Short-term memory effects in the phototactic behavior of microalgae.

Phototaxis, the directed motion in response to a light stimulus, is crucial for motile microorganisms that rely on photosynthesis, such as the unicellular microalga Chlamydomonas reinhardtii. It is well known that microalgae adapt to ambient light stimuli. On time scales of several dozen minutes, when stimulated long enough, the response of the microalga evolves as if the light intensity were decreasing [A. Mayer, Chlamydomonas: Adaptation phenomena in phototaxis, Nature, 1968, 217(5131), 875-876]. Here, we show experimentally that microalgae also have a short-term memory, on the time scale of a couple of minutes, which is the opposite of adaptation. At these short time scales, when stimulated consecutively, the response of C. reinhardtii evolves as if the light intensity were increasing. Our experimental results are rationalized by the introduction of a simplified model of phototaxis. Memory comes from the interplay between an internal biochemical time scale and the time scale of the stimulus; as such, these memory effects are likely to be widespread in phototactic microorganisms.

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.

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.

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.

Random network peristalsis in Physarum polycephalum organizes fluid flows across an individual.

Individuals can function as integrated organisms only when information and resources are shared across a body. Signals and substrates are commonly moved using fluids, often channeled through a network of tubes. Peristalsis is one mechanism for fluid transport and is caused by a wave of cross-sectional contractions along a tube. We extend the concept of peristalsis from the canonical case of one tube to a random network. Transport is maximized within the network when the wavelength of the peristaltic wave is of the order of the size of the network. The slime mold Physarum polycephalum grows as a random network of tubes, and our experiments confirm peristalsis is used by the slime mold to drive internal cytoplasmic flows. Comparisons of theoretically generated contraction patterns with the patterns exhibited by individuals of P. polycephalum demonstrate that individuals maximize internal flows by adapting patterns of contraction to size, thus optimizing transport throughout an organism. This control of fluid flow may be the key to coordinating growth and behavior, including the dynamic changes in network architecture seen over time in an individual.

Control parameter description of eukaryotic chemotaxis.

The chemotaxis of eukaryotic cells depends both on the average concentration of the chemoattractant and on the steepness of its gradient. For the social amoeba Dictyostelium discoideum, we test quantitatively the prediction by Ueda and Shibata [Biophys. J. 93, 11 (2007)] that the efficacy of chemotaxis depends on a single control parameter only, namely, the signal-to-noise ratio (SNR), determined by the stochastic fluctuations of (i) the binding of the chemoattractant molecule to the transmembrane receptor and (ii) the intracellular activation of the effector of the signaling cascade. For SNR < or approximately equal to 1, the theory captures the experimental findings well, while for larger SNR noise sources further downstream in the signaling pathway need to be taken into account.

Structural and Functional Mapping of Mesenchymal Bodies.

The formation of spheroids with mesenchymal stem/stromal cells (MSCs), mesenchymal bodies (MBs), is usually performed using bioreactors or conventional well plates. While these methods promote the formation of a large number of spheroids, they provide limited control over their structure or over the regulation of their environment. It has therefore been hard to elucidate the mechanisms orchestrating the structural organization and the induction of the trophic functions of MBs until now. We have recently demonstrated an integrated droplet-based microfluidic platform for the high-density formation and culture of MBs, as well as for the quantitative characterization of the structural and functional organization of cells within them. The protocol starts with a suspension of a few hundred MSCs encapsulated within microfluidic droplets held in capillary traps. After droplet immobilization, MSCs start clustering and form densely packed spherical aggregates that display a tight size distribution. Quantitative imaging is used to provide a robust demonstration that human MSCs self-organize in a hierarchical manner, by taking advantage of the good fit between the microfluidic chip and conventional microscopy techniques. Moreover, the structural organization within the MBs is found to correlate with the induction of osteo-endocrine functions (i.e., COX-2 and VEGF-A expression). Therefore, the present platform provides a unique method to link the structural organization in MBs to their functional properties. Graphic abstract: Droplet microfluidic platform for integrated formation, culture, and characterization of mesenchymal bodies (MBs). The device is equipped with a droplet production area (flow focusing) and a culture chamber that enables the culture of 270 MBs in parallel. A layer-by-layer analysis revealed a hierarchical developmental organization within MBs.

Mapping the structure and biological functions within mesenchymal bodies using microfluidics.

Organoids that recapitulate the functional hallmarks of anatomic structures comprise cell populations able to self-organize cohesively in 3D. However, the rules underlying organoid formation in vitro remain poorly understood because a correlative analysis of individual cell fate and spatial organization has been challenging. Here, we use a novel microfluidics platform to investigate the mechanisms determining the formation of organoids by human mesenchymal stromal cells that recapitulate the early steps of condensation initiating bone repair in vivo. We find that heterogeneous mesenchymal stromal cells self-organize in 3D in a developmentally hierarchical manner. We demonstrate a link between structural organization and local regulation of specific molecular signaling pathways such as NF-κB and actin polymerization, which modulate osteo-endocrine functions. This study emphasizes the importance of resolving spatial heterogeneities within cellular aggregates to link organization and functional properties, enabling a better understanding of the mechanisms controlling organoid formation, relevant to organogenesis and tissue repair.

Universal anchored-droplet device for cellular bioassays.

