Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8 T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs

In many cell types, migration can be oriented towards a chemical stimulus. In mammals, for example, embryonic cells migrate to follow developmental cues, immune cells migrate toward sites of inflammation, and cancer cells migrate away from the primary tumour and toward blood vessels during metastasis. Understanding how cells migrate in 3D environments in response to chemical cues is thus crucial to understanding directed migration in normal and disease states. To date, chemotaxis in mammalian cells has been primarily studied using 2D migration models. However, it is becoming increasingly clear that the mechanisms by which cells migrate in 2D and 3D environments dramatically differ, and cells in their native environments are confronted with a complex chemical milieu. To address these issues, we developed a microfluidic device to monitor the behaviour of cells embedded in a 3D collagen matrix in the presence of complex concentration fields of chemoattractants. This tuneable microsystem enables the generation of (1) homogeneous, stationary gradients set by a purely diffusive mechanism, or (2) spatially evolving, stationary gradients, set by a convection-diffusion mechanism. The device allows for stable gradients over several days and is large enough to study the behaviour of large cell aggregates. We observe that primary mature dendritic cells respond uniformly to homogeneous diffusion gradients, while cell behaviour is highly position-dependent in spatially variable convection-diffusion gradients. In addition, we demonstrate a directed response of cancer cells migrating away from tumour-like aggregates in the presence of soluble chemokine gradients. Together, this microfluidic device is a powerful system to observe the response of different cells and aggregates to tuneable chemical gradients.

Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.

Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

The sealing of microfluidic devices remains a complex and time-consuming process requiring specific equipment and protocols: a universal method is thus highly desirable. We propose here the use of a commercially available sealing tape as a robust, versatile, reversible solution, compatible with cell and molecular biology protocols, and requiring only the application of manually achievable pressures. The performance of the seal was tested with regards to the most commonly used chip materials. For most materials, the bonding resisted 5 bars at room temperature and 1 bar at 95 °C. This method should find numerous uses, ranging from fast prototyping in the laboratory to implementation in low technology environments or industrial production.

Fluidization, a process in which a granular solid phase behaves like a fluid under the influence of an imposed upward fluid flow, is routinely used in many chemical and biological engineering applications. It brings, to applications involving fluid–solid exchanges, advantages such as high surface to volume ratio, constant mixing, low flow resistance, continuous operation and high heat transfer. We present here the physics of a new miniaturized, microfluidic fluidized bed, in which gravity is replaced by a magnetic field created by an external permanent magnet, and the solid phase is composed of magnetic microbeads with diameters ranging from 1 to 5 μm. These beads can be functionalized with different ligands, catalysts or enzymes, in order to use the fluidized bed as a continuous purification column or bioreactor. It allows flow-through operations at flow rates ranging from 100 nL min−1 up to 5 μL min−1 at low driving pressures (<100 mbar) with intimate liquid/solid contact and a continuous recirculation of beads for enhanced target capture efficiencies. The physics of the system presents significant differences as compared to conventional fluidized beds, which are studied here. The effects of magnetic field profile, flow chamber shape and magnetic bead dipolar interactions on flow regimes are investigated, and the different regimes of operation are described. Qualitative rules to obtain optimal operation are deduced. Finally, an exemplary use as a platform for immunocapture is provided, presenting a limit of detection of 0.2 ng mL−1 for 200 μL volume samples.

Multiphase and droplet microfluidic systems are growing in relevance in bioanalytical-related fields, especially due to the increased sensitivity, faster reaction times and lower sample/reagent consumption of many of its derived bioassays. Often applied to homogeneous (liquid/liquid) reactions, innovative strategies for the implementation of heterogeneous (typically solid/liquid) processes have recently been proposed. These involve, for example, the extraction and purification of target analytes from complex matrices or the implementation of multi-step protocols requiring efficient washing steps. To achieve this, solid supports such as functionalized particles (micro or nanometric) presenting different physical properties (e.g. magnetic, optical or others) are used for the binding of specific entities. The manipulation of such supports with different microfluidic principles has both led to the miniaturization of existing biomedical protocols and the development of completely new strategies for diagnostics and research. In this review, multiphase and droplet-based microfluidic systems using solid suspensions are presented and discussed with a particular focus on: i) working principles and technological developments of the manipulation strategies and ii) applications, critically discussing the level of maturity of these systems, which can range from initial proofs of concept to real clinical validations.

We study long range density fluctuations (hyperuniformity) in two-dimensional jammed packings of bidisperse droplets. Taking advantage of microfluidics, we systematically span a large range of size and concentration ratios of the two droplet populations. We identify various defects increasing long range density fluctuations mainly due to organization of local particle environment. By choosing an appropriate bidispersity, we fabricate materials with a high level of hyperuniformity. Interesting transparency properties of these optimized materials are established based on numerical simulations.

The concept of using stimuli-responsive hydrogels to actuate fluids in microfluidic devices is particularly attractive, but limitations, in terms of spatial resolution, speed, reliability and integration, have hindered its development during the past two decades. By patterning and grafting poly(N-isopropylacrylamide) PNIPAM hydrogel films on plane substrates with a 2 μm horizontal resolution and closing the system afterward, we have succeeded in unblocking bottlenecks that thermo-sensitive hydrogel technology has been challenged with until now. In this paper, we demonstrate, for the first time with this technology, devices with up to 7800 actuated micro-cages that sequester and release solutes, along with valves actuated individually with closing and opening switching times of 0.6±0.1 and 0.25±0.15 s, respectively. Two applications of this technology are illustrated in the domain of single cell handling and the nuclear acid amplification test (NAAT) for the Human Synaptojanin 1 gene, which is suspected to be involved in several neurodegenerative diseases such as Parkinson’s disease. The performance of the temperature-responsive hydrogels we demonstrate here suggests that in association with their moderate costs, hydrogels may represent an alternative to the actuation or handling techniques currently used in microfluidics, that are, pressure actuated polydimethylsiloxane (PDMS) valves and droplets.

We study long range density fluctuations (hyperuniformity) in two-dimensional jammed packings of bidisperse droplets. Taking advantage of microfluidics, we systematically span a large range of size and concentration ratios of the two droplet populations. We identify various defects increasing long range density fluctuations mainly due to organization of local particle environment. By choosing an appropriate bidispersity, we fabricate materials with a high level of hyperuniformity. Interesting transparency properties of these optimized materials are established based on numerical simulations.