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.

Evaluating immune response in vitro in a relevant microenvironment: a high-throughput microfluidic model for clinical screening.

Aim: Functional screening of new pharmaceutical compounds requires clinically relevant models to monitor essential cellular and immune responses during cancer progression, with or without treatment. Beyond survival, the emergence of resistant tumor cell clones should also be considered, including specific properties related to plasticity, such as invasiveness, stemness, escape from programmed cell death, and immune response. Numerous pathways are involved in these processes. Defining the relevant ones in the context of a specific tumor type will be key to designing an appropriate combination of inhibitors. However, the diversity and potential redundancy of these pathways remain a challenge for therapy. Methods: A new microfluidic device developed by Okomera was dedicated to the screening of drug treatment for breast cancer. This microchip includes 150 droplet-trapping microwells, offering multi-chip settings and multiple treatment choices. Results: After validating the system with established cell lines and a panel of drugs used clinically at Gustave Roussy, preclinical experiments were initiated including patient-derived xenograft (PDX) and primary tumor cells-derived tumoroids with the collaboration of Gustave Roussy clinicians. Tumor-isolated lymphocytes were also added to the tumoroids, using secondary droplets in proof-of-concept experiments. Conclusions: These results show the relevance of the methodology for screening large numbers of drugs, a wide range of doses, and multiple drug combinations. This methodology will be used for two purposes: 1) new drug screening from the compound library, using the high throughput potential of the chip; and 2) pre-clinical assay for a two-weeks response for personalized medicine, allowing evaluation of drug combinations to flag an optimized treatment with potential clinical application.

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.

Individual Control and Quantification of 3D Spheroids in a High-Density Microfluidic Droplet Array.

As three-dimensional cell culture formats gain in popularity, there emerges a need for tools that produce vast amounts of data on individual cells within the spheroids or organoids. Here, we present a microfluidic platform that provides access to such data by parallelizing the manipulation of individual spheroids within anchored droplets. Different conditions can be applied in a single device by triggering the merging of new droplets with the spheroid-containing drops. This allows cell-cell interactions to be initiated for building microtissues, studying stem cells' self-organization, or observing antagonistic interactions. It also allows the spheroids' physical or chemical environment to be modulated, as we show by applying a drug over a large range of concentrations in a single parallelized experiment. This convergence of microfluidics and image acquisition leads to a data-driven approach that allows the heterogeneity of 3D culture behavior to be addressed across the scales, bridging single-cell measurements with population measurements.

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.

Quantifying the sol-gel process and detecting toxic gas in an array of anchored microfluidic droplets.

The detection of toxic gases is becoming an important element in tackling increased air pollution. This has led to the development of gas sensors based on porous solid materials, which are produced using sol-gel chemistry and functionalized to change their optical qualities when in contact with the gas. In this context it is interesting to explore how microfluidics can be used to miniaturize these sensors, to improve their sensitivity and dynamic range, or to multiplex many gas measurements on a single chip. In this article we show how the sol-gel process can be implemented using anchored droplet microfluidics. The sensor material is partitioned into droplets while in the sol phase and maintained using capillary anchors. The ability to hold the droplets in place first allows us to study the sol-gel process. We use an original rheology method, which consists of observing the flows within stationary droplets that are submitted to an external flow, to measure the gelation time of the droplets. These measurements show a gelation time that decreases from 50 minutes to below 10 minutes as the temperature increases from 20 to 50 °C. We also measure the shrinkage of individual gel beads after gelation and find that this syneresis process is nearly finished after about 12 hours, leading to a final bead size that is 50% smaller than the initial droplet. Finally, we show that the beads can be functionalized and used to detect the presence of formaldehyde. These results first provide a new way to observe the physics of the sol-gel process in a well-controlled and quantitative fashion. Moreover they highlight how the coupling of microfluidics and sol-gel chemistry can be used to detect toxic gases, in view of answering the challenges surrounding gas detection in real-world settings.

Proteins that control the geometry of microtubules at the ends of cilia.

Cilia, essential motile and sensory organelles, have several compartments: the basal body, transition zone, and the middle and distal axoneme segments. The distal segment accommodates key functions, including cilium assembly and sensory activities. While the middle segment contains doublet microtubules (incomplete B-tubules fused to complete A-tubules), the distal segment contains only A-tubule extensions, and its existence requires coordination of microtubule length at the nanometer scale. We show that three conserved proteins, two of which are mutated in the ciliopathy Joubert syndrome, determine the geometry of the distal segment, by controlling the positions of specific microtubule ends. FAP256/CEP104 promotes A-tubule elongation. CHE-12/Crescerin and ARMC9 act as positive and negative regulators of B-tubule length, respectively. We show that defects in the distal segment dimensions are associated with motile and sensory deficiencies of cilia. Our observations suggest that abnormalities in distal segment organization cause a subset of Joubert syndrome cases.

Tetrahymena RIB72A and RIB72B are microtubule inner proteins in the ciliary doublet microtubules.

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.

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.