Université PSL

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RECHERCHER

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The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire
Laboratoire Colloïdes et Matériaux Divisés - Author links open overlay panelMagdaRybczynskaaJeanBaudryaEyerKlaus
Vaccine - 38(33) 5337-5342 - https://doi.org/10.1016/j.vaccine.2020.05.066 - 2020
Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
Laboratoire Colloïdes et Matériaux Divisés - Yacine Bounab, Klaus Eyer, Sophie Dixneuf, Magda Rybczynska, Cécile Chauvel, Maxime Mistretta, Trang Tran, Nathan Aymerich, Guilhem Chenon, Jean-François Llitjos, Fabienne Venet, Guillaume Monneret, Iain A. Gillespie, Pierre Cortez, Virginie Moucadel, Al
Nature Protocols - 15 2920–2955 - https://www.nature.com/articles/s41596-020-0354-0 - 2020
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.
The Quantitative Assessment of the Secreted IgG Repertoire after Recall to Evaluate the Quality of Immunizations
Laboratoire Colloïdes et Matériaux Divisés - Klaus Eyer, Carlos Castrillon, Guilhem Chenon, Jérôme Bibette, Pierre Bruhns, Andrew D. Griffiths and Jean Baudry
The Journal of Immunology - 205 8 - DOI: https://doi.org/10.4049/jimmunol.2000112 - 2020
One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (<24 h) and this early response was free of naive IgG-SCs. We further confirmed that our phenotypic analysis of IgG-SCs after recall strongly correlated with the different employed immunization schemes. Additionally, a phenotypic comparison of IgG-SCs presented in the spleen during immunization or after recall revealed similarities but also significant differences. The developed approach introduced a novel (to our knowledge), quantitative, and functional highly resolved alternative to study the quality of immunizations.
Emulsification with rectangular tubes
Laboratoire Colloïdes et Matériaux Divisés - Erwan Crestel, Ladislav Derzsi, Hugo Bartolomei, Jérôme Bibette, and Nicolas Bremond*
Phys. Rev. Fluids - 4 073602 - DOI: 10.1103/PhysRevFluids.4.073602 - 2019
The flow of two immiscible liquids or fluids in bounded systems where confinement
geometry varies can lead to drop or bubble formation. This phenomenon has been reported
in the context of oil recovery and named snap-off, or exploited for making emulsions,
and then foams, by using microfluidic systems, namely, microchannel emulsification or
step emulsification. We report a comprehensive experimental investigation of such an
emulsification process occurring at the end of a glass rectangular tube filled with oil and
immersed in a water bath. This allows us to clearly visualize the breakup event of the
dispersed phase liquid finger at the capillary’s end. Below a critical flow rate, the drop size
varies slowly with the flow rate and it is linked to the pinching time of the dispersed phase.
A semiempirical law that gives the resulting drop size as a function of fluid and geometrical
properties is proposed. However, this feature is altered for an aspect ratio of the rectangular
tube below 2.5 where the forming drop hinders the counterflow of the continuous phase
leading to larger drops. Then, above a critical flow rate, or capillary number that weakly
depends on the viscosity ratio of the two liquids, the neck adopts a quasistatic shape well
accounted for by a model based on a Hele-Shaw flow. In that case, drop formation is
driven by gravity and a transition from a dripping regime to a jetting regime is observed
at higher flow rates. Monodisperse foam can also be formed by injecting air. While the
overall dynamics of bubble formation shares similarities with an incompressible fluid, the
bubble size and the critical capillary number do not follow the same scaling laws.
Convective dispersion of particles in a segmented flow
Laboratoire Colloïdes et Matériaux Divisés - Wafa Bouhlel,1,2 S. Danial Naghib,1 Jérôme Bibette,1 and Nicolas Bremond 1
Phys. Rev. Fluids - - DOI: 10.1103/PhysRevFluids.4.104303 - 2019
Convective dispersion of solutes is inherent to flow in channels because of the nonuniformity of the velocity profile. When diffusion is negligible, for large particles for example,
the trajectory of particles can be solely described by a kinematic approach. Here, we
investigate such a phenomenon for micrometer-size beads flowing in a circular pipe. We
show that the presence of large bubbles, namely in the case of a segmented flow, either
prevents the convective dispersion or leads to the accumulation of particles at the rear of
the bubble moving in front. The destabilization of the initially homogeneous suspension
occurs when liquid inertia comes into play. Indeed, for moderate Reynolds number of
the particles, particles move away from the wall, thus exploring different flow lines that
finally impact the axial dispersion features. Moreover, since the bubbles impose an axial
boundary condition of the mean velocity, a net flux of particles directed along the flow
direction is built up above a critical particle Reynolds number. This work is motivated by
the understanding of the flow behavior of biological samples, and especially in the context
of cell encapsulation.
