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Three-dimensional testicular cell co-culture model for reproductive toxicity screening

用于生殖毒性筛查的三维睾丸细胞共培养模型

基本信息

批准号：
9789886
负责人：
Lei Yin
金额：
$ 72.99万
依托单位：
REPROTOX BIOTECH, LLC
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-18 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9789886
关键词：
3-Dimensional Adverse effects Affect Animal Testing Animals Biological Assay Blood-Testis Barrier Cell Culture Techniques Cell Cycle Cell Line Cells Chemicals Classification Coculture Techniques Cytoskeleton DNA Damage Data Development Dimensions Dose Environment Evaluation Germ Cells Grant Health In Vitro Knowledge Laboratories Methods Modeling Modernization Molecular Morphology National Research Council Oxidative Stress Pathway interactions Pharmaceutical Preparations Phase Procedures Protocols documentation Quality Control Reporting Reproducibility Risk Assessment Screening procedure Sensitivity and Specificity Steroid biosynthesis Structure Testing Testis Time Toxic effect Toxicity Tests Toxicology United States Environmental Protection Agency Vision adverse outcome base commercialization cost design developmental disease developmental toxicity environmental chemical good laboratory practice in vitro Assay in vitro Model in vivo performance tests phase 1 study predictive modeling programs reproductive reproductive toxicity screening specific biomarkers toxicant

项目摘要

Summary Reproductive and developmental disorders caused by drugs or environmental chemicals are a prominent health issue worldwide. Current toxicity testing methods set by regulatory agencies predominately rely on animal testing. The cost and time associated with animal testing for risk assessment seriously limit efforts for extensive toxicity screening. These data gaps necessitate the implementation of a new vision for toxicity testing as reflected in the National Research Council’s report “Toxicity Testing in the 21st Century” and the restructuring of risk assessment procedures. Toxicity testing in the 21st century must move beyond classical methods of risk assessment by incorporating modern molecular knowledge, such as adverse outcome pathways (AOPs) with advanced in vitro models. Currently, in vitro reproductive toxicity testing models are actively being developed. Under the support of R43 ES027374 grant, ReproTOX established a Mini-Testis model from testicular cell lines and found this Mini-Testis model could identify reproductive toxicants. Both morphological and cell-specific biomarkers assessment demonstrated that this co-culture model created an in vitro-like niche, formed a three-dimensional cytoskeleton bundle structure and supported germ cells within the 3D environment. We initially validated this model by discriminating reproductive toxicants among 32 compounds and observed a strong correlation between the in vitro IC50 and in vitro rLOAEL (the lowest observed adverse effect level) for these selected compounds. The calculation of concordance, sensitivity, and specificity further supported the reliability of this model. Our results from the phase I study suggested that our in vitro Mini- Testis model might be a valuable screening tool for reproductive toxicant assessment and to prioritize chemicals for further testing. In this phase II proposal, we will further examine and optimize the protocol for the in vitro Mini-Testis model. We will test the Mini-Testis model with an expanding list of “reference testing compounds”, and validate blindly our predictive model of pathway-based high-content and high-throughput platform. The Specific Aims are (1) to complete the development of high-content assays based on the adverse outcome pathways associated with the reproductive toxicity in an in vitro Mini-testis model; (2) to apply the multi-parametric high-throughput and high-content analysis (HT/HCA) to examine the sensitivity and specificity through comparison between the IC50 obtained from the in vitro model and in vivo reproductive lowest observed adverse effect level (rLOAEL) to validate the in vitro Mini-testis model; (3) to conduct the intra- and inter-laboratory reproducibility study to validate the in vitro Mini-testis model; we will test the variability within-test and the reproducibility of the test within and among laboratories. Altogether, this proposed Phase II will focus on the continued development of this 3D Mini-Testis screening platform toward a validated quantitative pathway-based HT/HCA screen for reproductive toxicity. Commercialization of this in vitro platform and its ability to screen up to a thousand compounds via focusing on multiple endpoints associated with adverse reproductive effects caused by agents from a variety of chemical classes. This Mini- Testis model-based platform has the potential to offer predictive reproductive and developmental toxicity without the use of animals in a high-throughput format, and will be a critical part of an integrated testing strategy for Reproductive and developmental toxicity.

概括由药物或环境化学物质引起的生殖和发育障碍是一个突出的问题世界范围内的健康问题。目前监管机构制定的毒性测试方法主要依赖于动物测试。与风险评估相关的动物试验的成本和时间严重限制了广泛的毒性筛查。这些数据差距需要实施新的毒性测试愿景正如国家研究委员会的报告《21 世纪的毒性测试》和重组风险评估程序。 21 世纪的毒性测试必须超越传统的通过结合现代分子知识进行风险评估的方法，例如不良结果途径（AOP）具有先进的体外模型。目前，体外生殖毒性测试模型正在积极开展正在开发中。在R43 ES027374资助的支持下，ReproTOX建立了Mini-Testis模型睾丸细胞系，发现这种迷你睾丸模型可以识别生殖毒物。两者形态和细胞特异性生物标志物评估表明，这种共培养模型创造了一个类似体外的利基，形成三维细胞骨架束结构并在3D内支撑生殖细胞环境。我们最初通过区分 32 种化合物中的生殖毒物来验证该模型并观察到体外 IC50 和体外 rLOAEL（观察到的最低不良反应）之间存在很强的相关性。这些选定的化合物的效果水平）。进一步计算一致性、敏感性和特异性支持了该模型的可靠性。我们的第一阶段研究结果表明我们的体外 Mini- 睾丸模型可能是生殖毒物评估和优先排序的有价值的筛选工具化学物质以供进一步测试。在这个第二阶段提案中，我们将进一步检查和优化协议体外迷你睾丸模型。我们将通过扩展的“参考测试”列表来测试迷你睾丸模型化合物”，并盲目验证我们基于途径的高含量和高通量的预测模型平台。具体目标是 (1) 完成基于不良反应的高含量检测方法的开发体外迷你睾丸模型中与生殖毒性相关的结果途径； (2) 申请多参数高通量和高内涵分析（HT/HCA）以检查灵敏度和通过比较体外模型和体内生殖模型获得的 IC50 来确定特异性最低观察到的不良反应水平（rLOAEL）以验证体外迷你睾丸模型； (3) 进行实验室内和实验室间再现性研究，以验证体外迷你睾丸模型；我们将测试测试内的变异性以及实验室内部和实验室之间测试的可重复性。总而言之，这拟议的第二阶段将重点关注该 3D 迷你睾丸筛查平台的持续开发，以实现经验证的基于定量途径的 HT/HCA 生殖毒性筛查。将此商业化体外平台及其通过关注多个终点筛选多达一千种化合物的能力与多种化学物质引起的不良生殖影响有关。这个迷你基于睾丸模型的平台有可能提供预测生殖和发育毒性的潜力无需以高通量形式使用动物，并将成为综合测试的关键部分生殖和发育毒性策略。