Automated Microfluidic System for Dynamic Stimulation and Tracking of Single Cells.

Dynamic environments determine cell fate decisions and function. Understanding the relationship between extrinsic signals on cellular responses and cell fate requires the ability to dynamically change environmental inputs in vitro, while continuously observing individual cells over extended periods of time. This is challenging for nonadherent cells, such as hematopoietic stem and progenitor cells, because media flow displaces and disturbs such cells, preventing culture and tracking of single cells. Here, we present a programmable microfluidic system designed for the long-term culture and time-lapse imaging of nonadherent cells in dynamically changing cell culture conditions without losing track of individual cells. The dynamic, valve-controlled design permits targeted seeding of cells in up to 48 independently controlled culture chambers, each providing sufficient space for long-term cell colony expansion. Diffusion-based media exchange occurs rapidly and minimizes displacement of cells and eliminates shear stress. The chip was successfully tested with long-term culture and tracking of primary hematopoietic stem and progenitor cells, and murine embryonic stem cells. This system will have important applications to analyze dynamic signaling inputs controlling fate choices.

Spatiotemporal NF-κB dynamics encodes the position, amplitude, and duration of local immune inputs.

Infected cells communicate through secreted signaling molecules like cytokines, which carry information about pathogens. How differences in cytokine secretion affect inflammatory signaling over space and how responding cells decode information from propagating cytokines are not understood. By computationally and experimentally studying NF-κB dynamics in cocultures of signal-sending cells (macrophages) and signal-receiving cells (fibroblasts), we find that cytokine signals are transmitted by wave-like propagation of NF-κB activity and create well-defined activation zones in responding cells. NF-κB dynamics in responding cells can simultaneously encode information about cytokine dose, duration, and distance to the cytokine source. Spatially resolved transcriptional analysis reveals that responding cells transmit local cytokine information to distance-specific proinflammatory gene expression patterns, creating "gene expression zones." Despite single-cell variability, the size and duration of the signaling zone are tightly controlled by the macrophage secretion profile. Our results highlight how macrophages tune cytokine secretion to control signal transmission distance and how inflammatory signaling interprets these signals in space and time.

A microfluidic device for measuring cell migration towards substrate-bound and soluble chemokine gradients.

Cellular locomotion is a central hallmark of eukaryotic life. It is governed by cell-extrinsic molecular factors, which can either emerge in the soluble phase or as immobilized, often adhesive ligands. To encode for direction, every cue must be present as a spatial or temporal gradient. Here, we developed a microfluidic chamber that allows measurement of cell migration in combined response to surface immobilized and soluble molecular gradients. As a proof of principle we study the response of dendritic cells to their major guidance cues, chemokines. The majority of data on chemokine gradient sensing is based on in vitro studies employing soluble gradients. Despite evidence suggesting that in vivo chemokines are often immobilized to sugar residues, limited information is available how cells respond to immobilized chemokines. We tracked migration of dendritic cells towards immobilized gradients of the chemokine CCL21 and varying superimposed soluble gradients of CCL19. Differential migratory patterns illustrate the potential of our setup to quantitatively study the competitive response to both types of gradients. Beyond chemokines our approach is broadly applicable to alternative systems of chemo- and haptotaxis such as cells migrating along gradients of adhesion receptor ligands vs. any soluble cue.

Automated co-culture system for spatiotemporal analysis of cell-to-cell communication.

We present a microfluidic co-culture system that generates localized and precisely formulated immune signals among a population of cells, enabling spatiotemporal analysis of paracrine signal transmission between different cell types. The automated system allows us to create temporally modulated chemical inputs that can be delivered to single signal-transmitting and receiving cells in a highly controlled way. Using this system we stimulated a single macrophage with brief pulses of bacterial LPS and observed the macrophage transmitted TNF signal propagating in a population of fibroblasts via NF-κB activation. The signal receiving fibroblasts transformed the TNF signal into a spatiotemporally distributed NF-κB output, recapitulating the initiation of immune response to bacterial infection.

Real-time tracking, retrieval and gene expression analysis of migrating human T cells.

Dynamical analysis of single-cells allows assessment of the extent and role of cell-to-cell variability, however traditional dish-and-pipette techniques have hindered single-cell analysis in quantitative biology. We developed an automated microfluidic cell culture system that generates stable diffusion-based chemokine gradients, where cells can be placed in predetermined positions, monitored via single-cell time-lapse microscopy, and subsequently be retrieved based on their migration speed and directionality for further off-chip gene expression analysis, constituting a powerful platform for multiparameter quantitative studies of single-cell chemotaxis. Using this system we studied CXCL12-directed migration of individual human primary T cells. Spatiotemporally deterministic retrieval of T cell subsets in relation to their migration speed, and subsequent analysis with microfluidic droplet digital-PCR showed that the expression level of CXCR4 ­ the receptor of CXCL12 ­ underlies enhanced human T cell chemotaxis.

Flow-switching allows independently programmable, extremely stable, high-throughput diffusion-based gradients.

An automated microfluidic cell culture platform that creates and maintains independently programmable diffusion-based gradients is reported. Temporal modulation of the source and sink flow patterns allow generation of extremely stable spatial gradients. We developed a system that integrates 30 parallel gradients in a single device, with 10 different chemical formulations and 3 replicates. Mammalian fibroblast and macrophage cells were screened for NFκB pathway activity under gradients of TNFα, PDGF, and LPS, and multiparameter measurements were performed to demonstrate the capability of the device in dynamic single-cell analysis.