High-throughput assays using living cells have seen great advances, but the problem of evaporation makes reduction of volumes in microtiter plates difficult. The use of microfluidics eliminates evaporation, but other constraints of the system make it challenging to adequately isolate single living cells from one another so that each can be individually subjected to well-defined and independent assay conditions.

New research shows how combining microfluidics with emulsion technology allows manipulation and assaying of isolated single living cells and organisms. Emulsions are mixtures of two immiscible substances. In a water-in-oil emulsion, tiny droplets of water are surrounded by oil. By adding the components of a biochemical reaction to the water phase, each droplet can act as a tiny reaction vessel, allowing millions of individual reactions in a single tube.

Andrew Griffiths at Université Louis Pasteur has been using emulsions for years in directed evolution screens, but conventional emulsions have a problem. “The droplet sizes are quite polydisperse,” explains Griffiths. “This is problematic in directed evolution experiments because you can have droplets of different sizes with exactly the same gene inside but different phenotypes.” This can be problematic for other assays also, but work by others has shown that microfluidics devices can make monodisperse droplets. “Microfluidics allows you to do all sorts of clever things with droplets that are difficult to do otherwise,” says Griffiths. “For example you can split, fuse and sort droplets in a microfluidics chip and do it at very high speeds.”

Griffiths and his colleague Christoph Merten were interested in applying droplet-based microfluidics to miniaturize and increase the throughput of live-cell assays as part of the drug-discovery process. They started collaborating with David Weitz at Harvard University who was working on the physics of droplets in microfluidic systems.

Merten says: “The first interesting question was whether it is possible to encapsulate single cells and keep them alive in the drops.” This was highly dependent on the oil and surfactant used. They found that a perfluorocarbon oil and surfactant worked well (Fig. 1). Perfluorocarbon solvents provide several advantages. They do not cause swelling of the material in the microfluidic device, and they dissolve 20 times more oxygen than water. Finally, nonfluorinated molecules, such as the drug candidates they want to screen using droplet-based microfluidics, show almost no solubility in perfluorocarbons.

Figure 1: Creation of water droplets containing single living cells inside a microfluidic device.
figure 1

Image courtesy of Christoph Merten.

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Merten plans to use the system to screen for drugs that inhibit virus entry into cells. “For any cell-based assay you must guarantee that the cells survive during the assay period from a few hours to a few days,” notes Merten. Their results show that cells could survive and divide for several days in a single droplet in a microfluidic device.

The tiny size of the droplets greatly reduces the amount of reagents needed for assays. “The number of viral particles we need for a 96-well plate is sufficient to screen 107 samples in the droplet system,” remarks Merten. “We would not be able to produce enough viral particles to perform such a screen in a microtiter plate.” This nicely illustrates the value of this system for assays involving limiting reagents.

To test the limits of the system the researchers encapsulated living Caenorhabditis elegans worms within droplets in a small piece of tubing. A worm underwent a complete lifecycle in a droplet, showing that the droplet assay could be used for assays requiring testing of progeny.

This is just the first step on the way to using these microfluidic systems for doing high-throughput cell-based or small multicellular organism screens. So far all the droplets in the assays contain the same reagents, but Griffiths says “the next step is to establish strategies to allow us to encapsulate different drug candidates in the drops.”