞试剂。后续的研究将完善分析参数，支持分析的验证，从而降低公众接触有害化学物质的风险，并减少对动物试验的依赖。

DESCRIPTION (provided by applicant): The ultimate goal of this project is to develop an innovative in vitro assay for the classification of skin sensitizers that permits evaluation of chemicals within the highly relevant context of a three-dimensional model of human skin that more closely mimics the in vivo environment. Allergic contact dermatitis (ACD) is a wide- spread public health problem resulting from exposure of human skin to certain reactive chemicals in the workplace and in the home. Identifying which chemicals induce ACD is critical to reduce human exposure. There are currently no validated tests for skin sensitization that preclude animal use, yet compelling economic and ethical reasons support development of new in vitro methods that accurately predict the sensitizing potential of a chemical or mixture and eliminate the need for animals. The key technological innovation behind the proposed StrataScreenSENS 96-well assay is a unique three- dimensional human skin model that utilizes luciferase activity as a rapid, easy and accurate quantitative assay readout for skin sensitizing potential. An in vitro assay that provides accurate and robust responses to skin sensitizing chemicals in the context of a human skin model would 1) permit the application of test chemicals to a skin surface, mimicking the actual normal route of exposure in ACD; 2) model the in vivo physiological environment better than the two-dimensional, monolayer culture systems currently employed; 3) utilize a 96- well format to enhance screening throughput; and 4) reduce or replace the need for animal testing in skin sensitizer evaluation. The advantages of StrataScreenSENS directly address the challenges and limitations facing toxicology in the 21st century and speak to the Mission of the National Institute of Environmental Health Sciences. The goals of this Phase I SBIR proposal are to generate and characterize unique cellular reagents for production of the reporter skin model utilized in the StrataScreenSENS assay. This will be accomplished through use of Stratatech's platform technology, the NIKS cells, a consistent source of non-tumorigenic, pathogen-free human keratinocyte progenitors. The ability to genetically engineer NIKS keratinocytes and generate three- dimensional skin tissue that produces exogenous protein provides a unique opportunity to develop reporter skin models responsive to skin sensitizing chemicals. Successful completion of the Phase I Aims will demonstrate the feasibility of our approach and generate the cellular reagents employed in the StrataScreenSENS assay. Subsequent studies will refine assay parameters, supporting validation of an assay that will reduce the risk of public exposure to harmful chemicals and decrease the reliance on animal testing. PUBLIC HEALTH RELEVANCE: Allergic contact dermatitis (ACD) is a wide-spread public health problem resulting from exposure of human skin to certain reactive chemicals in the workplace and in the home. The identification of chemicals which induce ACD is important to reduce human exposure. Similarly, the elimination of tests requiring animals for this identification is important for economic and ethical reasons. Development of a non-animal-based test that identifies chemicals which induce ACD will fill a major need for the pharmaceutical, consumer products, and chemical Industries by decreasing reliance on animal testing and reducing the risk of public exposure to harmful chemicals.