Cytometrie multi-échelle de cultures cellulaires 3D dans des tableaux de billes de gel microfluidiques

Conventional 2D cell culture fails to reproduce emph{in vivo} conditions. In this PhD thesis, 3D cell culture is implemented into a highly integrated microfluidic platform. Adherent mammalian cells are encapsulated in droplets immobilized on a high density array of capillary traps called anchors. In each droplet, the cells reorganize into a single functional 3D microtissue called spheroid. The use of an hydrogel allows to extend the culturing time in microdroplets and to perfuse the array with aqueous solutions, for instance for immuno-cyto-chemistry. A single and viable spheroid can also be selectively retrieved from the microfluidic chip. High throughput and quantitative data is extracted at the population, spheroid (tens of thousands of spheroids) and cellular level emph{in situ} (hundreds of thousands of cells) thanks to fluorescent imaging and a custom image analysis software. As a first proof of concept, the viability, proliferation and functionality of hp sh s were demonstrated and correlated with morphological parameters. Drug toxicity experiments were also performed on this liver model. Then, human mesenchymal stem cell aggregates were produced and the spatial heterogeneities of the expression of proteins involved in their therapeutic properties were investigated. Finally, this technology was further developed to enable applying different biochemical conditions in each droplet. The production and culture of spheroids in this microfluidic platform could lead to major advances in many fields such as drug toxicity, high throughput drug screening, personalized cancer treatment, tissue engineering or disease modeling.Les conditions du corps humain ne sont pas reproduites fidèlement par la culture cellulaire traditionnelle en 2D. Dans cette thèse, des cultures cellulaires 3D sont réalisées dans une plateforme microfluidique hautement intégrée. Des cellules mammifères adhérentes sont encapsulées dans des gouttes immobilisées dans un tableau de pièges capillaires à haute densité. Dans chaque goutte, les cellules se réorganisent pour former un unique microtissu 3D et fonctionnel appelé sphéroïde. L'utilisation d'un hydrogel permet d'alonger le temps de culture et de perfuser le tableau avec des solutions aqueuses, par exemple pour de l'immuno-cyto-chimie. Un unique sphéroïde, viable, peut aussi être extrait de cette puce microfluidique. Des données quantitatives sont extraites à haut débit au niveau de la population, du sphéroïde (dizaines de miliers de sphéroïdes) et au niveau cellulaire emph{in situ} (centaines de miliers de cellules) grâce à de l'imagerie de fluorescence et au dévelopement d'un code d'analyse d'image. Une première preuve de concept a été obtenue en démontrant la viabilité, la prolifération et la fonctionalité de sphéroïdes d'hépatocytes et en les corrélant à des paramètres morphologiques. Ensuite, des aggrégats de cellules souches mésenchymales ont été produits et les hétérogénéités spatiales dans l'expression de protéines impliquées dans leurs propriétés thérapeutiques ont été étudiées. Enfin, cette technologie a été encore dévelopée pour permettre d'appliquer des conditions biochimiques différentes dans chaque goutte. La production et la culture de sphéroïdes dans cette plateforme microfluidique peut mener à des dévelopements importants dans beaucoup de domaines tels que l'analyse de la toxicité des médicaments, le criblage de médicaments à haut débit, le traitement personnalisé du cancer, l'ingénierie tissulaire ou la modélisation de maladies

Cytometrie multi-échelle de cultures cellulaires 3D dans des tableaux de billes de gel microfluidiques

Les conditions du corps humain ne sont pas reproduites fidèlement par la culture cellulaire traditionnelle en 2D. Dans cette thèse, des cultures cellulaires 3D sont réalisées dans une plateforme microfluidique hautement intégrée. Des cellules mammifères adhérentes sont encapsulées dans des gouttes immobilisées dans un tableau de pièges capillaires à haute densité. Dans chaque goutte, les cellules se réorganisent pour former un unique microtissu 3D et fonctionnel appelé sphéroïde. L'utilisation d'un hydrogel permet d'alonger le temps de culture et de perfuser le tableau avec des solutions aqueuses, par exemple pour de l'immuno-cyto-chimie. Un unique sphéroïde, viable, peut aussi être extrait de cette puce microfluidique. Des données quantitatives sont extraites à haut débit au niveau de la population, du sphéroïde (dizaines de miliers de sphéroïdes) et au niveau cellulaire emph{in situ} (centaines de miliers de cellules) grâce à de l'imagerie de fluorescence et au dévelopement d'un code d'analyse d'image. Une première preuve de concept a été obtenue en démontrant la viabilité, la prolifération et la fonctionalité de sphéroïdes d'hépatocytes et en les corrélant à des paramètres morphologiques. Ensuite, des aggrégats de cellules souches mésenchymales ont été produits et les hétérogénéités spatiales dans l'expression de protéines impliquées dans leurs propriétés thérapeutiques ont été étudiées. Enfin, cette technologie a été encore dévelopée pour permettre d'appliquer des conditions biochimiques différentes dans chaque goutte. La production et la culture de sphéroïdes dans cette plateforme microfluidique peut mener à des dévelopements importants dans beaucoup de domaines tels que l'analyse de la toxicité des médicaments, le criblage de médicaments à haut débit, le traitement personnalisé du cancer, l'ingénierie tissulaire ou la modélisation de maladies.Conventional 2D cell culture fails to reproduce emph{in vivo} conditions. In this PhD thesis, 3D cell culture is implemented into a highly integrated microfluidic platform. Adherent mammalian cells are encapsulated in droplets immobilized on a high density array of capillary traps called anchors. In each droplet, the cells reorganize into a single functional 3D microtissue called spheroid. The use of an hydrogel allows to extend the culturing time in microdroplets and to perfuse the array with aqueous solutions, for instance for immuno-cyto-chemistry. A single and viable spheroid can also be selectively retrieved from the microfluidic chip. High throughput and quantitative data is extracted at the population, spheroid (tens of thousands of spheroids) and cellular level emph{in situ} (hundreds of thousands of cells) thanks to fluorescent imaging and a custom image analysis software. As a first proof of concept, the viability, proliferation and functionality of hp sh s were demonstrated and correlated with morphological parameters. Drug toxicity experiments were also performed on this liver model. Then, human mesenchymal stem cell aggregates were produced and the spatial heterogeneities of the expression of proteins involved in their therapeutic properties were investigated. Finally, this technology was further developed to enable applying different biochemical conditions in each droplet. The production and culture of spheroids in this microfluidic platform could lead to major advances in many fields such as drug toxicity, high throughput drug screening, personalized cancer treatment, tissue engineering or disease modeling

Performances du positionnement de drone sous-marin autonome par communication acoustique

International audienc

Performances du positionnement de drone sous-marin autonome par communication acoustique

International audienc

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

International audienceAs 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

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

International audienceThe detection of toxic gases is becoming an important element in tackling increased air pollu- tion. 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 im- plemented 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 de- tect 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

Method for handling microdrops which include samples

A method for handling, in a microfluidic system, microdrops which include samples, including the steps of forming, in an oil, microdrops of an aqueous solution containing a sample, the oil and/or the aqueous solution containing a sample including a gelling agent; trapping the microdrops by means of surface-tension traps pre-arranged in a trapping area; and at least partially gelling the oil in the trapping area and/or at least partially gelling the trapped microdrops

Structural and Functional Mapping of Mesenchymal Bodies

International audienceThe 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

Mapping the structure and biological functions within mesenchymal bodies using microfluidics

International audienceOrganoids 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