The ability to encapsulate cells individually in droplets has many potential applications, for example for observing the heterogeneity of behaviors within a population. However, implementing operations on moving droplets require feedback control and instruments that provide precise timing. These technical difficulties impede the adoption of droplet microfluidic protocols in nonspecialist labs. In this chapter we describe an approach to produce and manipulate droplets that remain stationary within a microfluidic chamber, by fabricating a microfluidic device having three-dimensional topography. The method uses microchannels that confine the fluids everywhere except in predefined regions where the channels have a large height, a technique known as "rails and anchors." By relying on the natural tendency of droplets to minimize their surface area, the approach provides a wide range of droplet manipulation tools. This chapter shows how this can be used to produce droplets, and several biological applications are demonstrated.

Multiscale cytometry and regulation of 3D cell cultures on a chip.

Three-dimensional cell culture is emerging as a more relevant alternative to the traditional two-dimensional format. Yet the ability to perform cytometry at the single cell level on intact three-dimensional spheroids or together with temporal regulation of the cell microenvironment remains limited. Here we describe a microfluidic platform to perform high-density three-dimensional culture, controlled stimulation, and observation in a single chip. The method extends the capabilities of droplet microfluidics for performing long-term culture of adherent cells. Using arrays of 500 spheroids per chip, in situ immunocytochemistry and image analysis provide multiscale cytometry that we demonstrate at the population scale, on 104 single spheroids, and over 105 single cells, correlating functionality with cellular location within the spheroids. Also, an individual spheroid can be extracted for further analysis or culturing. This will enable a shift towards quantitative studies on three-dimensional cultures, under dynamic conditions, with implications for stem cells, organs-on-chips, or cancer research.3D cell culture is more relevant than the two-dimensional format, but methods for parallel analysis and temporal regulation of the microenvironment are limited. Here the authors develop a droplet microfluidics system to perform long-term culture of 3D spheroids, enabling multiscale cytometry of individual cells within the spheroid.

Corrigendum: Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography.

This corrects the article DOI: 10.1038/nnano.2017.111.

Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography.

Rare-earth phosphors exhibit unique luminescence polarization features originating from the anisotropic symmetry of the emitter ion's chemical environment. However, to take advantage of this peculiar property, it is necessary to control and measure the ensemble orientation of the host particles with a high degree of precision. Here, we show a methodology to obtain the photoluminescence polarization of Eu-doped LaPO4 nanorods assembled in an electrically modulated liquid-crystalline phase. We measure Eu3+ emission spectra for the three main optical configurations (σ, π and α, depending on the direction of observation and the polarization axes) and use them as a reference for the nanorod orientation analysis. Based on the fact that flowing nanorods tend to orient along the shear strain profile, we use this orientation analysis to measure the local shear rate in a flowing liquid. The potential of this approach is then demonstrated through tomographic imaging of the shear rate distribution in a microfluidic system.

Universal microfluidic platform for bioassays in anchored droplets.

In spite of the large number of droplet-based microfluidic tools that have appeared in recent years, their penetration into non-specialist labs remains limited to a small number of applications. This is partly due to the lack of a generic platform that integrates all of the necessary operations for end-users, and partly to the increasing complexity that emerges as several operations are combined together. Here we report the development of a platform that provides the capabilities of multiwell plates in a two-dimensional array of nanoliter droplets: encapsulation, time-resolved monitoring and variation of well contents, as well as the ability to selectively extract the contents of any of the wells. We demonstrate these capabilities by encapsulating thousands of individual bacterial cells in droplets that are stored on a two-dimensional array of surface-energy anchors. Bacterial culture can be performed either in liquid or hydrogel droplets, both of which allow precise quantification using either standard measurements or digital enumeration. Using hydrogels allows the removal of the external oil that surrounds the aqueous drops, for instance in order to apply a gradient of antibiotics across the droplet population. This defines a protocol to obtain an antibiogram in a single experiment. Finally, the liquid to gel transition provides a robust way to selectively extract any droplet from the array, by melting it with a focused laser. When combined with further off-chip culture or genotyping, this platform provides a unique culturing environment to relate phenotype and genotype measurements on monoclonal colonies.

A stochastic description of Dictyostelium chemotaxis.

Chemotaxis, the directed motion of a cell toward a chemical source, plays a key role in many essential biological processes. Here, we derive a statistical model that quantitatively describes the chemotactic motion of eukaryotic cells in a chemical gradient. Our model is based on observations of the chemotactic motion of the social ameba Dictyostelium discoideum, a model organism for eukaryotic chemotaxis. A large number of cell trajectories in stationary, linear chemoattractant gradients is measured, using microfluidic tools in combination with automated cell tracking. We describe the directional motion as the interplay between deterministic and stochastic contributions based on a Langevin equation. The functional form of this equation is directly extracted from experimental data by angle-resolved conditional averages. It contains quadratic deterministic damping and multiplicative noise. In the presence of an external gradient, the deterministic part shows a clear angular dependence that takes the form of a force pointing in gradient direction. With increasing gradient steepness, this force passes through a maximum that coincides with maxima in both speed and directionality of the cells. The stochastic part, on the other hand, does not depend on the orientation of the directional cue and remains independent of the gradient magnitude. Numerical simulations of our probabilistic model yield quantitative agreement with the experimental distribution functions. Thus our model captures well the dynamics of chemotactic cells and can serve to quantify differences and similarities of different chemotactic eukaryotes. Finally, on the basis of our model, we can characterize the heterogeneity within a population of chemotactic cells.