Date of Award

2018

Degree Type

Thesis

Degree Name

Master of Science in Chemical Engineering

Department

Chemical and Biomedical Engineering

First Advisor

Lee, Moo-Yeal

Subject Headings

Biomedical Engineering, Chemical Engineering

Abstract

The U.S Environmental Protection Agency (EPA) launched the Transform Tox Testing Challenge in 2016 with the goal of developing practical methods that can be integrated into conventional high-throughput screening (HTS) assays to better predict the toxicity of parent compounds and their metabolites in vivo. In response to this need and to retrofit existing HTS assays for assessing metabolism-induced toxicity of compounds, we have developed a 384-pillar plate that is complementary to traditional 384-well plates and ideally suited for culturing human cells in three dimensions (3D) at a microscale. Briefly, human embryonic kidney (HEK) 293 cells in a mixture of alginate and Matrigel were printed on the 384-pillar plates using a microarray spotter. These cells were then coupled with 384-well plates containing nine model compounds provided by the EPA, five representative Phase I and II drug metabolizing enzymes (DMEs), and one no enzyme control. Membrane integrity and viability of HEK 293 cells were measured with the calcein AM and CellTiter-Glo® kit, respectively, to determine the IC50 values of the nine parent compounds and DME generated metabolites. Out of the nine compounds tested, six compounds showed augmented toxicity with DMEs and one compound showed detoxification with a Phase II DME. This result indicates that the 384-pillar plate platform can be used to measure metabolism-induced toxicity of compounds with high predictivity. In addition, the Z’ factors and the coefficient of variation (CV) measured were above 0.6 and below 14%, respectively, indicating that the assays established on the 384-pillar plate are robust and reproducible.

Download
COinS