Behavior of liquid plugs at bifurcations in a microfluidic tree network

International audienceFlows in complex geometries, such as porous media or biological networks, often contain plugs of liquid flowing within air bubbles. These flows can be modeled in microfluidic devices in which the geometric complexity is well defined and controlled. We study the flow of wetting liquid plugs in a bifurcating network of micro-channels. In particular, we focus on the process by which the plugs divide as they pass each bifurcation. The key events are identified, corresponding to large modifications of the interface curvature, the formation of new interfaces, or the division of a single interface into two new ones. The timing of the different events and the amplitude of the curvature variations are analyzed in view of the design of an event-driven model of flow in branching micro-networks. They are found to collapse onto a master curve dictated by the network geometry. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4739072

In vivo dynamic characterization of heat shock response in Caenorhabditis elegans using high-throughput microfluidic systems and mathematical modelling

Caenorhabditis elegans is one of the most important and widely studied model organism in biological sciences. The high degree of genetic homology of this nematode with more complex organisms, including humans, makes it a primary model for studying fundamental, evolutionarily conserved processes in life, observable in a direct and simple manner. Particularly, the use of fluorescent protein reporters has enabled the direct localization and quantification of protein expression in a non-invasive manner at the cellular, tissue or organism level, further facilitating the visualization of dynamic processes in vivo. Recent years have seen much development of microfluidic technologies for the manipulation and processing of C. elegans in a precise, high-throughput and configurable manner. Importantly, fluorescence-based detection systems have been integrated with microfluidic platforms for phenotypic screens of mutagenized C. elegans encoding fluorescent proteins. Herein, we have developed microfluidic systems for high-throughput C. elegans in vivo screening and sorting based on the quantification of fluorescent protein expression. The rapid quantification of fluorescent proteins throughout the animal’s body allows for the extraction of fluorescence profiles of C. elegans, as well as basic characteristics of this expression, e.g. mean fluorescence value. Since the system allows processing of large numbers of C. elegans in a short amount of time (thousands of worms per hour), phenotypic variability among C. elegans populations can be assessed. This technology platform enables temporal dynamics of protein expression to be extracted and quantified with precision in a gentle manner, with no effect on C. elegans viability. Furthermore, the capability of the system of sorting populations in a quantitative manner, makes this microfluidic system suitable to be used for subsequent ageing and lifespan studies. In this thesis, we characterized and modelled the heat shock response of C. elegans at both individual and population levels, facilitated by the use of bespoke microfluidic systems. In living organisms, heat shock response is an ancient and highly conserved molecular mechanism that acts as a damage-control response to perturbations in protein homeostasis, restoring physiological levels of functional proteins, which is fundamental for preservation of cellular processes and essential to organismal health. In this study, we quantified the expression of the small heat shock protein HSP-16.2 in C. elegans in vivo following heat-shock stress using the transgenic strain TJ375 [hsp-16.2p::GFP], and observing the heterogeneous nature of this response. Our results uncovered significant variation in heat shock response dynamics at individual level, an effect that was also observable at population level and that can be directly related to differences in lifespan. We followed this dynamic variation of GFP expression in individual and population screening experiments along 48 hours after heat-shock, and demonstrated the diversity of dynamic expression profiles with sorting and subsequent screening experiments of groups of C. elegans expressing specific GFP expression levels at defined time points. We proposed a mathematical model of heat shock response in C. elegans, capable of explaining the observed individual-to-individual variability in our experiments. Notably, our results indicated that GFP expression variability is likely to arise from variability in individual protein turnover machinery, suggesting different protein translation and degradation rates amongst worms. Using these two parameters, we emulated the distribution of GFP expression profiles observed in large populations, finding good correlation between model and experimental data. Finally, we sorted a C. elegans population at relevant time points, collecting individuals with different dynamic expression profiles and we found statistical differences in the mean lifespan of extreme expression profile groups. This result encouraged our hypothesis that observable differences in GFP expression dynamics might provide valuable information on the protein homeostasis state of the studied animals

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

International audienceBackground: 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

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

International audienceBackground: 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

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

International audienceCulturing 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

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

Summary: 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

3D mechanical characterization of single cells and small organisms using acoustic manipulation and force microscopy

Quantitative micromechanical characterization of single cells and multicellular tissues or organisms is of fundamental importance to the study of cellular growth, morphogenesis, and cell-cell interactions. However, due to limited manipulation capabilities at the microscale, systems used for mechanical characterizations struggle to provide complete three-dimensional coverage of individual specimens. Here, we combine an acoustically driven manipulation device with a micro-force sensor to freely rotate biological samples and quantify mechanical properties at multiple regions of interest within a specimen. The versatility of this tool is demonstrated through the analysis of single Lilium longiflorum pollen grains, in combination with numerical simulations, and individual Caenorhabditis elegans nematodes. It reveals local variations in apparent stiffness for single specimens, providing previously inaccessible information and datasets on mechanical properties that serve as the basis for biophysical modelling and allow deeper insights into the biomechanics of these living systems

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

International audienceEvaluating 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

Acoustic Compressibility of Caenorhabditis elegans

The acoustic compressibility of Caenorhabditis elegans is a necessary parameter for further understanding the underlying physics of acoustic manipulation techniques of this widely used model organism in biological sciences. In this work, numerical simulations were combined with experimental trajectory velocimetry of L1 C. elegans larvae to estimate the acoustic compressibility of C. elegans. A method based on bulk acoustic wave acoustophoresis was used for trajectory velocimetry experiments in a microfluidic channel. The model-based data analysis took into account the different sizes and shapes of L1 C. elegans larvae (255 ± 26 μm in length and 15 ± 2 μm in diameter). Moreover, the top and bottom walls of the microfluidic channel were considered in the hydrodynamic drag coefficient calculations, for both the C. elegans and the calibration particles. The hydrodynamic interaction between the specimen and the channel walls was further minimized by acoustically levitating the C. elegans and the particles to the middle of the measurement channel. Our data suggest an acoustic compressibility κCe of 430 TPa-1 with an uncertainty range of ±20 TPa-1 for C. elegans, a much lower value than what was previously reported for adult C. elegans using static methods. Our estimated compressibility is consistent with the relative volume fraction of lipids and proteins that would mainly make up for the body of C. elegans. This work is a departing point for practical engineering and design criteria for integrated acoustofluidic devices for biological applications.status: publishe