Highly multiplexed single-cell quantitative PCR.

We present a microfluidic device for rapid gene expression profiling in single cells using multiplexed quantitative polymerase chain reaction (qPCR). This device integrates all processing steps, including cell isolation and lysis, complementary DNA synthesis, pre-amplification, sample splitting, and measurement in twenty separate qPCR reactions. Each of these steps is performed in parallel on up to 200 single cells per run. Experiments performed on dilutions of purified RNA establish assay linearity over a dynamic range of at least 104, a qPCR precision of 15%, and detection sensitivity down to a single cDNA molecule. We demonstrate the application of our device for rapid profiling of microRNA expression in single cells. Measurements performed on a panel of twenty miRNAs in two types of cells revealed clear cell-to-cell heterogeneity, with evidence of spontaneous differentiation manifested as distinct expression signatures. Highly multiplexed microfluidic RT-qPCR fills a gap in current capabilities for single-cell analysis, providing a rapid and cost-effective approach for profiling panels of marker genes, thereby complementing single-cell genomics methods that are best suited for global analysis and discovery. We expect this approach to enable new studies requiring fast, cost-effective, and precise measurements across hundreds of single cells.

Micro-ribonucleic acid-155 is a direct target of Meis1, but not a driver in acute myeloid leukemia.

Micro-ribonucleic acid-155 (miR-155) is one of the first described oncogenic miRNAs. Although multiple direct targets of miR-155 have been identified, it is not clear how it contributes to the pathogenesis of acute myeloid leukemia. We found miR-155 to be a direct target of Meis1 in murine Hoxa9/Meis1 induced acute myeloid leukemia. The additional overexpression of miR-155 accelerated the formation of acute myeloid leukemia in Hoxa9 as well as in Hoxa9/Meis1 cells in vivo However, in the absence or following the removal of miR-155, leukemia onset and progression were unaffected. Although miR-155 accelerated growth and homing in addition to impairing differentiation, our data underscore the pathophysiological relevance of miR-155 as an accelerator rather than a driver of leukemogenesis. This further highlights the complexity of the oncogenic program of Meis1 to compensate for the loss of a potent oncogene such as miR-155. These findings are highly relevant to current and developing approaches for targeting miR-155 in acute myeloid leukemia.

Scalable whole-genome single-cell library preparation without preamplification.

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates.

The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10(-6) and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.

Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures.

Heterogeneity in the clonal outputs of individual human embryonic stem cells (hESCs) confounds analysis of their properties in studies of bulk populations and how to manipulate them for clinical applications. To circumvent this problem we developed a microfluidic device that supports the robust generation of colonies derived from single ESCs. This microfluidic system contains 160 individually addressable chambers equipped for perfusion culture of individual hESCs that could be shown to match the growth rates, marker expression and colony morphologies obtained in conventional cultures. Use of this microfluidic device to analyze the clonal growth kinetics of multiple individual hESCs induced to differentiation revealed variable shifts in the growth rate, area per cell and expression of OCT4 in the progeny of individual hESCs. Interestingly, low OCT4 expression, a slower growth rate and low nuclear to cytoplasmic ratios were found to be correlated responses. This study demonstrates how microfluidic systems can be used to enable large scale live-cell imaging of isolated hESCs exposed to changing culture conditions, to examine how different aspects of their variable responses are correlated.

Methods for multiplex template sampling in digital PCR assays.

The efficient use of digital PCR (dPCR) for precision copy number analysis requires high concentrations of target molecules that may be difficult or impossible to obtain from clinical samples. To solve this problem we present a strategy, called Multiplex Template Sampling (MTS), that effectively increases template concentrations by detecting multiple regions of fragmented target molecules. Three alternative assay approaches are presented for implementing MTS analysis of chromosome 21, providing a 10-fold concentration enhancement while preserving assay precision.

Microfluidic-based manufacture of siRNA-lipid nanoparticles for therapeutic applications.

