Search

International audience

International audience; We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurement to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and lead to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa.s for leukocytes THP-1 cells, in agreement with literature data.

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like crawling of fibroblasts, relies on maturation of adhesion complexes and actin fiber traction, whereas the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on two-dimensional and in three-dimensional solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimental and computational evidence that leukocytes do swim, and that efficient propulsion is not fueled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell external membrane. Our model consists of a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. Furthermore, continuous paddling is enabled by a combination of external treadmilling and selective recycling by internal vesicular transport of cortex-bound transmembrane proteins. This mechanism explains observations that swimming is five times slower than the retrograde flow of cortex and also that lymphocytes are motile in nonadherent confined environments. Resultantly, the ubiquitous ability of mammalian amoeboid cells to migrate in two dimensions or three dimensions and with or without adhesion can be explained for lymphocytes by a single machinery of heterogeneous membrane treadmilling.

Collective cell migration is fundamental throughout development, during wound healing and in many diseases. Although much effort has focused on cell–cell junctions, a role for physical confinement in collective cell migration remains unclear. Here, we used adhesive microstripes of varying widths to mimic the spatial confinement experienced by follower cells within epithelial tissues. Our results reveal that the substrate area confinement is sufficient to modulate the three-dimensional cellular morphology without the need for intercellular adhesive cues. Our findings show a direct correlation between the migration velocity of confined cells and their cell–substrate adhesive area. Closer examination revealed that adhesive area confinement reduces lamellipodial protrusive forces, decreases the number of focal complexes at the leading edge and prevents the maturation of focal adhesions at the trailing edge, together leading to less effective forward propelling forces. The release of follower confinement required for the emergence of leader cells is associated with a threefold increase in contractile stress and a tenfold increase in protrusive forces, together providing a sufficient stress to generate highly motile mesenchymal cells. These findings demonstrate that epithelial confinement alone can induce follower-like behaviours and identify substrate adhesive area confinement as a key determinant of cell velocity in collective migration. Cells migrating within a collective naturally have restricted access to their surroundings. Experiments on micropatterned substrates now show that this confinement can regulate epithelial migration—governing cell morphology, forces and velocity.

