3D Skin Model to Test Toxic and Sensitizing Potentials of Environmental Chemicals

描述（由申请人提供）：本申请涉及广泛的挑战领域（06）使能技术和06-ES-102的特定挑战主题：减少研究中动物使用的3-D或虚拟模型：创建用于化学毒性试验高通量筛选的微型多细胞器官。虽然毒理学试验已经使用动物进行，但由于公众观点和监管要求的快速变化，迫切需要替代体外试验。在由环境化学品引起的许多疾病中，接触性皮炎是发病率最高的疾病-CDC已将接触性皮炎列为国家职业研究议程（诺拉）的21个研究优先领域之一。由于接触性皮炎可通过消除皮肤与病原体的接触来预防，因此开发用于鉴定此类病原体的体外测定法对全球公共卫生具有广泛且重大的影响。接触性皮炎分为刺激性皮炎（对刺激性化学物质的先天性炎症反应）和过敏性皮炎（对皮肤致敏剂的T细胞介导的适应性免疫反应）。我们已经确定ATP释放角质形成细胞（KCs）作为一个初始的和致病的事件在刺激性接触性皮炎的发病机制。由于已知几种不同的接触性过敏原诱导皮肤中树突状细胞（DC）表达IL-1 <$mRNA，我们通过工程化我们的皮肤来源的DC系XS 106以在IL-1 <$启动子的控制下表达黄色荧光蛋白（YFP）基因，开发了HTTP相容的DC生物传感器克隆。我们的目标是开发一种3D多通道皮肤生物传感器系统，该系统由KC、DC和成纤维细胞（FB）组成，可以同时检测皮肤刺激物（通过ATP释放测量）和皮肤致敏物（通过荧光信号测量）。在目标1下，我们将通过工程化Pam 212 KC系来构建KC生物传感器克隆，以在Nrf 2（能够触发Keep 1-Nrf 2-ARE细胞对环境胁迫的存活响应的转录因子）的控制下表达GFP基因。我们还将产生在Tfec（已知调节细胞因子和细胞因子受体基因表达的转录因子）控制下表达DsRed基因的第二DC生物传感器克隆。在目标2下，我们将通过以下方式重建3D皮肤模型：a）在水合胶原凝胶中培养NS FB系以形成真皮等效基质，B）在胶原基质的顶部培养KC生物传感器克隆，以及c）接种两个DC生物传感器克隆以形成具有KC的多细胞表皮结构。在目标3下，我们将在所得3D皮肤模型中测试18种皮肤刺激物和36种接触致敏物，以评估灵敏度和特异性。我们将测量ATP释放作为皮肤刺激潜力的指标，以及YFP、GFP和DsRed信号，以识别甚至分类皮肤致敏物。最后，我们将通过在96孔板格式中重建3D皮肤等效微培养物来提高HTP能力。一旦开发和验证，具有双重功能的3D皮肤模型可以使该行业消除使用动物进行皮肤毒理学研究。在由环境化学品引起的众多疾病中，接触性皮炎是发病率最高的疾病。我们将开发一种三维多细胞皮肤培养系统，以测试大量工业和环境化学品引起刺激性和过敏性接触性皮炎的潜力。一旦开发和验证，所产生的3D皮肤模型可能使该行业能够消除使用动物进行皮肤毒理学研究。

DESCRIPTION (provided by applicant): This application addresses the broad challenge area (06) Enabling Technologies and the specific Challenge Topic of 06-ES-102: 3-D or Virtual Models to Reduce Use of Animals in Research: Creation of Miniature Multi- Cellular Organs for High Throughput Screening for Chemical Toxicity Testing. Although toxicology tests have been performed using animals, there exists an urgent and critical need for alternative in vitro tests due to rapid changes in public views and regulatory requirements. Among many diseases caused by environmental chemicals, contact dermatitis is the leading disease in terms of the incidence - the CDC has listed contact dermatitis as one of the 21 research priority areas of the National Occupational Research Agenda (NORA). Because contact dermatitis is preventable by eliminating skin contact with causative agents, development of in vitro assays for identification of such agents has a broad and major impact on the global public health. Contact dermatitis is classified into irritant dermatitis (innate inflammatory responses to irritant chemicals) and allergic dermatitis (T cell-mediated adaptive immune responses to skin sensitizers). We have identified ATP release from keratinocytes (KCs) as an initial and causative event in the pathogenesis of irritant contact dermatitis. Because several different contact allergens are known to induce IL-1¿ mRNA expression by dendritic cells (DCs) in the skin, we have developed a HTP-compatible DC biosensor clone by engineering our skin-derived DC line XS106 to express the yellow fluorescence protein (YFP) gene under the control of the IL-1¿ promoter. Our objective is to develop a 3D multi-channel skin biosensor system that is composed of KCs, DCs, and fibroblasts (FBs) and that allows simultaneous detection of skin irritants (measured by ATP release) and skin sensitizers (measured by fluorescence signals). Under Aim 1, we will construct a KC biosensor clone by engineering the Pam 212 KC line to express the GFP gene under the control of Nrf2 (a transcription factor capable of triggering the Keep1-Nrf2-ARE cell survival response to environmental stresses). We will also generate a second DC biosensor clone expressing the DsRed gene under the control of Tfec (a transcription factor known to regulate cytokine and cytokine receptor gene expression). Under Aim 2, we will reconstitute a 3D skin model by: a) culturing the NS FB line in hydrated collagen gel to form a dermal-equivalent matrix, b) culturing a KC biosensor clone on the top of the collagen matrix, and c) seeding two DC biosensor clones to form a multi-cellular epidermal structure with KCs. Under Aim 3, we will test 18 skin irritants and 36 contact sensitizers in the resulting 3D skin model to evaluate the sensitivity and specificity. We will measure ATP release as an indicator of skin irritating potentials and YFP, GFP, and DsRed signals to identify and even classify skin sensitizers. Finally, we will improve the HTP capability by reconstituting 3D skin-equivalent micro- cultures in a 96 well-plate format. Once developed and validated, the 3D skin model with dual functionality may enable the industry to eliminate the use of animals for skin toxicology studies. Among many diseases caused by environmental chemicals, contact dermatitis is the leading disease in terms of the incidence. We will develop a three-dimensional, multi-cellular skin culture system to test a large number of industrial and environmental chemicals for their potentials to cause irritant and allergic contact dermatitis. Once developed and validated, the resulting 3D skin model may enable the industry to eliminate the use of animals for skin toxicology studies.