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Multispecies microbial communities drive most ecosystems on Earth. Chemical and biological interactions within these communities can affect the survival of individual members and the entire community. However, the prohibitively high number of possible interactions within a microbial community has made the characterization of factors that influence community development challenging. Here, we report a Microbial Community Interaction (µCI) device to advance the systematic study of chemical and biological interactions within a microbial community. The µCI creates a combinatorial landscape made up of an array of triangular wells interconnected with circular wells, which each contains either a different chemical or microbial strain, generating chemical gradients and revealing biological interactions. Bacillus cereus UW85 containing green fluorescent protein provided the "target" readout in the triangular wells, and antibiotics or microorganisms in adjacent circular wells are designated the "variables." The µCI device revealed that gentamicin and vancomycin are antagonistic to each other in inhibiting the target B. cereus UW85, displaying weaker inhibitory activity when used in combination than alone. We identified three-member communities constructed with isolates from the plant rhizosphere that increased or decreased the growth of B. cereus. The µCI device enables both strain-level and community-level insight. The scalable geometric design of the µCI device enables experiments with high combinatorial efficiency, thereby providing a simple, scalable platform for systematic interrogation of three-factor interactions that influence microorganisms in solitary or community life.

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All cells are subject to geometric constraints, such as surface area-to-volume (SA/V) ratio, that impact cell functions and force biological adaptations. Like the SA/V ratio of a sphere, it is generally assumed that the SA/V ratio of cells decreases as cell size increases. Here, we investigate this in near-spherical mammalian cells using single-cell measurements of cell mass and surface proteins, as well as imaging of plasma membrane morphology. We find that the SA/V ratio remains surprisingly constant as cells grow larger. This observation is largely independent of the cell cycle and the amount of cell growth. Consequently, cell growth results in increased plasma membrane folding, which simplifies cellular design by ensuring sufficient membrane area for cell division, nutrient uptake and deformation at all cell sizes.

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Electroencephalogram (EEG) studies have suggested compensatory brain overactivation in cognitively healthy (CH) older adults with pathological beta-amyloid(Aß42)/tau ratios during working memory and interference processing. However, the association between glutamatergic metabolites and brain activation proxied by EEG signals has not been thoroughly investigated. We aim to determine the involvement of these metabolites in EEG signaling. We focused on CH older adults classified under (1) normal CSF Aß42/tau ratios (CH-NATs) and (2) pathological Aß42/tau ratios (CH-PATs). We measured plasma glutamine, glutamate, pyroglutamate, and γ-aminobutyric acid concentrations using tandem mass spectrometry and conducted a correlational analysis with alpha frequency event-related desynchronization (ERD). Under the N-back working memory paradigm, CH-NATs presented negative correlations (r = ~-0.74--0.96, p = 0.0001-0.0414) between pyroglutamate and alpha ERD but positive correlations (r = ~0.82-0.95, p = 0.0003-0.0119) between glutamine and alpha ERD. Under Stroop interference testing, CH-NATs generated negative correlations between glutamine and left temporal alpha ERD (r = -0.96, p = 0.037 and r = -0.97, p = 0.027). Our study demonstrated that glutamine and pyroglutamate levels were associated with EEG activity only in CH-NATs. These results suggest cognitively healthy adults with amyloid/tau pathology experience subtle metabolic dysfunction that may influence EEG signaling during cognitive challenge. A longitudinal follow-up study with a larger sample size is needed to validate these pilot studies.

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Innate immune cell infiltration into neoplastic tissue is the first line of defense against cancer and can play a deterministic role in tumor progression. Here, we describe a series of assays, using a reconfigurable microscale assay platform (i.e. Stacks), which allows the study of immune cell infiltration in vitro with spatiotemporal manipulations. We assembled Stacks assays to investigate tumor-monocyte interactions, re-education of activated macrophages, and neutrophil infiltration. For the first time in vitro, the Stacks infiltration assays reveal that primary tumor-associated fibroblasts from specific patients differ from that associated with the benign region of the prostate in their ability to limit neutrophil infiltration as well as facilitate monocyte adhesion and anti-inflammatory monocyte polarization. These results show that fibroblasts play a regulatory role in immune cell infiltration and that Stacks has the potential to predict individual patients' cancer-immune response.

