High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.

Drops of water-in-fluorocarbon emulsions have great potential for compartmentalizing both in vitro and in vivo biological systems; however, surfactants to stabilize such emulsions are scarce. Here we present a novel class of fluorosurfactants that we synthesize by coupling oligomeric perfluorinated polyethers (PFPE) with polyethyleneglycol (PEG). We demonstrate that these block copolymer surfactants stabilize water-in-fluorocarbon oil emulsions during all necessary steps of a drop-based experiment including drop formation, incubation, and reinjection into a second microfluidic device. Furthermore, we show that aqueous drops stabilized with these surfactants can be used for in vitro translation (IVT), as well as encapsulation and incubation of single cells. The compatability of this emulsion system with both biological systems and polydimethylsiloxane (PDMS) microfluidic devices makes these surfactants ideal for a broad range of high-throughput, drop-based applications.

We use microfluidic devices to encapsulate, incubate, and manipulate individual cells in picoliter aqueous drops in a carrier fluid at rates of up to several hundred Hz. We use a modular approach with individual devices for each function, thereby significantly increasing the robustness of our system and making it highly flexible and adaptable to a variety of cell-based assays. The small volumes of the drops enables the concentrations of secreted molecules to rapidly attain detectable levels. We show that single hybridoma cells in 33 pL drops secrete detectable concentrations of antibodies in only 6 h and remain fully viable. These devices hold the promise of developing microfluidic cell cytometers and cell sorters with much greater functionality, allowing assays to be performed on individual cells in their own microenvironment prior to analysis and sorting.

Encapsulation of cells within picoliter-size monodisperse drops provides new means to perform quantitative biological studies on a single-cell basis for large cell populations. Variability in the number of cells per drop due to stochastic cell loading is a major barrier to these techniques. We overcome this limitation by evenly spacing cells as they travel within a high aspect-ratio microchannel; cells enter the drop generator with the frequency of drop formation.While drop-based microfluidics 1,2 promises breakthrough applications in biotechnology such as directed evolution, 3 tissue printing 4 and bead-based PCR in emulsions, 5 it also facilitates many quantitative studies of biology at the most fundamental level, that of single cells. Because each cell can be made to reside within its own picoliter-volume drop, chemically isolated from all other drops, cell-secreted molecules rapidly achieve detectable concentrations in the confined fluid surrounding the encapsulated cell. Similarly, uptake of trace chemicals specific to individual cells can be probed due to their depletion within the confined extracellular fluid. Moreover, highly monodisperse drops of water in an inert and immiscible carrier fluid can be formed at rates of several kHz using microfluidic techniques, 6 and these drops can be pairwise combined, 7 split in two, 8 and selected based on the contents of individual drops. 9,10 Despite their great potential, studies of single cells in drops suffer from an intrinsic limitation; because the process of loading cells into drops is purely random, the distribution is dictated by Poisson statistics. Thus, the probability of a drop containing k cells is λ k exp(−λ) / k!, where λ is the average number of cells per drop, so the ratio of drops containing one cell to those containing two is 2/λ. This means that to minimize the number of drops that contain more than a single cell requires very low average loading densities, meaning that most drops actually contain no cells whatsoever. This constraint significantly reduces the number of usable drops; for example, only 15.6 % of all drops will contain one cell if no more than one in ten of the occupied drops can be allowed to hold two or more cells.Recent work describes a method to passively sort drops containing single cells from smaller empty drops after each cell triggers its own encapsulation upon entering a narrow aqueous jet formed in a flow-focusing device. 11 This clever mechanism can also be used to sort cells based on their size since, for this system, drops are always slightly larger than the cell they contain; however, to overcome the inherent limitations of stochastic encapsulation of cells within controlled-size (monodisperse) drops, one (and only one) cell should be present whenever a drop is generated. This can be achieved manually for each drop, 12 or passively, and with a much higher throughput, by organizing cells in the direction of flow so that they enter the microfluidic nozzle with the same frequency at which drops form. * These autho...

We present a simple microfluidic device that uses an array of well-defined chambers to immobilize thousands of femtoliter- to picoliter-scale aqueous drops suspended in inert carrier oil. This device enables timelapse studies of large numbers of individual drops, while simultaneously enabling subsequent drop recovery.