A simple, efficient, and scalable manufacturing technique is required for developing siRNA-lipid nanoparticles (siRNA-LNP) for therapeutic applications. In this chapter we describe a novel microfluidic-based manufacturing process for the rapid manufacture of siRNA-LNP, together with protocols for characterizing the size, polydispersity, RNA encapsulation efficiency, RNA concentration, and total lipid concentration of the resultant nanoparticles.

Dissecting genealogy and cell cycle as sources of cell-to-cell variability in MAPK signaling using high-throughput lineage tracking.

Cells, even those having identical genotype, exhibit variability in their response to external stimuli. This variability arises from differences in the abundance, localization, and state of cellular components. Such nongenetic differences are likely heritable between successive generations and can also be influenced by processes such as cell cycle, age, or interplay between different pathways. To address the contribution of nongenetic heritability and cell cycle in cell-to-cell variability we developed a high-throughput and fully automated microfluidic platform that allows for concurrent measurement of gene expression, cell-cycle periods, age, and lineage information under a large number of temporally changing medium conditions and using multiple strains. We apply this technology to examine the role of nongenetic inheritance in cell heterogeneity of yeast pheromone signaling. Our data demonstrate that the capacity to respond to pheromone is passed across generations and that the strength of the response correlations between related cells is affected by perturbations in the signaling pathway. We observe that a ste50Δ mutant strain exhibits highly heterogeneous response to pheromone originating from a unique asymmetry between mother and daughter response. On the other hand, fus3Δ cells were found to exhibit an unusually high correlation between mother and daughter cells that arose from a combination of extended cell-cycle periods of fus3Δ mothers, and decreased cell-cycle modulation of the pheromone pathway. Our results contribute to the understanding of the origins of cell heterogeneity and demonstrate the importance of automated platforms that generate single-cell data on several parameters.

Microfluidic integration of parallel solid-phase liquid chromatography.

We report the development of a fully integrated microfluidic chromatography system based on a recently developed column geometry that allows for robust packing of high-performance separation columns in poly(dimethylsiloxane) microfluidic devices having integrated valves made by multilayer soft lithography (MSL). The combination of parallel high-performance separation columns and on-chip plumbing was used to achieve a fully integrated system for on-chip chromatography, including all steps of automated sample loading, programmable gradient generation, separation, fluorescent detection, and sample recovery. We demonstrate this system in the separation of fluorescently labeled DNA and parallel purification of reverse transcription polymerase chain reaction (RT-PCR) amplified variable regions of mouse immunoglobulin genes using a strong anion exchange (AEX) resin. Parallel sample recovery in an immiscible oil stream offers the advantage of low sample dilution and high recovery rates. The ability to perform nucleic acid size selection and recovery on subnanogram samples of DNA holds promise for on-chip genomics applications including sequencing library preparation, cloning, and sample fractionation for diagnostics.

Fabrication of high-quality microfluidic solid-phase chromatography columns.

Here we report a low-pressure bead packing technique for the robust integration of high-performance chromatography columns in poly(dimethylsiloxane) microfluidic devices made by multilayer soft lithography (MSL). A novel column geometry featuring micrometer-sized bypass channels along the entire length of the separation channel is used to achieve rapid packing of multiple high-quality bead bed columns in parallel with near-perfect yield. Pulse tests show that these microfluidic columns achieve exceptional reproducibility and efficiency, with measured plate counts of 1,650,000/m ± 7%, corresponding to a reduced plate height of h = 0.12 ± 7%. The combination of high-performance chromatography columns and valve-based microfluidics offers new opportunities for the integration of sample processing with preparative and analytical separations for biology and chemistry.

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core.

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.

Purification and quantitation of bacteriophage M13 using desalting spin columns and digital PCR.