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Macrophages within the tumor microenvironment (TME) exhibit a spectrum of protumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon autofluorescence imaging of NAD(P)H and FAD to nondestructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours poststimulation for mouse macrophages (RAW264.7) stimulated with IFNγ or IL4 plus IL13 in 2D culture, confirming that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse polyoma-middle T virus or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor cocultures exhibited significantly different FAD mean lifetimes and greater migration than monocultures at 24, 48, and 72 hours postseeding. In cocultures with primary human cancer cells, actively migrating monocyte-derived macrophages had greater redox ratios [NAD(P)H/FAD intensity] compared with passively migrating monocytes at 24 and 48 hours postseeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumor-immune cross-talk within the 3D TME. SIGNIFICANCE: Label-free metabolic imaging and microscale culture technologies enable monitoring of single-cell macrophage metabolism, migration, and function in the 3D tumor microenvironment.

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The study of intercellular signalling networks requires organotypic microscale systems that facilitate the culture, conditioning and manipulation of cells. Here, we describe a reconfigurable microfluidic cell-culture system that facilitates the assembly of three-dimensional tissue models by stacking layers that contain preconditioned microenvironments. By using principles of open and suspended microfluidics, the Stacks system is easily assembled or disassembled to provide spatial and temporal manoeuvrability in two-dimensional and three-dimensional assays of multiple cell types, enabling the modelling of sequential paracrine-signalling events, such as tumour-cell-mediated differentiation of macrophages and macrophage-facilitated angiogenesis. We used Stacks to recapitulate the in vivo observation that different prostate cancer tissues polarize macrophages with distinct gene-expression profiles of pro-inflammatory and anti-inflammatory cytokines. Stacks also enabled us to show that these two types of macrophages signal distinctly to endothelial cells, leading to blood vessels with different morphologies. Our proof-of-concept experiments exemplify how Stacks can efficiently model multicellular interactions and highlight the importance of spatiotemporal specificity in intercellular signalling.

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We present an open microfluidic platform that enables stable flow of an organic solvent over an aqueous solution. The device features apertures connecting a lower aqueous channel to an upper solvent compartment that is open to air, enabling easy removal of the solvent for analysis. We have previously shown that related open biphasic systems enable steroid hormone extraction from human cells in microscale culture and secondary metabolite extraction from microbial culture; here we build on our prior work by determining conditions under which the system can be used with extraction solvents of ranging polarities, a critical feature for applying this extraction platform to diverse classes of metabolites. We developed an analytical model that predicts the limits of stable aqueous-organic interfaces based on analysis of Laplace pressure. With this analytical model and experimental testing, we developed generalized design rules for creating stable open microfluidic biphasic systems with solvents of varying densities, aqueous-organic interfacial tensions, and polarities. The stable biphasic interfaces afforded by this device will enable on-chip extraction of diverse metabolite structures and novel applications in microscale biphasic chemical reactions.

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Double-exclusive liquid repellency (double-ELR) is an extreme wettability phenomenon in which adjacent regions selectively and completely repel immiscible liquids with different surface chemistries on a non-textured substrate (i.e., a substrate in absence of micro/nano-structures). Under double-ELR conditions, each liquid exhibits no physical contact (contact angle of 180°) with its non-preferred surface chemistry, thus enabling complete partitioning of adjacent fluidic volumes (e.g., between water and oil). This enables a new type of cell culture-based assay, where cell loss from common failure modes (e.g., biofouling from inadvertent cell adhesion, detrimental moisture loss/gain, and liquid handling dead volumes) is significantly mitigated. Importantly, the principles of double-ELR were leveraged to achieve underoil sweep patterning, a no-loss, robust and high-throughput distribution of sub-microliter volumes of aqueous media (and cells). In addition to high-efficiency distribution via sweep patterning, double-ELR can be used to construct "modular" (i.e., easily implemented and/or linked together with spatial and temporal control) higher-order architectures for in vitro imitation of physiologically relevant microenvironments that are of particular interest within the cell assay community, including multi-phenotype cultures with excellent spatial and temporal control, three-dimensional layered multi-phenotype cultures, cultures with selective mechanical cues of extracellular matrix (i.e., collagen fiber alignment), and spheroid cultures. Together, these features of double-ELR uniquely facilitate culture and high content analysis of limited cellular samples (e.g., a few hundred to a few thousand cells).

