Development of high throughput, mammalian cell-based metabolic rate assays for screening compounds

Abstract

Functional characterization of diseased cellular metabolic state is necessary to complement current methods of in vitro toxicity testing. This dissertation focused on development of high throughput assays in 24- and 96-well plates for the determination of metabolic rates of mammalian cells and demonstrated their uses for screening chemicals. First, a 90-min assay based on spectrophotometric detection of phenol red was developed in 24-well plates for measuring acidification rates. The assay is simple, inexpensive, and suitable for rapid screening of short-term metabolic effects of chemicals. Next, a second assay was developed for measuring CO2 production rate in a 24-well plate with wells sealed by custom made plugs. This assay was demonstrated to be more sensitive for toxicity testing than those based on cell death events, and has comparable sensitivity with cell counting to measure cell growth inhibition. Inspired by the finding that changes in metabolic rates can occur at lower concentrations than those causing cell death, a 96-well plate assay which combines resazurin reduction with lactate production and glucose consumption rate assays was designed to assess effects of compounds on both culture viability and metabolism during 6- or 24-hr exposure. The screen provides a simple and inexpensive way to determine concentrations over which a compound has lethal, sublethal metabolic, or no such effects. It should serve as a cost-effective and time-saving first step in a given research study, preceding more intricate experimental methods. Assays developed above paved the way for a more detailed investigation of changes in cellular metabolism resulting from toxic stress. Finally, a 24-well plate assay for simultaneously measuring changes in glucose, lactate, and CO2 / pH was synthesized based on the previously developed assays. It was used in conjunction with a carbon balance model for characterizing changes in the metabolic state of fibroblast cells treated with chemicals at sublethal levels. In response to four chemicals at different doses, cells responded by altering metabolic state in three different ways. Moreover, the sensitivities of responses of metabolic rates were associated with the closeness of the pathways to the action site of a compound.