High spatial heterogeneity and low connectivity of bacterial communities along a Mediterranean subterranean estuary.

Subterranean estuaries are biogeochemically active coastal sites resulting from the underground mixing of fresh aquifer groundwater and seawater. In these systems, microbial activity can largely transform the chemical elements that may reach the sea through submarine groundwater discharge (SGD), but little is known about the microorganisms thriving in these land-sea transition zones. We present the first spatially-resolved characterization of the bacterial assemblages along a coastal aquifer in the NW Mediterranean, considering the entire subsurface salinity gradient. Combining bulk heterotrophic activity measurements, flow cytometry, microscopy and 16S rRNA gene sequencing we find large variations in prokaryotic abundances, cell size, activity and diversity at both the horizontal and vertical scales that reflect the pronounced physicochemical gradients. The parts of the transect most influenced by freshwater were characterized by smaller cells and lower prokaryotic abundances and heterotrophic production, but some activity hotspots were found at deep low-oxygen saline groundwater sites enriched in nitrite and ammonium. Diverse, heterogeneous and highly endemic communities dominated by Proteobacteria, Patescibacteria, Desulfobacterota and Bacteroidota were observed throughout the aquifer, pointing to clearly differentiated prokaryotic niches across these transition zones and little microbial connectivity between groundwater and Mediterranean seawater habitats. Finally, experimental manipulations unveiled large increases in community heterotrophic activity driven by fast growth of some rare and site-specific groundwater Proteobacteria. Our results indicate that prokaryotic communities within subterranean estuaries are highly heterogeneous in terms of biomass, activity and diversity, suggesting that their role in transforming nutrients will also vary spatially within these terrestrial-marine transition zones.

Organotypic platform for studying cancer cell metastasis.

Metastasis is the leading cause of mortality in cancer patients. To migrate to distant sites, cancer cells would need to adapt their behaviour in response to different tissue environments. Thus, it is essential to study this process in models that can closely replicate the tumour microenvironment. Here, we evaluate the use of organotypic liver and brain slices to study cancer metastasis. Morphological and viability parameters of the slices were monitored daily over 3 days in culture to assess their stability as a realistic 3D tissue platform for in vitro metastatic assays. Using these slices, we evaluated the invasion of MDA-MB-231 breast cancer cells and of a subpopulation that was selected for increased motility. We show that the more aggressive invasion of the selected cells likely resulted not only from their lower stiffness, but also from their lower adhesion to the surrounding tissue. Different invasion patterns in the brain and liver slices were observed for both subpopulations. Cells migrated faster in the brain slices (with an amoeboid-like mode) compared to in the liver slices (where they migrated with mesenchymal or collective migration-like modes). Inhibition of the Ras/MAPK/ERK pathway increased cell stiffness and adhesion forces, which resulted in reduced invasiveness. These results illustrate the potential for organotypic tissue slices to more closely mimic in vivo conditions during cancer cell metastasis than most in vitro models.

Digital Manufacturing for Microfluidics.

The microfluidics field is at a critical crossroads. The vast majority of microfluidic devices are presently manufactured using micromolding processes that work very well for a reduced set of biocompatible materials, but the time, cost, and design constraints of micromolding hinder the commercialization of many devices. As a result, the dissemination of microfluidic technology-and its impact on society-is in jeopardy. Digital manufacturing (DM) refers to a family of computer-centered processes that integrate digital three-dimensional (3D) designs, automated (additive or subtractive) fabrication, and device testing in order to increase fabrication efficiency. Importantly, DM enables the inexpensive realization of 3D designs that are impossible or very difficult to mold. The adoption of DM by microfluidic engineers has been slow, likely due to concerns over the resolution of the printers and the biocompatibility of the resins. In this article, we review and discuss the various printer types, resolution, biocompatibility issues, DM microfluidic designs, and the bright future ahead for this promising, fertile field.

Coupling Flow, Heat, and Reactive Transport Modeling to Reproduce In Situ Redox Potential Evolution: Application to an Infiltration Pond.

