Cancer-on-a-chip model shows that the adenomatous polyposis coli mutation impairs T cell engagement and killing of cancer spheroids.

Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance, to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the 2 h preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction.

Cell Culture in Microfluidic Droplets.

Cell manipulation in droplets has emerged as one of the great successes of microfluidic technologies, with the development of single-cell screening. However, the droplet format has also served to go beyond single-cell studies, namely by considering the interactions between different cells or between cells and their physical or chemical environment. These studies pose specific challenges linked to the need for long-term culture of adherent cells or the diverse types of measurements associated with complex biological phenomena. Here we review the emergence of droplet microfluidic methods for culturing cells and studying their interactions. We begin by characterizing the quantitative aspects that determine the ability to encapsulate cells, transport molecules, and provide sufficient nutrients within the droplets. This is followed by an evaluation of the biological constraints such as the control of the biochemical environment and promoting the anchorage of adherent cells. This first part ends with a description of measurement methods that have been developed. The second part of the manuscript focuses on applications of these technologies for cancer studies, immunology, and stem cells while paying special attention to the biological relevance of the cellular assays and providing guidelines on improving this relevance.

Clogging of a Rectangular Slit by a Spherical Soft Particle.

The capture of a soft spherical particle in a rectangular slit leads to a nonmonotonic pressure-flow rate relation at low Reynolds number. Simulations reveal that the flow induced deformations of the trapped particle focus the streamlines and pressure drop to a small region. This increases the resistance to flow by several orders of magnitude as the driving pressure is increased. As a result, two regimes are observed in experiments and simulations: a flow-dominated regime for small particle deformations, where flow rate increases with pressure, and an elastic-dominated regime in which solid deformations block the flow.

Time-resolved microfluidics unravels individual cellular fates during double-strand break repair.

BACKGROUND: Double-strand break repair (DSBR) is a highly regulated process involving dozens of proteins acting in a defined order to repair a DNA lesion that is fatal for any living cell. Model organisms such as Saccharomyces cerevisiae have been used to study the mechanisms underlying DSBR, including factors influencing its efficiency such as the presence of distinct combinations of microsatellites and endonucleases, mainly by bulk analysis of millions of cells undergoing repair of a broken chromosome. Here, we use a microfluidic device to demonstrate in yeast that DSBR may be studied at a single-cell level in a time-resolved manner, on a large number of independent lineages undergoing repair. RESULTS: We used engineered S. cerevisiae cells in which GFP is expressed following the successful repair of a DSB induced by Cas9 or Cpf1 endonucleases, and different genetic backgrounds were screened to detect key events leading to the DSBR efficiency. Per condition, the progenies of 80-150 individual cells were analyzed over 24 h. The observed DSBR dynamics, which revealed heterogeneity of individual cell fates and their contributions to global repair efficacy, was confronted with a coupled differential equation model to obtain repair process rates. Good agreement was found between the mathematical model and experimental results at different scales, and quantitative comparisons of the different experimental conditions with image analysis of cell shape enabled the identification of three types of DSB repair events previously not recognized: high-efficacy error-free, low-efficacy error-free, and low-efficacy error-prone repair. CONCLUSIONS: Our analysis paves the way to a significant advance in understanding the complex molecular mechanism of DSB repair, with potential implications beyond yeast cell biology. This multiscale and multidisciplinary approach more generally allows unique insights into the relation between in vivo microscopic processes within each cell and their impact on the population dynamics, which were inaccessible by previous approaches using molecular genetics tools alone.

Griottes: a generalist tool for network generation from segmented tissue images.

