Author

Pranav Joshi

Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering

Department

Washkewicz College of Engineering

First Advisor

Moo-Yeal Lee

Subject Headings

Biomedical Engineering, Neurosciences, Toxicology

Abstract

Only a few hundred of compounds, among tens of thousands of commercially available compounds, have been tested for developmental neurotoxicity (DNT) due to the limitations of current guidelines for DNT which are based entirely on in vivo experiments. In vivo studies are highly expensive and time-consuming, which often do not correlate to human outcomes. There is a key gap in our ability to predict in vivo outcomes accurately and robustly using in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brains. Conventional in vitro assays are typically performed in two-dimensional (2D) cell culture systems and use cytotoxicity assays that do not provide the information on mechanisms of toxicity. High-content imaging (HCI) assays performed on three-dimensional (3D) cell cultures can provide better understanding of mechanisms of toxicity needed to predict DNT in humans. However, current 3D cell culture systems lack the throughput required for screening DNT against a large number of chemicals. Thus, there is a need for cost-effective, high-throughput, alternative in vitro test methods based on mechanisms of toxicity. In this study, we first developed a miniaturized, 3D human NSC culture with ReNcell VM on the micropillar chip platform and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on human NSC spheroids to assess cell viability, self-renewal, and differentiation. Next, we identified major ion channels and ABC-transporters expressed in ReNcell VM via RNA-seq analysis and established high-throughput ion channel and ABC-transporter assays in 3D-cultured ReNcell VM on the 384-pillar plate. In the third step, we established high-content imaging (HCI) assays in 3D-cultured ReNcell VM with multiple assays which were tested with four model compounds. Finally, we established a high-throughput metabolism-mediated neurotoxicity testing system by combining 3D-cultured ReNcell VM on the 384PillarPlate and HepaRG spheroids in a ULA 384-well plate. Alternative in vitro systems for high-throughput neurotoxicity assessment established in this study will enable researchers to screen a library of test compounds with high confidence in terms of predictability of adverse reactions in vivo from those compounds.

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