Redox potential (Eh) measurements are widely used as indicators of the dominant reduction-oxidation reactions occurring underground. Yet, Eh data are mostly used in qualitative terms, as actual values cannot be used to distinguish uniquely the dominant redox processes at a sampling point and should therefore be combined with a detailed geochemical characterization of water samples. In this work, we have intensively characterized the redox potential of the first meter of soil in an infiltration pond recharged with river water using a set of in situ sensors measuring every 12 min during a 1 year period. This large amount of data combined with hydrogeochemical campaigns allowed developing a reactive transport model capable of reproducing the redox potential in space and time together with the site hydrochemistry. Our results showed that redox processes were mainly driven by the amount of sedimentary organic matter in the system as well as by seasonal variation of temperature. As a subsidiary result, our work emphasizes the need to use a fully coupled model of flow, heat transport, solute transport, and the geochemical reaction network to fully reproduce the Eh observations in the topsoil.

Are dominant microbial sub-surface communities affected by water quality and soil characteristics?

Subsurface microorganisms must deal with quite extreme environmental conditions. The lack of light, oxygen, and potentially nutrients are the main environmental stresses faced by subsurface microbial communities. Likewise, environmental disruptions providing an unbalanced positive input of nutrients force microorganisms to adapt to varying conditions, visible in the changes in microbial community diversity. In order to test microbial community adaptation to environmental changes, we performed a study in a surface Managed Aquifer Recharge facility, consisting of a settlement basin (two-day residence time) and an infiltration pond. Data on groundwater hydrochemistry, soil texture, and microbial characterization was compiled from surface water, groundwater, and soil samples at two distinct recharge operation conditions. Multivariate statistics by means of Principal Component Analysis (PCA) was the technique used to map the relevant dimensionality reduced combinations of input variables that properly describe the system behavior. The methodology selected allows including variables of different nature and displaying very different range values. Strong differences in the microbial assemblage under recharge conditions were found, coupled to hydrochemistry and grain-size distribution variables. Also, some microbial groups displayed correlations with either carbon or nitrogen cycles, especially showing abundant populations of denitrifying bacteria in groundwater. A significant correlation was found between Methylotenera mobilis and the concentrations of NO3 and SO4, and also between Vogesella indigofera and the presence of DOC in the infiltrating water. Also, microbial communities present at the bottom of the pond correlated with representative descriptors of soil grain size distribution.

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range.

Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general "systems level" mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step.

3D-Printed Microfluidics.

The advent of soft lithography allowed for an unprecedented expansion in the field of microfluidics. However, the vast majority of PDMS microfluidic devices are still made with extensive manual labor, are tethered to bulky control systems, and have cumbersome user interfaces, which all render commercialization difficult. On the other hand, 3D printing has begun to embrace the range of sizes and materials that appeal to the developers of microfluidic devices. Prior to fabrication, a design is digitally built as a detailed 3D CAD file. The design can be assembled in modules by remotely collaborating teams, and its mechanical and fluidic behavior can be simulated using finite-element modeling. As structures are created by adding materials without the need for etching or dissolution, processing is environmentally friendly and economically efficient. We predict that in the next few years, 3D printing will replace most PDMS and plastic molding techniques in academia.

Genetic elimination of GABAergic neurotransmission reveals two distinct pacemakers for spontaneous waves of activity in the developing mouse cortex.

Many structures of the mammalian CNS generate propagating waves of electrical activity early in development. These waves are essential to CNS development, mediating a variety of developmental processes, such as axonal outgrowth and pathfinding, synaptogenesis, and the maturation of ion channel and receptor properties. In the mouse cerebral cortex, waves of activity occur between embryonic day 18 and postnatal day 8 and originate in pacemaker circuits in the septal nucleus and the piriform cortex. Here we show that genetic knock-out of the major synthetic enzyme for GABA, GAD67, selectively eliminates the picrotoxin-sensitive fraction of these waves. The waves that remain in the GAD67 knock-out have a much higher probability of propagating into the dorsal neocortex, as do the picrotoxin-resistant fraction of waves in controls. Field potential recordings at the point of wave initiation reveal different electrical signatures for GABAergic and glutamatergic waves. These data indicate that: (1) there are separate GABAergic and glutamatergic pacemaker circuits within the piriform cortex, each of which can initiate waves of activity; (2) the glutamatergic pacemaker initiates waves that preferentially propagate into the neocortex; and (3) the initial appearance of the glutamatergic pacemaker does not require preceding GABAergic waves. In the absence of GAD67, the electrical activity underlying glutamatergic waves shows greatly increased tendency to burst, indicating that GABAergic inputs inhibit the glutamatergic pacemaker, even at stages when GABAergic pacemaker circuitry can itself initiate waves.