BACKGROUND: Microscopy techniques and image segmentation algorithms have improved dramatically this decade, leading to an ever increasing amount of biological images and a greater reliance on imaging to investigate biological questions. This has created a need for methods to extract the relevant information on the behaviors of cells and their interactions, while reducing the amount of computing power required to organize this information. RESULTS: This task can be performed by using a network representation in which the cells and their properties are encoded in the nodes, while the neighborhood interactions are encoded by the links. Here, we introduce Griottes, an open-source tool to build the "network twin" of 2D and 3D tissues from segmented microscopy images. We show how the library can provide a wide range of biologically relevant metrics on individual cells and their neighborhoods, with the objective of providing multi-scale biological insights. The library's capacities are demonstrated on different image and data types. CONCLUSIONS: This library is provided as an open-source tool that can be integrated into common image analysis workflows to increase their capacities.

Tip Streaming of a Lipid-Stabilized Double Emulsion Generated in a Microfluidic Channel.

Water/oil/water (w/o/w) double emulsions (DEs) are multicompartment structures which can be used in many technological applications and in fundamental studies as models of cell like microreactors or templates for other materials. Herein, we study the flow dynamics of water/oil/water double emulsions generated in a microfluidic device and stabilized with the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). We show that by varying the concentration of lipids in the oil phase (chloroform) or by modulating the viscosity of the aqueous continuous phase, the double emulsions under flow exhibit a rich dynamic behavior. An initial deformation of the double emulsions is followed by tube extraction at the rear end, relative to the flow direction, resulting in pinch off at the tube extremity by which small aqueous compartments are released. These compartments are phospholipid vesicles as deduced from fluorescence experiments. The overall process can thus be of help to shed light on the mechanical aspects of phenomena such as the budding and fusion in cell membranes.

Intermittent dynamics of bubble dissolution due to interfacial growth of fat crystals.

Foams are inherently unstable objects, that age and disappear over time. The main cause of foam aging is Ostwald ripening: smaller air bubbles within the foam empty their gas content into larger ones. One strategy to counter Ostwald ripening consists in creating armored bubbles, where solid particles adsorbed at the air/liquid interface prevent bubbles from shrinking below a given size. Here, we study the efficiency of coating air bubbles with fat crystals to prevent bubble dissolution. A monoglyceride, monostearin, is directly crystallized at the air/oil interface. Experiments on single bubbles in a microfluidic device show that the presence of monostearin fat crystals slows down dissolution, with an efficiency that depends on the crystal size. Bubble ripening in the presence of crystals exhibits intermittent dissolution dynamics, with phases of arrest, when crystals jam at the interface, followed by phases of dissolution, when monostearin crystals are ejected from the interface. In the end, crystals do not confer enough mechanical strength to the bubbles to prevent them from fully dissolving.

High-Throughput Measurements of Intra-Cellular and Secreted Cytokine from Single Spheroids Using Anchored Microfluidic Droplets.

While many single-cell approaches have been developed to measure secretions from anchorage-independent cells, these protocols cannot be applied to adherent cells, especially when these cells require to be cultured in 3D formats. Here, a platform to measure secretions from individual spheroids of human mesenchymal stem cells, cultured within microfluidic droplets is introduced. The platform allows to quantify the secretions from hundreds of individual spheroids in each device, by using a secondary droplet to bring functionalized micro-beads in proximity to each spheroid. Vascular endothelial growth factor (VEGF-A) is measured on and a broad distribution of secretion levels within the population of spheroids is observed. The intra-cellular level of VEGF-A on each spheroid, measured through immuno-staining, correlates well with the extra-cellular measurement, indicating that the heterogeneities observed at the spheroid level result from variations at the intra-cellular level. Further, the molecular accumulation within the droplets is modeled and it is found that physical confinement is crucial for measurements of protein secretions. The model predicts that the time to achieve a measurement scales with droplet volume. These first measurements of secretions from individual spheroids provide several new biological and technological insights.

Arresting dissolution by interfacial rheology design.