Axonal guidance is a very important process during brain development, allowing to give it its structure and organization. The neuroscience scientific community has a growing interest in it during the last years. Several tools belonging to the field of microtechnologies, microfluidics and micropatterning are of important help to study axonal guidance in vitro. They allow to confine neurons and their axons and to apply gradients of guidance molecules. During this thesis, my goal was to develop a system to study the effect of guidance molecules gradients on axonal guidance. For that, I tested several configurations of microfluidic devices, micropatterns and combinations of both.First, we used two approaches to isolate dissociated neurons axons from their somas. Our goal was to study the effect of the molecular environment on neurons growth cones, with a high throughput. The first approach consisted in growing neurons on different proteins patterns. It also allowed to show their capacity to adhere on these patterns. The second one consisted in seeding neurons in a microfluidic device in which, during their growth, axons are separated from somas by microchannels. Then we studied the effect, on the axons, of guidance molecules gradients. To begin, we measured the effect of two guidance molecules: ephrin and semaphorin, by culturing neurons in the presence of patterned gradients of these two molecules. After that, we studied another model where neurons are closer from their environment in vivo, explants growing on laminin patterns containing a gradient. To help the explant positioning, we polymerized hydrogels. Then, we put explants next to patterned gradients of ephrin. Finally, we tried to obtain a soluble gradient of guidance molecules, over a long period of time (days), closer to existing gradients in vivo. In that goal, we wanted to build a microfluidic device enabling the application of a soluble gradient of guidance molecules on neurons. To obtain a constant gradient, we also cultured neurons next to cells expressing netrin, another guidance molecule. Finally, we cultured dissociated neurons and glial cells to study their interactions.All these experiments did not allow to obtain a reliable device to study the effect of molecules on axons growth and guidance. Nevertheless, the configuration consisting in a coculture of neurons next to cells releasing netrin allows us to obtain promising preliminary results. We thus drew up a group of methods that will enable us to finalize the development of a system to study axonal guidance, functional and efficient., Le guidage axonal est un processus très important dans le développement du cerveau, permettant de lui donner sa structure et son organisation. La communauté scientifique des neurosciences lui porte un intérêt grandissant ces dernières années. Plusieurs outils appartenant au domaine des microtechnologies, que sont la microfluidique et le micropatterning, sont d’une aide importante pour étudier le guidage axonal in vitro. Ils permettent de confiner les neurones et leurs axones et de leur appliquer des gradients de molécules de guidage. Lors de ce travail de thèse, j’ai voulu développer un système pour étudier l’effet de gradients de molécules de guidage sur le guidage axonal. J’ai pour cela testé plusieurs configurations de dispositifs microfluidiques, de micromotifs (micropatterns) et de combinaisons de ces derniers.Nous avons d’abord utilisé deux approches pour isoler les axones de neurones dissociés de leurs somas afin de pouvoir étudier, à haut débit, l’effet de l’environnement moléculaire sur les cônes de croissance des neurones. La première approche consistait à faire pousser des neurones sur des motifs (patterns) de différentes protéines. Elle a permis de montrer leur capacité d’adhésion spécifique sur ces motifs. La seconde consistait à ensemencer des neurones dans un dispositif microfluidique dans lequel, lors de leur pousse, les axones sont séparés des somas par des microcanaux. Nous avons ensuite étudié l’effet, sur les axones, de gradients de molécules de guidage. Pour commencer, nous avons mesuré l’effet de deux molécules de guidage : l’éphrine et la sémaphorine, en cultivant des neurones en présence de gradients patternés de ces deux molécules. Par la suite, nous avons étudié un autre modèle où les neurones sont plus proches de leur environnement in vivo, des explants poussant sur des motifs de laminine contenant un gradient. Pour aider au positionnement de l’explant, nous avons polymérisé des hydrogels. Ensuite, nous avons mis des explants à côté de gradients patternés d’éphrine. Enfin, nous avons cherché à obtenir un gradient soluble de molécules de guidage entretenu sur des temps longs, plus proche des gradients existant in vivo. Dans ce but, nous avons voulu fabriquer un dispositif microfluidique permettant d’appliquer un gradient soluble de molécules de guidage sur des neurones. Pour obtenir un gradient stable dans le temps, nous avons aussi cultivé des neurones à côté de cellules exprimant la nétrine, une autre molécule de guidage. Pour finir, nous avons cultivé des neurones et des glies dissociés pour étudier leurs interactions.L’ensemble de ces recherches n’a pas permis d’obtenir un dispositif fiable pour étudier l’effet de molécules sur la pousse et le guidage des axones. Néanmoins, la configuration consistant en une coculture de neurones à proximité de cellules relargant de la nétrine nous a permis d’obtenir des premiers résultats encourageants. Nous avons ainsi mis au point un ensemble de méthodes qui pourront nous permettre de finaliser le développement d’un système pour étudier le guidage axonal, fonctionnel et efficace.

The recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with mechanisms only partially deciphered. We studied here the guidance by flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins LFA-1 (αLβ2) and VLA-4 (α4β1), and showed that cells segregated in two populations of opposite orientation for combined adhesion. Sharp decisions of orientation were shown to rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-dominant downstream phenotypes. At the molecular level, bistability results from a differential front-rear polarization of both integrins affinity, combined with an inhibiting crosstalk of LFA-1 toward VLA-4. At the cellular level, directivity with or against the flow is mechanically mediated by the passive orientation of detached uropod or lamellipod by flow. This complete chain of logical events provides a unique mechanistic picture of a guiding mechanism, from stimuli to cell orientation.SignificanceCellular guidance is crucial to many biological functions, but the precise mechanisms remain unclear. We have analyzed here an original phenotype of flow-guided cells mimicking leukocytes crawling into the blood vessels and showed that thecontrolling parameterof cells decision to migrate upstream or downstream was therelative numberof two specific adhesion molecules, the integrins LFA-1 and VLA-4. The spatial polarisation of integrins affinity and an intermutually feedback of their activation create a bistable system where cells adhere either by their tip or their tail and orient respectively downstream or upstream. This mechanism therefore proposes a complete chain of event from stimuli to cell orientation and differs strongly from the chemotaxis paradigm because stimuli trigger no signaling.

International audience; A wide variety of cells migrate directionally in response to chemical or mechanical cues, however the mechanisms involved in cue detection and translation into directed movement are debatable. Here, we investigate a model of lymphocyte migration on the inner surface of blood vessels. Cells orient their migration against fluid flow, suggesting the existence of an adaptive mechano-tranduction mechanism. We find that flow detection may not require molecular mechano-sensors of shear stress and detection of flow direction can be achieved by the orientation in the flow of the non-adherent cell rear, the uropod. Uropods act as microscopic wind vanes that can transmit detection of flow direction into cell steering via the on-going machinery of polarity maintenance, without need for novel internal guidance signalling triggered by flow. Contrary to chemotaxis, which implies active regulation of cue-dependant signalling, upstream flow mechanotaxis of lymphocytes may only rely on a passive self-steering mechanism.