Ultrarapid detection of pathogenic bacteria using a 3D immunomagnetic flow assay.

We developed a novel 3D immunomagnetic flow assay for the rapid detection of pathogenic bacteria in a large-volume food sample. Antibody-functionalized magnetic nanoparticle clusters (AbMNCs) were magnetically immobilized on the surfaces of a 3D-printed cylindrical microchannel. The injection of a Salmonella-spiked sample solution into the microchannel produced instant binding between the AbMNCs and the Salmonella bacteria due to their efficient collisions. Nearly perfect capture of the AbMNCs and AbMNCs-Salmonella complexes was achieved under a high flow rate by stacking permanent magnets with spacers inside the cylindrical separator to maximize the magnetic force. The concentration of the bacteria in solution was determined using ATP luminescence measurements. The detection limit was better than 10 cfu/mL, and the overall assay time, including the binding, rinsing, and detection steps for a 10 mL sample took less than 3 min. To our knowledge, the 3D immunomagnetic flow assay described here provides the fastest high-sensitivity, high-capacity method for the detection of pathogenic bacteria.

An assessment tool to improve rural groundwater access: Integrating hydrogeological modelling with socio-technical factors.

Sustainable exploitation of groundwater resources for drinking water provision in rural communities in sub-Sahara Africa remains elusive due to the limited knowledge of these hydrogeological systems. This is exacerbated by poor maintenance of existing infrastructure, limited technical capacity, the socio-economic characteristics of the area and poor governance. Assessing the likelihood of a given individual user experiencing water shortage calls for an interdisciplinary approach. After a preliminary multifactorial analysis incorporating a range of variables from technical to societal, it was found that most of the overall risk of water shortage for an individual household could be attributed to three factors; (1) Proximity, specified as the distance to the closest supply well (determined by geographical parameters), (2) Availability of good quality water in the wells (determined by hydrogeological understanding and modelling), and (3) Sustainability (determined by socio-technical and socio-economic parameters). In the latter case, a distinction was made between hardware functionality- the water point's performance considering a sufficient yield and reliability through time- and software functionality, based on a combination of socioeconomic data from surveys and analysed using Multiple Factor Analysis (MFA). All three factors are eventually mapped onto indicators in the range of [0-1] and then represented in a Geographical Information System based on the partition of the entire spatial domain (e.g., counties, villages, and neighbourhoods). The three indicators are then combined in a final index based on the product of the three factors, thus mapping time-dependent overall risk and allowing the assessment of temporal risk-evolution scenarios. The methodology is applied to Kwale County, Kenya, where community handpumps and groundwater points comprise the main water supply system. Apart from mapping the present situation, the methodology is finally used to assess the impact of future climate scenarios.

Drug testing of monodisperse arrays of live microdissected tumors using a valved multiwell microfluidic platform.