A tuneable microfluidic system for long duration chemotaxis experiments in a 3D collagen matrix
Laboratoire Colloïdes et Matériaux Divisés - Aizel K, Clark AG, Simon A, Geraldo S, Funfak A, Vargas P, Bibette J, Vignjevic DM, Bremond N.
Lab. Chip - 7;17(22): 3851-3861 - DOI: 10.1039/c7lc00649g - 2019
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.
A conductive hydrogel based on alginate and carbon nanotubes for probing microbial electroactivity
Laboratoire Colloïdes et Matériaux Divisés - Leopold Mottet, Domitille Le Cornec, a Jean-Marc Noe, Frederic Kanoufi, Brigitte Delord, Philippe Poulin, Jerome Bibettea and Nicolas Bremond
Soft Matter - 14 1434 - DOI: 10.1039/c7sm01929g - 2018
Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential
of generating electrical current. Despite their high value for sustainable energy, organic compound
production and bioremediation, a tool to probe the natural biodiversity and to select most efficient
microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but
the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we
develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes.
Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant
assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the
mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain
hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the
bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads
or the inner wall of the conductive capsules’ shell that function as an anode from which electrons
produced by the cells are collected.
Osmotic pressure in polyelectrolyte solutions: cell-model and bulk simulations
Laboratoire Colloïdes et Matériaux Divisés - Magnus Ullner, Khawla Qamhieh and Bernard Cabane
Soft Matter - 14 5832-5846 - https://doi.org/10.1039/C8SM00654G - 2018
The osmotic pressure of polyelectrolyte solutions as a function of concentration has been calculated by Monte Carlo simulations of a spherical cell model and by molecular dynamics simulations with periodic boundary conditions. The results for the coarse-grained polyelectrolyte model are in good agreement with experimental results for sodium polyacrylate and the cell model is validated by the bulk simulations. The cell model offers an alternative perspective on osmotic pressure and also forms a direct link to even simpler models in the form of the Poisson–Boltzmann approximation applied to cylindrical and spherical geometries. As a result, the non-monotonic behaviour of the osmotic coefficient seen in simulated salt-free solutions is shown not to rely on a transition between a dilute and semi-dilute regime, as is often suggested when the polyion is modelled as a linear flexible chain. The non-monotonic behaviour is better described as the combination of a finite-size effect and a double-layer effect. Parameters that represent the linear nature of the polyion, including an alternative to monomer concentration, make it possible to display a generalised behaviour of equivalent chains, at least at low concentrations. At high concentrations, local interactions become significant and the exact details of the model become important. The effects of added salt are also discussed and one conclusion is that the empirical additivity rule, treating the contributions from the polyelectrolyte and any salt separately, is a reasonable approximation, which justifies the study of salt-free solutions.
A conductive hydrogel based on alginate and carbon nanotubes for probing microbial electroactivity
Laboratoire Colloïdes et Matériaux Divisés - Leopold Mottet, Domitille Le Cornec, Jean-Marc Noel, Frederic Kanoufi, Brigitte Delord, Philippe Poulin, Jerome Bibette, and Nicolas Bremond
Soft Matter - 14 1434 - DOI: 10.1039/c7sm01929g - 2018
Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential of generating electrical current. Despite their high value for sustainable energy, organic compound production and bioremediation, a tool to probe the natural biodiversity and to select most efficient microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes. Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads or the inner wall of the conductive capsules' shell that function as an anode from which electrons produced by the cells are collected.

Micropipette-powered droplet based microfluidics
Laboratoire Colloïdes et Matériaux Divisés - Krzysztof Langer, Nicolas Bremonda, Laurent Boitard, Jean Baudry, and Jérôme Bibette
Biomicrofluidics - 12 044106 - https://doi.org/10.1063/1.5037795 - 2018
Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.
Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase.
