Development of a multiplexed, high content imaging-based assay for assessing chemical effects on proliferation and...

Collaborative research between the US EPA, European research institutes, and the OECD has resulted in a developmental neurotoxicity in vitro testing battery (DNT-IVB) that assesses multiple biological processes that are critical for neurodevelopment. The DNT-IVB was developed to address the large number of chemicals that have not been tested in time- and resource-intensive in vivo DNT guideline studies. In keeping with recommendations that the DNT-IVB should evolve with the science, this work has taken two 96-well DNT-IVB assays that independently measure human neural progenitor cell proliferation or apoptosis and combined them into a multiplexed, 384-well assay that simultaneously measures proliferation, apoptosis, and cell viability. The 384-well assay and accompanying data analysis pipeline were developed and optimized, then a total of 315 chemicals were screened. Robust Z-prime and strictly standardized mean difference values indicated that the 384-well assay was excellent for both proliferation and apoptosis endpoints, improving upon the 96-well assays. Out of the 315 chemicals, 158 had been assessed in the original 96-well assays. The multiplexed assay produced highly comparable results to the original 96-well assays in terms of activity, potency, sensitivity and specificity, and identified more chemicals as selective for the proliferation endpoint. Multiplexed assay activity calls generally matched 96-well assay activity calls. With comparable performance to the 96-well assays but with significantly improved throughput, the multiplexed, 384-well assay is proposed as an updated alternative to the existing 96-well proliferation and apoptosis assays that are included in the DNT-IVB.