Cancer drug testing in animals is an extremely poor predictor of the drug's safety and efficacy observed in humans. Hence there is a pressing need for functional testing platforms that better predict traditional and immunotherapy responses in human, live tumor tissue or tissue constructs, and at the same time are compatible with the use of mouse tumor tissue to facilitate building more accurate disease models. Since many cancer drug actions rely on mechanisms that depend on the tumor microenvironment (TME), such platforms should also retain as much of the native TME as possible. Additionally, platforms based on miniaturization technologies are desirable to reduce animal use and sensitivity to human tissue scarcity. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, microdissected tumors (µDTs) or "spheroids" that retain an intact TME have shown promising responses to immunomodulators acting on native immune cells. However, difficult tissue handling after microdissection has reduced the throughput of drug testing on µDTs, thereby constraining the inherent advantages of producing numerous TME-preserving units of tissue for drug testing. Here we demonstrate a microfluidic 96-well platform designed for drug treatment of hundreds of similarly-sized, cuboidal µDTs ("cuboids") produced from a single tumor sample. The platform organizes a monodisperse array of four cuboids per well in 384 hydrodynamic traps. The microfluidic device, entirely fabricated in thermoplastics, features 96 microvalves that fluidically isolate each well after the cuboid loading step for straightforward multi-drug testing. Since our platform makes the most of scarce tumor tissue, it can potentially be applied to human biopsies that preserve the human TME while minimizing animal testing.

Label-Free, Real-Time Monitoring of Cytochrome C Responses to Drugs in Microdissected Tumor Biopsies with a Multi-Well Aptasensor Platform.

Functional assays on intact tumor biopsies can potentially complement and extend genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key determinants of therapeutic response, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Currently, most of these assays rely on fluorescent labeling, a semi-quantitative method best suited to be a single-time-point terminal assay or labor-intensive terminal immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of increases of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Since the aptamer probe can be easily exchanged to recognize different targets, the platform could be adapted for multiplexed monitoring of various biomarkers, providing critical information on the tumor and its microenvironment. This approach could not only help develop more advanced cancer disease models but also apply to other complex in vitro disease models, such as organs-on-chips and organoids.

Micromanipulation of Live Microdissected Tissues with a Low-Cost Integrated Robotic Platform.

The small amount of human tissue available for testing is a paramount challenge in cancer drug development, cancer disease models, and personalized oncology. Technologies that combine the microscale manipulation of tissues with fluid handling offer the exciting possibility of miniaturizing and automating drug evaluation workflows. This approach minimizes animal testing and enables inexpensive, more efficient testing of samples with high clinical biomimicry using scarce materials. We have developed an inexpensive platform based on an off-the-shelf robot that can manipulate microdissected tissues (µDTs) into user-programmed positions without using intricate microfluidic designs nor any other accessories such as a microscope or a pneumatic controller. The robot integrates complex functions such as vision and fluid actuation by incorporating simple items including a USB camera and a rotary pump. Through the robot's camera, the platform software optically recognizes randomly-seeded µDTs on the surface of a petri dish and positions a mechanical arm above the µDTs. Then, a custom rotary pump actuated by one of the robot's motors generates enough microfluidic lift to hydrodynamically pick and place µDTs with a pipette at a safe distance from the substrate without requiring a proximity sensor. The platform's simple, integrated construction is cost-effective and compact, allowing placement inside a tissue culture hood for sterile workflows. The platform enables users to select µDTs based on their size, place them in user-programmed arrays, such as multi-well plates, and control various robot motion parameters. As a case application, we use the robotic system to conduct semi-automated drug testing of mouse and human µDTs in 384-well plates. Our user-friendly platform promises to democratize microscale tissue research to clinical and biological laboratories worldwide.

Microdissected tumor cuboids: a microscale cancer model that retains a complex tumor microenvironment.

Present cancer disease models - typically based on cell cultures and animal models that lack the human tumor microenvironment (TME) - are extremely poor predictors of human disease outcomes. Microscale cancer models that combine the micromanipulation of tissues and fluids offer the exciting possibility of miniaturizing the drug testing workflow, enabling inexpensive, more efficient tests of high clinical biomimicry that maximize the use of scarce human tissue and minimize animal testing. Critically, these microscale models allow for precisely addressing the impact of the structural features of the heterogeneous TME to properly target and understand the contributions of these unique zones to therapeutic response. We have recently developed a precision slicing method that yields large numbers of cuboidal micro-tissues ("cuboids", ∼ (400 µm) 3 ) from a single tumor biopsy. Here we evaluate cuboids from syngeneic mouse tumor models and human tumors, which contain native immune cells, as models for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as their cellular architecture (immune cells and vasculature), cytokine secretion, proteomics profiles, and their response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong functional responses such as cytokine and drug responses. Overall, our results suggest that cuboids make an excellent model for applications that require the TME, such as immunotherapy drug evaluations, including for clinical trials and personalized oncology approaches.

