Numerous opportunities exist to apply microfluidic technology to high-throughput and high-content cell-based assays. However, maximizing the value of these assays for drug discovery, screening or toxicity evaluation, for example, will require validation of assay performance due to within device, day-to-day, and interlaboratory variability. This report involves a study of the repeatability, reproducibility, and robustness of a cell-based assay for reporting ribosome activity in microfluidic devices made of poly(dimethylsiloxane) (PDMS). A previously described toxicity assay that involves expression of green fluorescent protein (GFP) is employed as a reporter of intracellular protein synthesis and degradation. Variability of GFP loss due to ribosome inhibition by cycloheximide (CHX) was quantified with real-time quantitative imaging across 8 duplicate columns of chambers (8 rows in each column) within a 64-chamber microfluidic device. Assay performance across a range of cell loading densities within a single device was assessed, as was replication of measurements in microfluidics devices prepared on different days. Assay robustness was tested by using different fluorescence illumination sources and reservoir-to-device tubing choices, which we found could affect assay readout. The microfluidic measurements were also compared with results from large volume conditions in conventional petri dishes. Overall, the GFP expression by the indicator cell line under CHX toxicity was found to be equivalent within microfluidic devices and rather insensitive to cell-plating density. Even though proliferation rates between microfluidic and the dish cultures were different, replicate microfluidic devices and bare and PDMS-coated dish culture systems showed comparable GFP decay rates upon toxicity. In each case, the mean exponential decay time constant was found to be approximately 240 minutes, though the response to toxin could be easily detected within the first 60 minutes.
Citation: Analytical Chemistry
Pub Type: Journals
reproducibility, repeatability, replicate, microfluidics, lab-on-a-chip, image analysis, validation, quantitative, in vitro, cytotoxicity, reporter cell line, cell-based assay