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

总结 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。目前由监管机构设定的毒性测试方法主要依赖于 动物实验与用于风险评估的动物试验相关的成本和时间严重限制了 广泛的毒性筛选。这些数据缺口需要实施新的毒性测试愿景 正如国家研究理事会的报告“21世纪世纪的毒性测试”和 调整风险评估程序。世纪的毒性测试必须超越传统的 结合现代分子学知识的风险评估方法，如不良后果途径 （AOPs）与先进的体外模型。目前，体外生殖毒性试验模型正在积极研究中， 正在开发中。在R43 ES 027374基金的支持下，ReproTOX建立了一个Mini-Testis模型， 睾丸细胞系，并发现这种迷你睾丸模型可以识别生殖毒物。两种形态 并且细胞特异性生物标志物评估表明该共培养模型产生了体外类小生境， 形成了三维细胞骨架束结构，并在三维空间内支持生殖细胞。 环境我们通过区分32种化合物中的生殖毒物初步验证了该模型 并观察到体外IC 50和体外rLOAEL之间的强相关性（观察到的最低不良反应）， 效果水平）。一致性、灵敏度和特异性的计算进一步 证明了这个模型的可靠性。我们的I期研究结果表明，我们的体外迷你- 睾丸模型可能是一种有价值的生殖毒性评估筛选工具， 化学品进行进一步测试。在第二阶段的提案中，我们将进一步审查和优化协议， 体外小睾丸模型。我们将测试迷你睾丸模型与扩展列表的“参考测试 化合物”，并盲目验证我们的预测模型的途径为基础的高含量和高通量 平台具体目标是（1）完成基于不良反应的高含量测定的开发 在体外微型睾丸模型中与生殖毒性相关的结果途径;（2）应用 多参数高通量和高含量分析（HT/HCA），以检查灵敏度和 通过比较从体外模型和体内生殖模型获得的IC 50的特异性 最低观察到的不良反应水平（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现性研究，以验证体外微型睾丸模型;我们将测试 试验内变异性和试验在实验室内和实验室间的重现性。总之，这 拟议的第二阶段将集中于继续开发这个3D迷你睾丸筛查平台， 经验证的基于定量途径的HT/HCA生殖毒性筛查。商业化，在 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 与由各种化学类别的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有可能提供预测生殖和发育毒性 在高通量模式下不使用动物，并且将成为综合测试的关键部分。 生殖和发育毒性策略。

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.