Single cell analysis

Genetic and biochemical analysis of single cells in areas of basic biology where it is difficult or impossible to use conventional methods: stem cells, other primary cells, unculturable microbes, and developing organisms. There is anecdotal evidence from a number of labs that microfluidic environments provide a more “in-vivo like” environment than conventional cell culture, especially for primary cells. Suggestions to explain this phenomenon range from high surface area to volume ratios yielding more efficient aeration and gas transfer to confined environments having higher effective concentrations of extracellular signaling molecules. We would like to rigorously understand these effects and try to use them in microfluidic tools that could be applied to virtually all areas of cell biology. We would also like to explore the use of microfluidic tools to eliminate the need for cell culture altogether, especially with respect to the large fraction (estimated at 99%) of microbial diversity that cannot be grown in culture. We are beginning to address questions of this nature in collaborations with investigators at the U.S.C. Medical School , Stanford Medical School , and Caltech, and have been able to demonstrate microfluidic chips that perform parallel single cell gene expression measurements (1), as well as chips that perform parallel single cell biochemical analysis (2). (See Figure 1 for an example of the latter.)

Figure 1: A. Optical micrograph of the Multiwell High Throughput Screening Chip (MHTSC), also referred to as a comparator array. This chip has 2,056 valves with only 18 valve control lines needed to interface to the outside world. It is used in high throughput screening applications and allows a complex sample to be segmented into many compartments; each compartment can then be assayed individually by pairwise mixing with another set of compartments containing, for example, fluorescent substrate. After interrogation of the chip, any single chamber can be selected and its contents recovered from the chip. The chip occupies a total area of one square inch. In this image, the various channels of the chip are filled with food dyes. This chip (without food dyes) was shown on the cover of the October 18, 2002 issue of Science . B. Portion of an image of the MHTSC chip taken with a DNA array scanner. A dilute sample of E. Coli bacteria expressing cytochrome C peroxidase (CCP) was segmented and then allowed to mix pairwise with the substrate Amplex Red. When an input chamber contains cells expressing CCP, nonfluorescent Amplex Red is converted to the fluorescent product resorufin. Arrows indicate compartments having single bacteria, as verified by optical microscopy. Chambers without cells show low fluorescence, while those containing cells have high fluorescence. The chip thus functions as a comparator to measure CCP activity.

1. J.W. Hong, V. Studer, G. Hang, W.F. Anderson, and S.R. Quake, “A nanoliter-scale nucleic acid processor with parallel architecture”, Nature Biotech. 22: 435-439 (2004).

2. T. Thorsen, S.J. Maerkl, and S.R. Quake, "Microfluidic Large Scale Integration", Science 298: 580-584 (2002).