1-Microevaporation - Microfluidics is the branch of fluid mechanics dedicated to the study of flows in the channel withdimensions between 1 micron and 100 micron. The object of this chapter is to illustrate the basicprinciples and possible applications of microfluidic chip, called microevaporator. In the first part ofthe chapter, we present a detailed description of the physics of microevaporators using analyticalarguments, and describe some applications. In the second part of the chapter, we present theexperimental protocol of engineering of micro evaporator and different type of microfluidics device.2- On-chip microspectroscopy - The object of this chapter is to illustrate a method to measure absorption spectra during theprocess of growth of our materials in our microfluidic tools. The aim is to make an opticalcharacterization of our micro materials and to carry-out a spatio-temporal study of kineticproperties of our dispersion under study. This instrumental chapter presents the theoretical basis !of the method we used.3-Role of colloidal stability in the growth of micromaterials - We used combined microspectroscopy and videomicroscopy to follow the nucleation and growth ofmaterials made of core-shell Ag@SiO2 NPs in micro evaporators.!We evidence that the growth is actually not always possible, and instead precipitation may occurduring the concentration process. This event is governed by the concentration of dispersion in thereservoir and we assume that its origin come from ionic species that are concentrated all togetherwith the NPs and may alter the colloidal stability en route towards high concentration. 4-Microfluidic-induced growth and shape-up of three-dimensional extended arrays of denselypacked nano particles - In this chapter I present in details microfluidic evaporation experiments to engineer various denselypacked 3D arrays of NPs.5-Bulk metamaterials assembled by microfluidic evaporation - In this chapter I introduced the technique we used (microspot ellipsometry) in close collaborationswith V.Kravets and A.Grigorenko(University of Manchester) and with A.Aradian, P.Barois, A.Baron,K.Ehrhardt(CRPP, Pessac) to characterized the solids made of densely packed NPs. I describe theconstraints that emerge from the coupling between the small size of our materials and the opticalrequirements, the analysis and interpretation of the ellipsometry experiments show that for thematerial with high volume fraction of metal exists the strong electrical coupling between the NPsand the materials display an extremely high refraction index in the near infra-red regime.; Les métamatériaux sont définis comme étant des matériaux artificiels présentant des propriétés exotiques qui modifient la propagation des ondes électromagnétiques. À la fin des années 90, Pendry et al. démontrèrent théoriquement qu'il est possible de générer de tels métamatériaux, grâce à des structures particulières au sein du matériau (le fameux "splitring resonator"). Les métamatériaux sont donc structurés à une échelle inférieure à la longueur d'onde incidente, et décrits par une permittivité et une perméabilité effective. En 2000, Smith et al. fabriquèrent le premier métamatériau mais dans la gamme micro-onde. Les perspectives dans le domaine de l'optique (300800 nm) sont très prometteuses, mais le transfert des technologies utilisées en micro-ondes rencontre des obstacles. Un des défis dans le domaine émergent des métamatériaux est d'assembler à grande échelle des nanoparticules NPs (10-50 nm) en des super-réseaux présentant des propriétés collectives. Des nanostructures tridimensionnelles de matériaux nobles, ayant de fortes réponses plasmoniques, peuvent en effet générer des matériaux aux nouvelles propriétés optiques. Cette thèse fait partie du projet européen METACHEM, dont le but est de fabriquer des métamatériaux dans le domaine de l'infrarouge et du visible, en se basant sur l'utilisation de la nanochimie et de l'assemblage de matériaux. Plus précisément, ce travail de thèse se situe à l'interface entre les groupes de chimie qui synthétisent des nanoparticules en dispersion, et les groupes de caractérisation optique des matériaux. Dans ce travail de thèse, nous utilisons une technique originale la microévaporation basée sur les outils microfluidiques, afin de générer de façon contrôlée des assemblées 3D de nanoparticules (dimensions typiques 1 mm10 m 50 m).

International audience; Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in Acute Respiratory Distress Syndrome (ARDS). We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (Latrunculin A, Jasplakinolide and Blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in absence of myosin II activity, showing the major role of molecular motors on microvillus wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments.