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Publication Title | Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells

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Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells

Max Chabert*† and Jean-Louis Viovy*†‡

*Institut Curie, Section de Recherche, 26, rue d’Ulm, 75005 Paris, France; and †Centre National de la Recherche Scientifique, Unité Mixte de Recherche 168, 75005 Paris, France

Edited by Howard Brenner, Massachusetts Institute of Technology, Cambridge, MA, and approved December 28, 2007 (received for review September 3, 2007)

We present a purely hydrodynamic method for the high-through- put encapsulation of single cells into picoliter droplets, and spon- taneous self-sorting of these droplets. Encapsulation uses a cell-triggered Rayleigh–Plateau instability in a flow-focusing geo- metry, and self-sorting puts to work two extra hydrodynamic mechanisms: lateral drift of deformable objects in a shear flow, and sterically driven dispersion in a compressional flow. Encapsulation and sorting are achieved on-flight in continuous flow at a rate up to 160 cells per second. The whole process is robust and cost- effective, involving no optical or electrical discrimination, active sorting, flow switching, or moving parts. Successful encapsulation and sorting of 70 – 80% of the injected cell population into drops containing one and only one cell, with <1% contamination by empty droplets, is demonstrated. The system is also applied to the direct encapsulation and sorting of cancerous lymphocytes from a whole blood mixture, yielding individually encapsulated cancer cells with a >10,000-fold enrichment as compared with the initial mix. The method can be implemented in simple ‘‘soft lithography’’ chips, allowing for easy downstream coupling with microfluidic cell biology or molecular biology protocols.

droplets Rayleigh–Plateau size-fractionnation digital microfluidics

The postgenome era is stimulating a strong demand for high-throughput cell assays. Because genetically identical cells may display very heterogeneous behaviors (1, 2), bulk measurements on cell populations provide only partial informa- tion on cell metabolism, in particular from a dynamic point of view. Cell-to-cell variability is also of paramount importance for cancer research, developmental biology, drug screening (3), and stem cell research. Recent trends in cell biology thus put strong emphasis on studies at the single-cell level (4).

Microtechnologies raise the hope of dramatic breakthroughs in this field (5). For instance, compartmentalization of single cells in microchambers allows the analysis of stochastic protein expression at the single molecule level (6). In another approach, the capture of cells in microdroplets within double emulsions enables the screening of enzyme libraries with an unprecedented resolution and speed (7). In this perspective, combining encap- sulation within droplets with microfluidic techniques may allow the observation and analysis of individual cells (e.g., with drugs or reagents) in a fully automated, time-resolved manner. These operations can be achieved using optical traps, but this approach remains complex and is hardly amenable to high throughput (8). In contrast, the use of classical continuous microdroplets gene- ration techniques such as flow-focusing (9) or break-up at a T-junction (10) from a cell suspension offers potential for very high throughput, but could so far not warrant a controlled distribution of the cells in the drops (11). In these methods, the number of cells contained in the formed drops is dictated by the probability that a given volume of the initial cell suspension contains a given number of cells, following a Poisson distribu- tion. It imposes a rather unfavorable compromise between the rate of encapsulation (total number of ‘‘positive drops,’’ con- taining one and only one cell, created per second) and the yield

(fraction of the initial cell population ending up in a positive drop). A suspension containing one cell per three drops volume on average is generally used, resulting in an 22% rate of encapsulation for a 75% yield. To obtain reasonably pure populations of ‘‘positive’’ droplets, the steady-state generation of droplets from the suspension of cells is thus coupled with a detection and sorting of ‘‘positive’’ droplets by flow-cytometry- like technologies (12, 13). Active sorting can operate at very high frequencies, so that the presence of negative droplets does not raise serious problems regarding throughput, but it considerably increases the complexity and cost of the process. Also, in real systems, a perfectly random cell suspension is hard to achieve because of rapid sedimentation of cells in containers and pinning effects in microchannels (14). Therefore, the main issue when using these encapsulation devices in a routine mode lies in the number of droplets that contain no more than one cell. In summary, despite their strong interest for cell biology, current single-cell encapsulation processes remain delicate to tune and to maintain.

Here, we propose a different approach, relying entirely on passive hydrodynamic effects, in which picoliter droplets con- taining a single cell are prepared and self-sorted with high purity and yield. This system works even with highly concentrated suspensions, whatever the distribution of cells in the solution. It uses a triggered Rayleigh–Plateau instability in a jet flow (15), followed by shear-induced drift and excluded-volume-driven dispersion of the individual droplets. Its performance is char- acterized experimentally using a pure population of T- lymphocytes and described semi-quantitatively. We also dem- onstrate the robustness of the method for practical applications by directly encapsulating and sorting cancerous T-lymphocytes out of a whole-blood mixture. This method opens the route toward robust, low-cost and high-throughput molecular biology assays and diagnosis on single cells.

Design Principles

Fluidic Design. The device consists in (i) a flow focusing region (9); (ii) a narrowed straight channel; and (iii) an expansion finally splitting into two symmetric outlets (Fig. 1A). By convention, ‘‘right’’ and ‘‘left’’ sides of the microfluidic channels are refe- renced to the flow direction. The stream of culture medium containing the cells is introduced in the center channel and is focused into the narrow channel downstream by two co-flowing streams of oil. The flow rate of the left oil stream Qo is twice that of the right oil stream Qo/2, and the flow rate of the central

Author contributions: M.C. and J.-L.V. designed research; M.C. performed research; M.C. and J.-L.V. analyzed data; and M.C. and J.-L.V. wrote the paper.

Conflict of interest statement: The authors have a pending patent on the technology presented in this manuscript.

This article is a PNAS Direct Submission.

‡To whom correspondence should be addressed. E-mail: jean-louis.viovy@curie.fr.

This article contains supporting information online at www.pnas.org/cgi/content/full/ 0708321105/DC1.

© 2008 by The National Academy of Sciences of the USA

ENGINEERING CELL BIOLOGY

www.pnas.org cgi doi 10.1073 pnas.0708321105

PNAS March 4, 2008 vol. 105 no. 9 3191–3196

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