High-throughput DNT testing with engineered brain organoids

Project Summary/Abstract Despite the potential vulnerability of developing brains to environmental toxicants, only a few hundred compounds, among tens of thousands of commercially used chemicals, have been tested for developmental neurotoxicity (DNT) due to the limitations of current US EPA and OECD guidelines for DNT assessment which rely entirely on animal tests. Therefore, there is a critical need for predictive cellular models for the assessment of DNT to rapidly prioritize potential environmental toxicants. To address this unmet need, Bioprinting Laboratories Inc. (BPL) proposes to establish genetically engineered, induced pluripotent stem cells (iPSCs) with a promoter-reporter assay system on a high- throughput, pillar/perfusion plate platform and differentiate them into brain organoids to mimic the complexity of brain development and accurately predict DNT potential by chemical exposure. Our pillar/perfusion plate platform consisting of a 144-pillar plate with sidewalls and slits (144PillarPlate) and a complementary 144-perfusion well plate with reservoirs and microchannels (144PerfusionPlate) can combine unique features of 3D bioprinting and microfluidic perfusion culture of organoids, while supporting high throughput and user-friendliness. In particular, the pillar/perfusion plate platform built on the footprint of standard 384-well plates is compatible with existing high-throughput screening (HTS) equipment such as automated fluorescent microscopes and microtiter well plate readers, which is an important feature for organoid-based compound screening. Unlike traditional microfluidic devices, the pillar plate with brain organoids can be easily detached from the perfusion plate and sandwiched onto a 384-well plate containing cell-staining reagents for high-throughput DNT assays. Our core hypotheses are: (i) iPSCs with a promoter-reporter assay system can be differentiated into engineered brain organoids to rapidly assess the expression levels of neural cell types, including neurons, astrocytes, and oligodendrocytes; (2) the pillar/perfusion plate can facilitate robust dynamic culture of brain organoids by eliminating the necrotic core of organoids and enable high-throughput, high-content cell function analysis. The specific aims of the proposed research are to: (1) generate human iPSCs with integrated promoter-reporter assay systems for rapid assessment of brain organoid differentiation; (2) generate engineered brain organoids with the promoter-reporter assay system in the pillar/perfusion plate and evaluate DNT potential of various compounds. We envision that engineered brain organoids with the promoter-reporter assay system in the pillar/perfusion plate could be used for predictive screening of environmental chemicals, which in turn can impact human toxicology, and ultimately, safer chemical product development and use.