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We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying ($99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing ($70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems).The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency E patt was .75% during lengthy in chip culture, with y85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.

We present a rapid, reproducible and sensitive neurotoxicity testing platform that combines the benefits of neurite outgrowth analysis with cell patterning. This approach involves patterning neuronal cells within a hexagonal array to standardize the distance between neighbouring cellular nodes, and thereby standardize the length of the neurite interconnections. This feature coupled with defined assay coordinates provides a streamlined display for rapid and sensitive analysis. We have termed this the network formation assay (NFA). To demonstrate the assay we have used a novel cell patterning technique involving thin film poly(dimethylsiloxane) (PDMS) microcontact printing. Differentiated human SH-SY5Y neuroblastoma cells colonized the array with high efficiency, reliably producing pattern occupancies above 70%. The neuronal array surface supported neurite outgrowth, resulting in the formation of an interconnected neuronal network. Exposure to acrylamide, a neurotoxic reference compound, inhibited network formation. A dose-response curve from the NFA was used to determine a 20% network inhibition (NI(20)) value of 260 microM. This concentration was approximately 10-fold lower than the value produced by a routine cell viability assay, and demonstrates that the NFA can distinguish network formation inhibitory effects from gross cytotoxic effects. Inhibition of the mitogen-activated protein kinase (MAPK) ERK1/2 and phosphoinositide-3-kinase (PI-3K) signaling pathways also produced a dose-dependent reduction in network formation at non-cytotoxic concentrations. To further refine the assay a simulation was developed to manage the impact of pattern occupancy variations on network formation probability. Together these developments and demonstrations highlight the potential of the NFA to meet the demands of high-throughput applications in neurotoxicology and neurodevelopmental biology.

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Continuous flow microreactors with an annular microchannel for cyclical chemical reactions were fabricated by either bulk micromachining in silicon or by rapid prototyping using EPON SU-8. Fluid propulsion in these unusual microchannels was achieved using AC magnetohydrodynamic (MHD) actuation. This integrated micropumping mechanism obviates the use of moving parts by acting locally on the electrolyte, exploiting its inherent conductive nature. Both silicon and SU-8 microreactors were capable of MHD actuation, attaining fluid velocities of the order of 300 microm s(-1) when using a 500 mM KCl electrolyte. The polymerase chain reaction (PCR), a thermocycling process, was chosen as an illustrative example of a cyclical chemistry. Accordingly, temperature zones were provided to enable a thermal cycle during each revolution. With this approach, fluid velocity determines cycle duration. Here, we report device fabrication and performance, a model to accurately describe fluid circulation by MHD actuation, and compatibility issues relating to this approach to chemistry.