Development of a multiplexed, high content imaging-based assay for assessing chemical effects on proliferation and apoptosis in human neural progenitor cells

Abstract

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.