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Cell–cell communication and physical interactions play a vital role in cancer initiation, homeostasis, progression, and immune response. Here, we report a system that combines live capture of different cell types, co-incubation, time-lapse imaging, and gene expression profiling of doublets using a microfluidic integrated fluidic circuit that enables measurement of physical distances between cells and the associated transcriptional profiles due to cell–cell interactions. We track the temporal variations in natural killer—triple-negative breast cancer cell distances and compare them with terminal cellular transcriptome profiles. The results show the time-bound activities of regulatory modules and allude to the existence of transcriptional memory. Our experimental and bioinformatic approaches serve as a proof of concept for interrogating live-cell interactions at doublet resolution. Together, our findings highlight the use of our approach across different cancers and cell types. A microfluidic system combines the capture, co-incubation, time-lapse imaging, and gene expression profiling of doublets to measure the distances between cells and associated transcriptional profiles due to cell–cell interactions. [ABSTRACT FROM AUTHOR]

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (∼50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells. [ABSTRACT FROM AUTHOR]

A major challenge for the lab-on-a-chip (LOC) community is to develop point-of-care diagnostic chips that do not use instruments. Such instruments include pumping or liquid handling devices for distribution of patient’s nucleic-acid test sample among an array of reactors and microvalves or mechanical parts to seal these reactors. In this paper, we report the development of a primer pair pre-loaded PCR array chip, in which the loading of the PCR mixture into an array of reactors and subsequent sealing of the reactors were realized by a novel capillary-based microfluidics with a manual two-step pipetting operations. The chip is capable of performing simultaneous (parallel) analyses of multiple gene targets and its performance was tested by amplifying twelve different gene targets against cDNA template from human hepatocellular carcinoma using SYBR Green I fluorescent dye. The versatility and reproducibility of the PCR-array chip are demonstrated by real-time PCR amplification of the BNI-1 fragment of SARS cDNA cloned in a plasmid vector. The reactor-to-reactor diffusion of the pre-loaded primer pairs in the chip is investigated to eliminate the possibility of primer cross-contamination. Key technical issues such as PCR mixture loss in gas-permeable PDMS chip layer and bubble generation due to different PDMS-glass bonding methods are investigated. [ABSTRACT FROM AUTHOR]

High-throughput microchip devices used for nucleic-acid amplification require sealed reactors. This is to prevent evaporative loss of the amplification mixture and cross-contamination, which may occur among fluidically connected reactors. In most high-throughput nucleic-acid amplification devices, reactor sealing is achieved by microvalves. Additionally, these devices require micropumps to distribute amplification mixture into an array of reactors, thereby increasing the device cost, and adding complexity to the chip fabrication and operation processes. To overcome these limitations, we report microfluidic devices harboring open (unsealed) reactors in conjunction with a single-step capillary based flow scheme for sequential distribution of amplification mixture into an array of reactors. Concern about evaporative loss in unsealed reactors have been addressed by optimized reactor design, smooth internal reactor surfaces, and incorporation of a localized heating scheme for the reactors, in which isothermal, real-time helicase-dependent amplification (HDA) was performed. [ABSTRACT FROM AUTHOR]

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3′-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5′-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-β of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization. Single-cell transcriptomic profiling allows the exploration of cellular heterogeneity but commonly focuses on the 3′-end of the transcript. Here the authors introduce C1 CAGE, which detects the 5′ transcript end in a multiplexed microfluidic system. [ABSTRACT FROM AUTHOR]