Laboratoire Colloïdes et Matériaux Divisés - Rouzeau C Dagkesamanskaya A Langer K Bibette J Baudry J Pompon D Anton-Leberre V
Res Microbiol - 169(6) 335-342 - DOI : 10.1016/j.resmic.2018.06.002 - 2018
Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.
Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase.
Laboratoire Colloïdes et Matériaux Divisés - Rouzeau C Dagkesamanskaya A Langer K Bibette J Baudry J Pompon D Anton-Leberre V
Res Microbiol - 169(6) 335-342 - DOI : 10.1016/j.resmic.2018.06.002 - 2018
Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.
Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening
Laboratoire Colloïdes et Matériaux Divisés - Adilya Dagkesamanskaya 1, Krzysztof Langer , Alexandra S Tauzin , Catherine Rouzeau , Delphine Lestrade , Gabrielle Potocki-Veronese , Laurent Boitard , Jérôme Bibette , Jean Baudry , Denis Pompon , Véronique Anton-Leberre
J Microbiol Methods. - 147 59-65 - DOI: 10.1016/j.mimet.2018.03.001 - 2018
Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.
Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.
Laboratoire Colloïdes et Matériaux Divisés - Eyer K, Doineau RCL,Castrillon C, Briseño-Roa L, Menrath V, Mottet G, England P, Godina A, Brient-Litzler E, Nizak C, Jensen A, Griffiths AD, Bibette J, Bruhns P, Baudry J.
Nat Biotechnol. - 35(10) 977-982 - doi: 10.1038/nbt.3964. - 2017
Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening.
Hydrophobization of Silica Nanoparticles in Water: Nanostructure and Response to Drying Stress
Laboratoire Colloïdes et Matériaux Divisés - Solenn Moro, Caroline Parneix, Bernard Cabane†, Nicolas Sanson, and Jean-Baptiste d’Espinose de Lacaillerie
Langmuir - 33, 19 4709-4719 - DOI: 10.1021/acs.langmuir.6b04505 - 2017
We report on the impact of surface hydrophobization on the structure of aqueous silica dispersions and how this structure resists drying stress. Hydrophilic silica particles were hydrophobized directly in water using a range of organosilane precursors, with a precise control of the grafting density. The resulting nanostructure was precisely analyzed by a combination of small-angle X-ray scattering (SAXS) and cryo-microscopy (cryo-TEM). Then, the dispersion was progressively concentrated by drying, and the evolution of the nanostructures as a function of the grafting density was followed by SAXS. At the fundamental level, because the hydrophobic character of the silica surfaces could be varied continuously through a precise control of the grafting density, we were able to observe how the hydrophobic interactions change particles interactions and aggregates structures. Practically, this opened a new route to tailor the final structure, the residual porosity, and the damp-proof properties of the fully dried silica. For example, regardless of the nature of the hydrophobic precursor, a grafting density of 1 grafter per nm2 optimized the interparticle interactions in solution in view to maximize the residual porosity in the dried material (0.9 cm3/g) and reduced the water uptake to less than 4% in weight compared to the typical value of 13% for hydrophilic particles (at T = 25 °C and relative humidity = 80%).
Interparticle Capillary Forces at a Fluid − Fluid Interface with Strong Polymer-Induced Aging
Laboratoire Colloïdes et Matériaux Divisés - Stefano Cappelli, Arthur M. de Jon, Jean Baudry, and Menno W. J. Prins
Langmuir - 33 (3) 696–705 - DOI: 10.1021/acs.langmuir.6b03910 - 2017
We report on a measurement of forces between particles adsorbed at a water–oil interface in the presence of an oil-soluble polymer. The cationic polymer interacts electrostatically with the negatively charged particles, thereby modulating the particle contact angle and the magnitude of capillary attraction between the particles. However, polymer adsorption to the interface also generates an increase in the apparent interfacial viscosity over several orders of magnitude in a time span of a few hours. We have designed an experiment in which repeated motion trajectories are measured on pairs of particles. The experiment gives an independent quantification of the interfacial drag coefficient (10–7–10–4 Ns/m) and of the interparticle capillary forces (0.1–10 pN). We observed that the attractive capillary force depends on the amount of polymer in the oil phase and on the particle pair. However, the attraction appears to be independent of the surface rheology, with changes over a wide range of apparent viscosity values due to aging. Given the direction (attraction), the range (∼μm), and the distance dependence (∼1/S5) of the observed interparticle force, we interpret the force as being caused by quadrupolar deformations of the fluid–fluid interface induced by particle surface roughness. The results suggest that capillary forces are equilibrated in the early stages of interface aging and thereafter do not change anymore, even though strong changes in surface rheology still occur. The described experimental approach is powerful for studying dissipative as well as conservative forces of micro- and nanoparticles at fluid–fluid interfaces for systems out of equilibrium.
