Massive Parallel Analysis of Single Cells in an ... - ACS Publications

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Massive Parallel Analysis of Single Cells in an Integrated Microfluidic Platform Rocio J. Jimenez-Valdes, Roberto Rodriguez-Moncayo, Diana F. Cedillo-Alcantar, and Jose L. Garcia-Cordero* Unidad Monterrey, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Via del Conocimiento 201, Parque PIIT, Apodaca, Nuevo León CP 66628, Mexico S Supporting Information *

ABSTRACT: New tools that facilitate the study of cell-to-cell variability could help uncover novel cellular regulation mechanisms. We present an integrated microfluidic platform to analyze a large number of single cells in parallel. To isolate and analyze thousands of individual cells in multiplexed conditions, our platform incorporates arrays of microwells (7 pL each) in a multilayered microfluidic device. The device allows the simultaneous loading of cells into 16 separate chambers, each containing 4640 microwells, for a total of 74 240 wells per device. We characterized different parameters important for the operation of the microfluidic device including flow rate, solution exchange rate in a microchamber, shear stress, and time to fill up a single microwell with molecules of different molecular weight. In general, after ∼7.5 min of cell loading our device has an 80% microwell occupancy with 1−4 cells, of which 36% of wells contained a single cell. To test the functionality of our device, we carried out a cell viability assay with adherent and nonadherent cells. We also studied the production of neutrophil extracellular traps (NETs) from single neutrophils isolated from peripheral blood, observing the existence of temporal heterogeneity in NETs production, perhaps having implications in the type of the neutrophil response to an infection or inflammation. We foresee our platform will have a variety of applications in drug discovery and cellular biology by facilitating the characterization of phenotypic differences in a monoclonal cell population.

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techniques, microwells enable different types of assays; for example, microwells have been used (i) to measure protein secretion from individual cells by interfacing them to a glass slide coated with antibodies,9 (ii) to measure DNA damage with a comet assay,11 (iii) to analyze the cytoplasmic contents of cells using patterned electrodes,12 (iv) to control cell trapping with dielectrophoresis,13,14 or simply (v) to culture cells.13 These applications demonstrate that the microwell platform offers versatility to study different parameters from single cells. Despite the many advantages offered by microwells, and even when operating times have been reduced, aided by vacuum15 or centrifugation,16 operation of these devices still involves several manual handling steps. Attempts have been made to integrate microwells in a microfluidic environment but the majority of devices remain largely unsophisticated, with only a single fluidic chamber placed on top of a few microwells.13,14,17−21 A seamless integration of microwells in a microfluidic environment, without losing throughput capabilities (>1000 wells per

great deal of knowledge about cellular mechanisms and processes has been obtained mainly with data gathered from two approaches, either employing cell populationaveraging techniques or measuring a few cells and generalizing the findings to all cells.1,2 However, to gain insight into cellular signal processing and into the complexity of biological circuits it is important to study the causes that give rise to cell-to-cell differences in otherwise genetically identical cells of the same cell type.3−5 To capture population statistics, such heterogeneity studies require molecular and phenotypic measurements of a large number of single cells over long periods of time in controlled microenvironments.1,3−5 Thus, experimental techniques that provide a wide spectrum of measurements from single cells are poised to have significant impact in molecular cell biology. Microwell arrays (