A strategy to halt dissolution of particle-coated air bubbles in water based on interfacial rheology design is presented. Whereas previously a dense monolayer was believed to be required for such an "armored bubble" to resist dissolution, in fact engineering a 2D yield stress interface suffices to achieve such performance at submonolayer particle coverages. We use a suite of interfacial rheology techniques to characterize spherical and ellipsoidal particles at an air-water interface as a function of surface coverage. Bubbles with varying particle coverages are made and their resistance to dissolution evaluated using a microfluidic technique. Whereas a bare bubble only has a single pressure at which a given radius is stable, we find a range of pressures over which bubble dissolution is arrested for armored bubbles. The link between interfacial rheology and macroscopic dissolution of [Formula: see text] 100 [Formula: see text]m bubbles coated with [Formula: see text] 1 [Formula: see text]m particles is presented and discussed. The generic design rationale is confirmed by using nonspherical particles, which develop significant yield stress at even lower surface coverages. Hence, it can be applied to successfully inhibit Ostwald ripening in a multitude of foam and emulsion applications.

Order to Disorder Transition in a Coarsening Two-Dimensional Foam.

We quantify the spatiotemporal transformation of a monodisperse and well-ordered monolayer of bubbles, as they undergo Ostwald ripening, by tracking the size polydispersity of the bubbles and local ordering of the foam. After nuclei of disorder appear at random locations, the transition takes place through two successive phases: first, the disordered regions grow while the value of polydispersity increases slowly, then the polydispersity grows rapidly once the disordered zones begin to merge together. The transition is captured by a modified logistic model.

Capture of colloidal particles by a moving microfluidic bubble.

Foams can be stabilized for long periods by the adsorption of solid particles on the liquid-gas interfaces. Although such long-term observations are common, mechanistic descriptions of the particle adsorption process are scarce, especially in confined flows, in part due to the difficulty of observing the particles in the complex gas-liquid dispersion of a foam. Here, we characterise the adsorption of micron-scale particles onto the interface of a bubble flowing in a colloidal aqueous suspension within a microfluidic channel. Three parameters are systematically varied: the particle size, their concentration, and the mean velocity of the colloidal suspension. The bubble coverage is found to increase linearly with position in the channel for all conditions but with a slope that depends on all three parameters. The optimal coverage is found for 1 µm particles at low flow rates and high concentrations. In this regime the particles pass the bubbles through the gutters between the interface and the channel corners, where the complex 3D flow leads them onto the interface. The largest particles cannot enter into the gutters and therefore provide very poor coverage. In contrast, particle aggregates can sediment onto the microchannel floor ahead of the bubble and get swept up by the advancing interface, thus improving the coverage for both large and medium particle sizes. These observations provide new insight on the influence of boundaries for particle adsorption at an air-liquid interface.

Droplet microfluidics driven by gradients of confinement.

The miniaturization of droplet manipulation methods has led to drops being proposed as microreactors in many applications of biology and chemistry. In parallel, microfluidic methods have been applied to generate monodisperse emulsions for applications in the pharmaceuticals, cosmetics, and food industries. To date, microfluidic droplet production has been dominated by a few designs that use hydrodynamic forces, resulting from the flowing fluids, to break drops at a junction. Here we present a platform for droplet generation and manipulation that does not depend on the fluid flows. Instead, we use devices that incorporate height variations to subject the immiscible interfaces to gradients of confinement. The resulting curvature imbalance along the interface causes the detachment of monodisperse droplets, without the need for a flow of the external phase. Once detached, the drops are self-propelled due to the gradient of surface energy. We show that the size of the drops is determined by the device geometry; it is insensitive to the physical fluid properties and depends very weakly on the flow rate of the dispersed phase. This allows us to propose a geometric theoretical model that predicts the dependence of droplet size on the geometric parameters, which is in agreement with experimental measurements. The approach presented here can be applied in a wide range of standard applications, while simplifying the device operations. We demonstrate examples for single-droplet operations and high-throughput generation of emulsions, all of which are performed in simple and inexpensive devices.

Airway reopening through catastrophic events in a hierarchical network.