Separation of small molecules such as biotinylated baits from solutions of filamentous bacteriophage is achieved generally through polyethylene glycol precipitation of the phage and centrifugation prior to affinity selection or panning. This method is laborious and time-consuming and is accompanied frequently by significant loss of virions, especially when performed at low phage concentrations. Similarly, accurate quantitation of phage is performed typically by counting plaques, a method that is tedious, low-throughput, and not amenable easily to high titers. In this report it is demonstrated that commercially available Zeba Spin Desalting Columns are useful devices for the efficient separation of small molecules from bacteriophage, which pass through almost unimpeded and remain infectious. It is shown further that digital PCR on microfluidic chips is a fast and accurate high-throughput technique to determine phage genome concentrations precisely.

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities.

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by "flow-controlled wetting," a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.

Microfluidic single cell analysis: from promise to practice.

Methods for single-cell analysis are critical to revealing cell-to-cell variability in biological systems, especially in cases where relevant minority cell populations can be obscured by population-averaged measurements. However, to date single cell studies have been limited by the cost and throughput required to examine large numbers of cells and the difficulties associated with analyzing small amounts of starting material. Microfluidic approaches are well suited to resolving these issues by providing increased senstitivity, economy of scale, and automation. After many years of development microfluidic systems are now finding traction in a variety of single-cell analytics including gene expression measurements, protein analysis, signaling response, and growth dynamics. With newly developed tools now being applied in fields ranging from human haplotyping and drug discovery to stem cell and cancer research, the long-heralded promise of microfluidic single cell analysis is now finally being realized.

Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA.

Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.

Parylene C coating for high-performance replica molding.

This paper presents an improvement to the soft lithography fabrication process that uses chemical vapor deposition of poly(chloro-p-xylylene) (parylene C) to protect microfabricated masters and to improve the release of polymer devices following replica molding. Chemical vapor deposition creates nanometre thick conformal coatings of parylene C on silicon wafers having arrays of 30 µm high SU8 pillars with densities ranging from 278 to 10,040 features per mm(2) and aspect ratios (height : width) from 1 : 1 to 6 : 1. A single coating of parylene C was sufficient to permanently promote poly(dimethyl)siloxane (PDMS) mold release and to protect masters for an indefinite number of molding cycles. We also show that the improved release properties of parylene treated masters allow for fabrication with hard polymers, such as poly(urethane), that would otherwise not be compatible with SU8 on silicon masters. Parylene C provides a robust and high performance mold release coating for soft lithography microfabrication that extends the life of microfabricated masters and improves the achievable density and aspect ratio of replicated features.

Ontogeny stage-independent and high-level clonal expansion in vitro of mouse hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor.

Achieving high-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Here, we couple the ability of engineered NUP98-HOXA10hd expression to stimulate > 1000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin(-)Sca-1(+)c-kit(+) cells, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. We find that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at ∼ 60% to 90% unit efficiency in cultures initiated with single cells. Clonally expanded HSCs consistently show balanced long-term contributions to the lymphoid and myeloid lineages without evidence of leukemogenic activity. Although effects on fetal and adult HSCs were indistinguishable, NUP98-HOXA10hd-transduced adult HSCs did not thereby gain a competitive advantage in vivo over freshly isolated fetal HSCs. Live-cell image tracking of single transduced HSCs cultured in a microfluidic device indicates that NUP98-HOXA10hd does not affect their proliferation kinetics, and flow cytometry confirmed the phenotype of normal proliferating HSCs and allowed reisolation of large numbers of expanded HSCs at a purity of 25%. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.

High-throughput microfluidic single-cell RT-qPCR.

A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity. We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells. The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed.

Megapixel digital PCR.

We present a microfluidic 'megapixel' digital PCR device that uses surface tension-based sample partitioning and dehydration control to enable high-fidelity single DNA molecule amplification in 1,000,000 reactors of picoliter volume with densities up to 440,000 reactors cm(-2). This device achieves a dynamic range of 10(7), single-nucleotide-variant detection below one copy per 100,000 wild-type sequences and the discrimination of a 1% difference in chromosome copy number.