Controlled production of sub-millimeter liquid core hydrogel capsules for parallelized 3D cell culture
Laboratoire Colloïdes et Matériaux Divisés - Hugo Doméjean, Mathieu de la Motte Saint Pierre, Anette Funfak, Nicolas Atrux-Tallau, Kevin Alessandri, Pierre Nassoy, Jérôme Bibettea and Nicolas Bremond
Lab. Chip - 17 110-119 - DOI: 10.1039/C6LC00848H - 2017
Liquid core capsules having a hydrogel membrane are becoming a versatile tool for three-dimensional culture of micro-organisms and mammalian cells. Making sub-millimeter capsules at a high rate, via the breakup of a compound jet in air, opens the way to high-throughput screening applications. However, control of the capsule size monodispersity, especially required for quantitative bioassays, was still lacking. Here, we report how the understanding of the underlying hydrodynamic instabilities that occur during the process can lead to calibrated core–shell bioreactors. The requirements are: i) damping the shear layer instability that develops inside the injector arising from the co-annular flow configuration of liquid phases having contrasting viscoelastic properties; ii) controlling the capillary instability of the compound jet by superposing a harmonic perturbation onto the shell flow; iii) avoiding coalescence of drops during jet fragmentation as well as during drop flight towards the gelling bath; iv) ensuring proper engulfment of the compound drops into the gelling bath for building a closed hydrogel shell. We end up with the creation of numerous identical compartments in which cells are able to form multicellular aggregates, namely spheroids. In addition, we implement an intermediate composite hydrogel layer, composed of alginate and collagen, allowing cell adhesion and thus the formation of epithelia or monolayers of cells.
Osmotic pressures of lysozyme solutions from gas-like to crystal states
Laboratoire Colloïdes et Matériaux Divisés - Coralie Pasquier,ab Sylvie Beaufils,b Antoine Bouchoux, Sophie Rigault, Bernard Cabane, Mikael Lund, Valérie Lechevalier, Cécile Le Floch-Fouéré, Maryvonne Pasco, Gilles Pabœuf, Javier Pérezf and Stéphane Pezennec
Phys. Chem. - 18 28458-28465 - DOI: 10.1039/C6CP03867K - 2016
We obtained osmotic pressure data of lysozyme solutions, describing their physical states over a wide concentration range, using osmotic stress for pressures between 0.05 bar and about 40 bar and volume fractions between 0.01 and 0.61. The osmotic pressure vs. volume fraction data consist of a dilute, gas-phase regime, a transition regime with a high-compressibility plateau, and a concentrated regime where the system is nearly incompressible. The first two regimes are shifted towards a higher protein volume fraction upon decreasing the strength or the range of electrostatic interactions. We describe this shift and the overall shape of the experimental data in these two regimes through a model accounting for a steric repulsion, a short-range van der Waals attraction and a screened electrostatic repulsion. The transition is caused by crystallization, as shown by small-angle X-ray scattering. We verified that our data points correspond to thermodynamic equilibria, and thus that they consist of the reference experimental counterpart of a thermodynamic equation of state.
Power transduction of actin filaments ratcheting in vitro against a load
Laboratoire Colloïdes et Matériaux Divisés - Démoulin D., Carlier M.F, Bibette J. and Baudry J.
Proc. Nat. Acad. Sci. USA - Vol.111(n°50) 17845-50 - DOI: 10.1073/pnas.1414184111 - 2014
The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.
Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro
Laboratoire Colloïdes et Matériaux Divisés - K. Alessandri, Bibhu Ranjan Sarangi, V. Valérïévitch Gurchenkov, B. Sinha, T. Kissling, L. Fetler, F. Rico, Simon Scheuring, Christophe Lamaze, S. Geraldo, D. Vignjević, H. Doméjean, L. Rolland, A. Funfak, Jérôme Bibette, Nicolas Bremond, Pierre Nas
Proc. Nat. Acad. Sci. USA - vol.110(n°37) 14843–48 - DOI: 10.1073/pnas.1309482110 - 2013
Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell–cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.

53 publications.