When you reach with your straw for the final drops of a milkshake, the liquid forms a train of plugs that flow slowly initially because of the high viscosity. They then suddenly rupture and are replaced with a rapid airflow with the characteristic slurping sound. Trains of liquid plugs also are observed in complex geometries, such as porous media during petroleum extraction, in microfluidic two-phase flows, or in flows in the pulmonary airway tree under pathological conditions. The dynamics of rupture events in these geometries play the dominant role in the spatial distribution of the flow and in determining how much of the medium remains occluded. Here we show that the flow of a train of plugs in a straight channel is always unstable to breaking through a cascade of ruptures. Collective effects considerably modify the rupture dynamics of plug trains: Interactions among nearest neighbors take place through the wetting films and slow down the cascade, whereas global interactions, through the total resistance to flow of the train, accelerate the dynamics after each plug rupture. In a branching tree of microchannels, similar cascades occur along paths that connect the input to a particular output. This divides the initial tree into several independent subnetworks, which then evolve independently of one another. The spatiotemporal distribution of the cascades is random, owing to strong sensitivity to the plug divisions at the bifurcations.

Measuring Fast and Slow Enzyme Kinetics in Stationary Droplets.

We present a new microfluidic platform for the study of enzymtatic reactions using static droplets on demand. This allows us to monitor both fast and slow reactions with the same device and minute amounts of reagents. The droplets are produced and displaced using confinement gradients, which allows the experiments to be performed without having any mean flow of the external phase. Our device is used to produce six different pairs of drops, which are placed side by side in the same microfluidic chamber. A laser pulse is then used to trigger the fusion of each pair, thus initiating a chemcial reaction. Imaging is used to monitor the time evolution of enzymatic reactions. In the case of slow reactions, the reagents are completely mixed before any reaction is detected. This allows us to use standard Michaelis-Menten theory to analyze the time evolution. In the case of fast reactions, the time evolution takes place through a reaction-diffusion process, for which we develop a model that incorporates enzymatic reactions in the reaction terms. The theoretical predictions from this model are then compared to experiments in order to provide measurements of the chemical kinetics. The approach of producing droplets through confinement gradients and analyzing reactions within stationary drops provides an ultralow consumption platform. The physical principles are simple and robust, which suggests that the platform can be automated to reach large throughput analyses of enzymes.

Trapping and release of giant unilamellar vesicles in microfluidic wells.

We describe the trapping and release of giant unilamellar vesicles (GUVs) in a thin and wide microfluidic channel, as they cross indentations etched in the channel ceiling. This trapping results from the reduction of the membrane elastic energy, which is stored in the GUV as it squeezes to enter into the thin channel. We demonstrate that GUVs whose diameter is slightly larger than the channel height can be trapped and that they can be untrapped by flowing the outer fluid beyond a critical velocity. GUVs smaller than the channel height flow undisturbed while those much larger cannot squeeze into the thin regions. Within the range that allows trapping, larger GUVs are anchored more strongly than smaller GUVs. The ability to trap vesicles provides optical access to the GUVs for extended periods of time; this allows the observation of recirculation flows on the surface of the GUVs, in the forward direction near the mid-plane of the channel and in the reverse direction elsewhere. We also obtain the shape of GUVs under different flow conditions through confocal microscopy. This geometric information is used to derive a mechanical model of the force balance that equates the viscous effects from the outer flow with the elastic effects based on the variation of the membrane stretching energy. This model yields good agreement with the experimental data when values of the stretching moduli are taken from the scientific literature. This microfluidic approach provides a new way of storing a large number of GUVs at specific locations, with or without the presence of an outer flow. As such, it constitutes a high-throughput alternative to micropipette manipulation of individual GUVs for chemical or biological applications.

Transitions between three swimming gaits in Paramecium escape.