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Micromilling is an underutilized technique for fabricating microfluidic platforms that is well-suited for the diverse needs of the biologic community. This technique, however, produces culture surfaces that are considerably rougher than in commercially available culture platforms and the hydrophilicity of these surfaces can vary considerably depending on the choice of material. In this study, we evaluated the impact of surface topography and hydrophilicity in milled microfluidic devices on the cellular phenotype and function of primary human macrophages. We found that the rough culture surface within micromilled systems affected the phenotype of macrophages cultured in these devices. However, the presence, type, and magnitude of this effect was dependent on the surface hydrophilicity as well as exposure to chemical polarization signals. These findings confirm that while milled microfluidic systems are an effective platform for culture and analysis of primary macrophages, the topography and hydrophilicity of the culture surface within these systems should be considered in the planning and analysis of any macrophage experiments in which phenotype is relevant.

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The concept of high liquid repellency in multi-liquid-phase systems (e.g., aqueous droplets in an oil background) has been applied to areas of biomedical research to realize intrinsic advantages not available in single-liquid-phase systems. Such advantages have included minimizing analyte loss, facile manipulation of single-cell samples, elimination of biofouling, and ease of use regarding loading and retrieving of the sample. In this paper, we present generalized design rules for predicting the wettability of solid-liquid-liquid systems (especially for discrimination between exclusive liquid repellency (ELR) and finite liquid repellency) to extend the applications of ELR. We then apply ELR to two model systems with open microfluidic design in cell biology: (1) in situ underoil culture and combinatorial coculture of mammalian cells in order to demonstrate directed single-cell multiencapsulation with minimal waste of samples as compared to stochastic cell seeding and (2) isolation of a pure population of circulating tumor cells, which is required for certain downstream analyses including sequencing and gene expression profiling.

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Angiogenesis (the formation of blood vessels from existing blood vessels) plays a critical role in many diseases such as cancer, benign tumors, and macular degeneration. There is a need for cell culture methods capable of dissecting the intricate regulation of angiogenesis within the microenvironment of the vasculature. We have developed a microscale cell-based assay that responds to complex pro- and antiangiogenic soluble factors with an in vitro readout for vessel formation. The power of this system over traditional techniques is that we can incorporate the whole milieu of soluble factors produced by cells in situ into one biological readout (vessel formation), even if the identity of the factors is unknown. We have currently incorporated macrophages, endothelial cells, and fibroblasts into the assay, with the potential to include additional cell types in the future. Importantly, the microfluidic platform is simple to operate and multiplex to test drugs targeting angiogenesis in a more physiologically relevant context. As a proof of concept, we tested the effect of an enzyme inhibitor (targeting matrix metalloproteinase 12) on vessel formation; the triculture microfluidic assay enabled us to capture a dose-dependent effect entirely missed in a simplified coculture assay (p < 0.0001). This result underscores the importance of cell-based assays that capture chemical cross-talk occurring between cell types. The microscale dimensions significantly reduce cell consumption compared to conventional well plate platforms, enabling the use of limited primary cells from patients in future investigations and offering the potential to screen therapeutic approaches for individual patients in vitro.

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Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the major cell-cycle checkpoint that regulates chromosome segregation during mitosis, the mitotic checkpoint (also known as the spindle assembly checkpoint). During mitosis, Mad1 recruits Mad2 to unattached kinetochores, where Mad2 is converted into an inhibitor of the anaphase-promoting complex/cyclosome bound to its specificity factor, Cdc20. During interphase, Mad1 remains tightly bound to Mad2, and both proteins localize to the nucleus and nuclear pores, where they interact with Tpr (translocated promoter region). Recently, it has been shown that interaction with Tpr stabilizes both proteins and that Mad1 binding to Tpr permits Mad2 to associate with Cdc20. However, interphase functions of Mad1 that do not directly affect the mitotic checkpoint have remained largely undefined. Here we identify a previously unrecognized interphase distribution of Mad1 at the Golgi apparatus. Mad1 colocalizes with multiple Golgi markers and cosediments with Golgi membranes. Although Mad1 has previously been thought to constitutively bind Mad2, Golgi-associated Mad1 is Mad2 independent. Depletion of Mad1 impairs secretion of α5 integrin and results in defects in cellular attachment, adhesion, and FAK activation. Additionally, reduction of Mad1 impedes cell motility, while its overexpression accelerates directed cell migration. These results reveal an unexpected role for a mitotic checkpoint protein in secretion, adhesion, and motility. More generally, they demonstrate that, in addition to generating aneuploidy, manipulation of mitotic checkpoint genes can have unexpected interphase effects that influence tumor phenotypes.

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