Culbreth Megan, Nyffeler Johanna, Willis Clinton, Harrill Joshua A
Center for Computational Toxicology and Exposure, Office of Research and Development, U.S. Environmental Protection Agency, Durham, NC, United States.
Oak Ridge Institute for Science and Education (ORISE) Postdoctoral Fellow, Oak Ridge, TN, United States.
Front Toxicol. 2022 Feb 16;3:803987. doi: 10.3389/ftox.2021.803987. eCollection 2021.
Studies in rodent models have been the accepted approach by regulatory agencies to evaluate potential developmental neurotoxicity (DNT) of chemicals for decades. These studies, however, are inefficient and cannot meet the demand for the thousands of chemicals that need to be assessed for DNT hazard. As such, several new approach methods (NAMs) have been developed to circumvent limitations of these traditional studies. The DNT NAMs, some of which utilize human-derived cell models, are intended to be employed in a testing battery approach, each focused on a specific neurodevelopmental process. The need for multiple assays, however, to evaluate each process can prolong testing and prioritization of chemicals for more in depth assessments. Therefore, a multi-endpoint higher-throughput approach to assess DNT hazard potential would be of value. Accordingly, we have adapted a high-throughput phenotypic profiling (HTPP) approach for use with human-derived neural progenitor (hNP1) cells. HTPP is a fluorescence-based assay that quantitatively measures alterations in cellular morphology. This approach, however, required optimization of several laboratory procedures prior to chemical screening. First, we had to determine an appropriate cell plating density in 384-well plates. We then had to identify the minimum laminin concentration required for optimal cell growth and attachment. And finally, we had to evaluate whether addition of antibiotics to the culture medium would alter cellular morphology. We selected 6,000 cells/well as an appropriate plating density, 20 µg/ml laminin for optimal cell growth and attachment, and antibiotic addition in the culture medium. After optimizing hNP1 cell culture conditions for HTPP, it was then necessary to select appropriate in-plate assay controls from a reference chemical set. These reference chemicals were previously demonstrated to elicit unique phenotypic profiles in various other cell types. Aphidicolin, bafilomycin A1, berberine chloride, and cucurbitacin I induced robust phenotypic profiles as compared to dimethyl sulfoxide vehicle control in the hNP1 cells, and thus can be employed as in-plate assay controls for subsequent chemical screens. We have optimized HTPP for hNP1 cells, and consequently this approach can now be assessed as a potential NAM for DNT hazard evaluation and results compared to previously developed DNT assays.
几十年来，啮齿动物模型研究一直是监管机构评估化学品潜在发育神经毒性（DNT）的公认方法。然而，这些研究效率低下，无法满足对数千种需要评估DNT危害的化学品的评估需求。因此，已经开发了几种新方法（NAMs）来规避这些传统研究的局限性。DNT NAMs中的一些利用人源细胞模型，旨在用于测试组合方法，每种方法都专注于特定的神经发育过程。然而，评估每个过程需要多种测定，这可能会延长化学品测试和更深入评估的优先级排序。因此，一种用于评估DNT危害潜力的多终点高通量方法将具有价值。因此，我们采用了高通量表型分析（HTPP）方法用于人源神经祖细胞（hNP1）。HTPP是一种基于荧光的测定方法，可定量测量细胞形态的变化。然而，这种方法在化学筛选之前需要优化几个实验室程序。首先，我们必须确定384孔板中合适的细胞接种密度。然后，我们必须确定最佳细胞生长和附着所需的最低层粘连蛋白浓度。最后，我们必须评估向培养基中添加抗生素是否会改变细胞形态。我们选择每孔6000个细胞作为合适的接种密度，20μg/ml层粘连蛋白用于最佳细胞生长和附着，并在培养基中添加抗生素。在为HTPP优化hNP1细胞培养条件后，接下来有必要从参考化学品组中选择合适的板内测定对照。这些参考化学品先前已证明在各种其他细胞类型中引发独特的表型谱。与hNP1细胞中的二甲基亚砜载体对照相比，阿非科林、巴弗洛霉素A1、氯化小檗碱和葫芦素I诱导了强大的表型谱，因此可作为后续化学筛选的板内测定对照。我们已经为hNP1细胞优化了HTPP，因此现在可以将这种方法评估为一种潜在的NAM用于DNT危害评估，并将结果与先前开发的DNT测定进行比较。
