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

摘要 由药物或环境化学品引起的生殖和发育障碍是一个突出的问题 全球健康问题。目前监管机构制定的毒性测试方法主要依赖于 动物实验。与动物试验相关的风险评估的成本和时间严重限制了 广泛的毒性筛查。这些数据差距使得实施毒性测试的新愿景成为必要 国家研究委员会的报告《21世纪的毒性测试》和 调整风险评估程序。21世纪的毒性测试必须超越经典 结合现代分子知识进行风险评估的方法，如不良结局途径 (AOPS)与先进的体外模型。目前，体外生殖毒性测试模型正在活跃。 正在开发中。在R43 ES027374拨款的支持下，ReproTOX从 并发现这种迷你睾丸模型可以识别生殖毒物。两者都在形态上 细胞特异性生物标志物的评估表明，这种共培养模式创造了一个类似于体外的利基环境， 形成了三维的细胞骨架束结构，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这一模型。 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 对这些选定的化合物)。协调性、敏感度和特异度的计算 支持了这一模型的可靠性。我们一期研究的结果表明，我们的体外迷你- 睾丸模型可能是一种有价值的筛查工具，用于生殖毒性评估和优先排序 用于进一步测试的化学物质。在这个第二阶段的提案中，我们将进一步审查和优化议定书，以 体外迷你睾丸模型。我们将测试Mini-Testis模型，并提供一个扩展的参考测试列表 化合物“，并盲目验证我们基于途径的高含量和高通量预测模型 站台。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 在体外迷你睾丸模型中与生殖毒性相关的结局途径；(2)应用 多参数高通量和高含量分析(HT/HCA)以检查灵敏度和 比较体外模型和体内生殖模型获得的IC50的特异性 最低观察不良反应水平(RLOAEL)以验证体外微型睾丸模型；(3)进行 实验室内和实验室间的重复性研究，以验证体外微型睾丸模型；我们将测试 测试内的可变性和实验室内和实验室间测试的重复性。总而言之，这 拟议的第二阶段将侧重于继续开发这一3D迷你睾丸筛查平台，以实现 验证了基于定量途径的HT/HCA生殖毒性筛查。将这一技术商业化 体外平台及其通过关注多个终点筛选多达1000个化合物的能力 与多种化学物质引起的不良生殖影响有关。这辆迷你- 基于睾丸模型的平台具有提供预测性生殖和发育毒性的潜力 在没有使用动物的情况下，高通量的格式将是综合测试的关键部分 生殖和发育毒性战略。

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.