Paramecium and other protists are able to swim at velocities reaching several times their body size per second by beating their cilia in an organized fashion. The cilia beat in an asymmetric stroke, which breaks the time reversal symmetry of small scale flows. Here we show that Paramecium uses three different swimming gaits to escape from an aggression, applied in the form of a focused laser heating. For a weak aggression, normal swimming is sufficient and produces a steady swimming velocity. As the heating amplitude is increased, a higher acceleration and faster swimming are achieved through synchronized beating of the cilia, which begin by producing oscillating swimming velocities and later give way to the usual gait. Finally, escape from a life-threatening aggression is achieved by a "jumping" gait, which does not rely on the cilia but is achieved through the explosive release of a group of trichocysts in the direction of the hot spot. Measurements through high-speed video explain the role of trichocysts in defending against aggressions while showing unexpected transitions in the swimming of microorganisms. These measurements also demonstrate that Paramecium optimizes its escape pattern by taking advantage of its inertia.

Using a micro-device with a deformable ceiling to probe stiffness heterogeneities within 3D cell aggregates.

Recent advances in the field of mechanobiology have led to the development of methods to characterise single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional (3D) multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single polydimethylsiloxane piece cast on a 3D-printed mould and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chamber's ceiling. The device can be used in static or dynamic modes over time scales of seconds to hours, with displacement amplitudes from a fewµm to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labelling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighbourhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behaviour with the global mechanical response of the aggregates.

Generation of embryo-like structures from mouse embryonic stem cells treated with a chemical inhibitor of SUMOylation and cultured in microdroplets.

The field of stem cell-based embryo-like models is rapidly evolving, providing in vitro models of in utero stages of mammalian development. Here, we detail steps to first establish adherent spheroids composed of three cell types from mouse embryonic stem cells solely treated with a chemical inhibitor of SUMOylation. We then describe procedures for generating highly reproducible gastruloids from these dissociated spheroid cells, as well as embryo-like structures comprising anterior neural and trunk somite-like regions using an optimized microfluidics platform. For complete details on the use and execution of this protocol, please refer to Cossec et al. (2023).1.

Combinatorial drug screening on 3D Ewing sarcoma spheroids using droplet-based microfluidics.

Culturing and screening cells in microfluidics, particularly in three-dimensional formats, has the potential to impact diverse areas from fundamental biology to cancer precision medicine. Here, we use a platform based on anchored droplets for drug screening. The response of spheroids of Ewing sarcoma (EwS) A673 cells to simultaneous or sequential combinations of etoposide and cisplatin was evaluated. This was done by culturing spheroids of EwS cells inside 500 nL droplets then merging them with secondary droplets containing fluorescent-barcoded drugs at different concentrations. Differences in EwS spheroid growth and viability were measured by microscopy. After drug exposure such measurements enabled estimation of their IC50 values, which were in agreement with values obtained in standard multiwell plates. Then, synergistic drug combination was evaluated. Sequential combination treatment of EwS with etoposide applied 24 h before cisplatin resulted in amplified synergistic effect. As such, droplet-based microfluidics offers the modularity required for evaluation of drug combinations.

Transient suppression of SUMOylation in embryonic stem cells generates embryo-like structures.

Recent advances in synthetic embryology have opened new avenues for understanding the complex events controlling mammalian peri-implantation development. Here, we show that mouse embryonic stem cells (ESCs) solely exposed to chemical inhibition of SUMOylation generate embryo-like structures comprising anterior neural and trunk-associated regions. HypoSUMOylation-instructed ESCs give rise to spheroids that self-organize into gastrulating structures containing cell types spatially and functionally related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in a droplet microfluidic device form elongated structures that undergo axial organization reminiscent of natural embryo morphogenesis. Single-cell transcriptomics reveals various cellular lineages, including properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures. Transient SUMOylation suppression gradually increases DNA methylation genome wide and repressive mark deposition at Nanog. Interestingly, cell-to-cell variations in SUMOylation levels occur during early embryogenesis. Our approach provides a proof of principle for potentially powerful strategies to explore early embryogenesis by targeting chromatin roadblocks of cell fate change.