Drug discovery is an extremely lengthy and expensive process. On average, drugs today cost more than $5 billion to develop, and take more than a decade to reach the market. Most drugs today are discovered using high throughput screening, in which millions of trial compounds are assayed against cells to determine if they interact with a disease target. This is typically performed using well-level screening techniques, such as fluorescence, chemiluminescence, and optical absorbance. While these techniques are high throughput, they lack the ability to interrogate wells at the single-cell level, which provides a much more comprehensive picture of the drug-cell interaction than population-averaged measurements. Flow cytometry is a well established and widely used technique used in most areas of cell biology that provides multi-parameter, cell-level information by measuring optical scatter and fluorescence information from individual cells in flow at a high throughput (~10,000 cells/second). While this cellular throughput is high, the sample throughput of flow cytometers is relatively low, when compared to fluorescence plate readers, for example, due to the serial sample handling approach typically employed by modern flow cytometers. If the sample throughput could be increased to appropriate levels, it would enable multi-parameter drug screening assays using flow cytometry, which would obviate certain further downstream assays in the drug discovery workflow (e.g. cytotoxicity assays). This improvement would increase the efficiency of drug discovery by better elucidating the complex drug-cell interactions, reducing the overall cost and time-to-market of new drugs. We propose here to develop a parallel flow cytometer system using a combination of two technologies developed in the laboratories of the co-PI's. Fluorescence Imaging using Radiofrequency-tagged Emission (FIRE) is a high-speed optical technique that enables a single photomultiplier tube detector to measure fluorescence or scatter from multiple points on a sample using radiofrequency-domain multiplexing. We will use a modified FIRE optical system to probe fluorescence and scatter from cells flowing in multiple parallel intertially focused streams, created by an Inertial Microfluidic Parallel Stream (IMPS) microfluidic chip. IMPS chips create ordered streams of cells using inertial flow field shaping without the complexity of multiple sheath fluids to direct cells, allowing high numerical aperture optics to detect fluorescence from all streams simultaneously. Combining these techniques, we will develop an optical and fluidic system capable of measuring multi-color flow cytometry data from 10 samples of cells at the same time. This order of magnitude increase in the sample throughput will transform the utility of flow cytometry in drug discovery, ultimately enabling its widespread use in high throughput screening (>100,000 wells/day). We will characterize the system (precision, linearity, sensitivity) using standard protocols, and perform a basic two-color apoptosis time course assay and compare our results in this assay to those obtained using a conventional commercial flow cytometer.
A flow cytometer capable of performing high content screening assays on large (>1 million) libraries of compounds by screening 10 samples in parallel overcomes a bottleneck in high throughput screening and drug discovery, speeding up the drug development process, allowing drugs to reach market more quickly to help patients and save lives.