Tunable resins with PDMS-like elastic modulus for stereolithographic 3D-printing of multimaterial microfluidic actuators.

Stereolithographic 3D-printing (SLA) permits facile fabrication of high-precision microfluidic and lab-on-a-chip devices. SLA photopolymers often yield parts with low mechanical compliancy in sharp contrast to elastomers such as poly(dimethyl siloxane) (PDMS). On the other hand, SLA-printable elastomers with soft mechanical properties do not fulfill the distinct requirements for a highly manufacturable resin in microfluidics (e.g., high-resolution printability, transparency, low-viscosity). These limitations restrict our ability to print microfluidic actuators containing dynamic, movable elements. Here we introduce low-viscous photopolymers based on a tunable blend of the monomers poly(ethylene glycol) diacrylate (PEGDA, Mw ∼ 258) and the monoacrylate poly(ethylene glycol methyl ether) methacrylate (PEGMEMA, Mw ∼ 300). In these blends, which we term PEGDA-co-PEGMEMA, tuning the PEGMEMA content from 0% to 40% (v/v) alters the elastic modulus of the printed plastics by ∼400-fold, reaching that of PDMS. Through the addition of PEGMEMA, moreover, PEGDA-co-PEGMEMA retains desirable properties of highly manufacturable PEGDA such as low viscosity, solvent compatibility, cytocompatibility and low drug absorptivity. With PEGDA-co-PEGMEMA, we SLA-printed drastically enhanced fluidic actuators including microvalves, micropumps, and microregulators with a hybrid structure containing a flexible PEGDA-co-PEGMEMA membrane within a rigid PEGDA housing. These components were built using a custom "Print-Pause-Print" protocol, referred to as "3P-printing", that allows for fabricating high-resolution multimaterial parts with a desktop SLA printer without the need for post-assembly. SLA-printing of multimaterial microfluidic actuators addresses the unmet need of high-performance on-chip controls in 3D-printed microfluidic and lab-on-a-chip devices.

A 'print-pause-print' protocol for 3D printing microfluidics using multimaterial stereolithography.

Methods to make microfluidic chips using 3D printers have attracted much attention because these simple procedures allow rapid fabrication of ready-to-use products from digital 3D designs with minimal human intervention. Printing high-resolution chips that are simultaneously transparent, biocompatible and contain regions of dissimilar materials is an ongoing challenge. Transparency allows for the optical inspection of specimens containing cells and labeled biomolecules inside the chip. Being able to use different materials for different layers in the product increases the number of potential applications. In this 'print-pause-print' protocol, we describe detailed strategies for fabricating transparent biomicrofluidic devices and multimaterial chips using stereolithographic 3D printing. To print transparent biomicrofluidic chips, we developed a transparent resin based on poly(ethylene glycol) diacrylate (PEG-DA) (average molecular weight: 250 g/mol, PEG-DA-250) and a smooth chip surface technique achieved using glass. Cells can be successfully cultured and visualized on PEG-DA-250 prints and inside PEG-DA-250 microchannels. The multimaterial potential of the technique is exemplified using a molecular diffusion device that comprises parts made of two different materials: the channel walls, which are water impermeable, and a porous barrier structure, which is permeable to small molecules that diffuse through it. The two materials were prepared from two different molecular-weight PEG-DA-based printing resins. Alignment of the two dissimilar material structures is performed automatically by the printer during the printing process, which only requires a simple pause step to exchange the resins. The procedure takes less than 1 h and can facilitate chip-based applications including biomolecule analysis, cell biology, organ-on-a-chip and tissue engineering.

Chemicals of emerging concern in coastal aquifers: Assessment along the land-ocean interface.

