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“The past decade has seen the rapid development of microfluidic devices for a variety of single-cell applications including live cell studies and molecular analysis.  In conjunction with time-lapse imaging, microfluidic systems provide a highly parallel platform for studying the behavior of individual cells in microchambers. “

Microfluidic Devices for Single-Cell Analysis

by Adam White

Genome Science and Technology, University of British Columbia, Vancouver, BC, Canada

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Cell-to-cell variability is one of the hallmarks of biological systems, from multi-cellular humans to microbial communities. This heterogeneity is present even in populations of genetically identical or functionally similar cells, which can exhibit fluctuations in protein production and asynchronous responses to stimulation. Despite cells being distinct units, much of our biological understanding is the result of techniques such as DNA sequencing or mass spectrometry which often require large numbers of cells and yield population-averaged measurements. Ensemble measurements obscure cellular heterogeneity and are blind to minority cell populations that are particularly relevant in systems such as stem cell development and the progression of disease. Currently single-cell analysis faces challenges overcoming cell handling, the limited amount of sample, and the labour and reagent costs associated with performing a large number of single-cell experiments [1]. Consequently, methods for high-throughput, cost-effective, single-cell analysis are needed in both research and clinical settings.

Advances in microfabrication techniques have given rise to integrated microfluidic circuits, capable of performing thousands of fluid operations in parallel and automating chemical reactions [2,3]. Microfluidic systems have a number of properties that make them well suited to analysis of individual cells [4]. By reducing the reaction vessels from conventional microliter (10-6 L) volumes to nanoliter (10-9 L) or picoliter (10-12 L) sizes, the concentration of contained molecules is increased. For example, a single molecule of DNA in a picoliter is at the equivalent concentration of 1 million DNA molecules in a microliter. This concentration enhancement leads to increased sensitivity and efficiency of many chemical reactions, such as the amplification of DNA by polymerase chain reaction. Decreasing reaction volumes reduces reagent consumption and associated costs, further facilitating high-throughput applications. Although the field of microfluidics is still maturing, components have been developed for a wide variety of fluid manipulations including microchannel pipes, valves [5], pumps, mixers, and droplet generators [6]. Borrowing processes developed in the microelectronics industry, microfluidic structures can be fabricated in materials ranging from glass and silicon, to plastics and elastic polymers.

The past decade has seen the rapid development of microfluidic devices for a variety of single-cell applications including live cell studies and molecular analysis. In conjunction with time-lapse imaging, microfluidic systems provide a highly parallel platform for studying the behavior of individual cells in microchambers. In particular, the fluid handling of these systems permits precise spatial control of the chemical environment inside each chamber. This approach has been used to study how cells respond to spatial gradients of pheromone signals, or to stimulation conditions that change over time [7–9]. For example, Lecault et al. exposed hematopoietic (blood-cell generating) stem cells to time-varying concentrations of a signaling molecule and found that it was required for survival specifically when cells entered the cell-cycle [10]. Microfluidic devices can also aid in the development of pharmaceutical agents by exposing large numbers of single cells to different drug formulations in a high-throughput fashion [11]. An important consequence of containing cells in sub-nanoliter volumes is that they are able to rapidly affect their own environment with secreted proteins. This has been valuable in studying immune cells, where secreted proteins (such as antibodies) from individual cells are quickly able to reach analyzable concentrations [12].

In addition to live cell experiments, microfluidic devices are increasingly being applied to high-throughput genetic analysis of single cells. Adoption of this technology has been spurred by commercially available microfluidic systems (e.g. the Dynamic Array™ from Fluidigm) that perform thousands of low-volume polymerase chain reactions (PCR), and permit the cost-effective measurement of multiple genes from single cells. Single-cell genetic analysis is particularly applicable to studying diseases such as cancer, where cellular heterogeneity may reveal genetic patterns relevant to the origins of disease or treatment strategies [13,14]. For example, White et al. used microfluidic single-cell PCR to measure the fraction of cells in a breast cancer tumor sample that have a specific mutation in their DNA sequence [15]. Similarly, single-cell analysis has also revealed distinct cell sub-populations within colon cancer tissues [16], and that a subset of breast tumor cells exhibit increased activity of genes associated with resistance to radiation therapy [17]. Recently, White et al. developed a microfluidic device that incorporated cell trapping and lysis components into an integrated system for performing PCR analysis on hundreds of individual cells (in a design that can be scaled to thousands) (Figure 1) [15]. In addition to increased sensitivity in small volumes, mixing samples and reagents within a closed microfluidic device suppresses contamination, and reduces technical noise associated with pipetting [18]. Reaction products from microfluidic devices may be recovered for downstream analysis such as DNA sequencing. This approach has been demonstrated in the establishment of a patient’s genetic sequence from single cell chromosome fragments [19], as well as uncovering the previously unknown genome of a microorganism from a complex environmental sample [20].

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Figure 1. (A) A microfluidic device for performing 300 single cell gene expression measurements . (B) View of microfluidic architecture from (A) with dyes highlighting fluid paths and reaction chambers (blue) and channels on an adjacent layer (red) used to valve the fluid paths. Scale bar: 1 mm.

In summary, microfluidic systems provide numerous advantages to single cell analysis including precise fluid and cell manipulation, increased sensitivity, economy of scale, automation and parallelization. Additional developments involve integration of microfluidics with surface patterning and active electronics (such as heating elements and chemical or optical sensors), as well as using microfluidic single-cell processing as a front-end to a variety of analytical techniques. Further innovation is currently needed in order to perform high-throughput measurements of proteins, metabolites, and epigenetic states in single cells, however the pace of development is encouraging. The demonstration of microfluidic single-cell analysis in applications ranging from human genotyping and drug screening, to genetic measurements in stem cell development and cancer progression, should lead to widespread adoption of this ‘lab-on-a-chip’ technology.

References: visit INK journal website

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