Submarine Groundwater Discharge (SGD) is recognized as a relevant source of pollutants to the sea, but little is known about its relevance as a source of chemicals of emerging concern (CECs). Here, both the presence and distribution of a wide range of CECs have been evaluated in the most comprehensive manner to date, in a well-characterized Mediterranean coastal aquifer near Barcelona (Spain). Samples from coastal groundwater and seawater allowed for the unique spatial characterization of the pollutants present in the land-ocean interface, an outstanding research gap that required attention. The main goals were (1) to determine CECs in the aquifer, so as to evaluate the SGD as a relevant source of marine pollution, and (2) to identify new tracers to improve our understanding of SGD dynamics. To this end, 92 CECs were located in the aquifer by using wide-scope analytical target methodologies (>2000 chemicals). Among them, the perfluoroalkyl and polyfluoroalkyl substances (PFAS), along with the pharmaceuticals carbamazepine and topiramate, were revealed to be good markers for tracing anthropogenic contamination in ground- and seawater, in concrete situations (e.g., highly contaminated sites). Additionally, non-target analysis expanded the number of potential tracers, making it a promising tool for identifying both the source and the fate of pollutants.

Identification and quantification of chemical reactions in a coastal aquifer to assess submarine groundwater discharge composition.

In coastal aquifers, two opposite but complementary processes occur: Seawater intrusion (SWI), which may salinize heavily exploited aquifers, and Submarine groundwater discharge (SGD) which transports oligo-elements to the sea. Aquifers are expected to be chemically reactive, both because they provide abundant surfaces to catalyze reactions and the mixing of very different Fresh Water (FW) and Sea Water (SW) promote numerous reactions. Characterizing and quantifying these reactions is essential to assess the quality and composition of both aquifer water, and SGD. Indeed, sampling SGD is difficult, so its composition is usually uncertain. We propose a reactive end-member mixing analysis (rEMMA) methodology based on principal component analysis (PCA) to (i) identify the sources of water and possible reactions occurring in the aquifer and (ii) quantify mixing ratios and the extent of chemical reactions. We applied rEMMA to the Argentona coastal aquifer located North of Barcelona that contains fluvial sediments of granitic origin and overlies weathered granite. The identification of end members (FW and SW) and the spatial distribution of their mixing ratios illustrate the application procedure. The extent of reactions and their spatial distribution allow us to distinguish reactions that occur as a result of mixing from those caused by sediment disequilibrium, which are relevant to recirculated saltwater SGD. The most important reaction is cation exchange, especially between Ca and Na, which promotes other reactions such as Gypsum and Fluorite precipitation. Iron and Manganese are mobilized in the SW portion but oxidized and precipitated in the mixing zone, so that Fe (up to 15 µEq/L) and Mn (up to 10 µEq/L) discharge is restricted to SW SGD. Nitrate is reduced in the mixing zone. The actual reaction amounts are site-specific, but the processes are not, which leads us to conjecture the importance of these reactions to understand the SGD discharge elsewhere.

Microdissected "cuboids" for microfluidic drug testing of intact tissues.

As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a multi-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or "cuboids" with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.

Large-scale investigation of the olfactory receptor space using a microfluidic microwell array.

The mammalian olfactory system is able to discriminate among tens of thousands of odorant molecules. In mice, each odorant is sensed by a small subset of the approximately 1000 odorant receptor (OR) types, with one OR gene expressed by each olfactory sensory neuron (OSN). However, the sum of the large repertoire of OR-OSN types and difficulties with heterologous expression have made it almost impossible to analyze odorant-responsiveness across all OR-OSN types. We have developed a microfluidic approach that allowed us to screen over 20,000 single cells at once in microwells. By using calcium imaging, we were able to detect and analyze odorant responses of about 2900 OSNs simultaneously. Importantly, this technique allows for both the detection of rare responding OSNs as well as the identification of OSN populations broadly responsive to odorants of unrelated structures. This technique is generally applicable for screening large numbers of single cells and should help to characterize rare cell behaviors in fields such as toxicology, pharmacology, and cancer research.