Mechanical behavior of multi-cellular spheroids under osmotic compression

The internal and external mechanical environment plays an important role in tumorogenesis. As a proxy of an avascular early state tumor, we use multicellular spheroids, a composite material made of cells, extracellular matrix and permeating fluid. We characterize its effective rheology at the timescale of minutes to hours by compressing the aggregates with osmotic shocks and modeling the experimental results with an active poroelastic material that reproduces the stress and strain distributions in the aggregate. The model also predicts how the emergent bulk modulus of the aggregate as well as the hydraulic diffusion of the percolating interstitial fluid are modified by the preexisting active stress within the aggregate. We further show that the value of these two phenomenological parameters can be rationalized by considering that, in our experimental context, the cells are effectively impermeable and incompressible inclusions nested in a compressible and permeable matrix

VIDÉO-MICROSCOPIE SANS LENTILLE POUR LA BIOLOGIE CELLULAIRE 2D ET 3D

International audienceL'étude de l'évolution et de l'organisation de populations de cellules cultivées in vitro intéresse les biologistes depuis plusieurs dizaines d'années. À ces fins, d'importants progrès ont été réalisés dans les méthodes d'imagerie à l'échelle microscopique. Cependant, certaines informations demeurent inaccessibles, notamment à l'échelle mésoscopique, en raison du champ de vue réduit, ainsi que la complexité et le coût pour réaliser des acquisitions hors incubateur en temps réel sur de longues périodes. En réponse à ces limitations, nous avons développé la vidéo-microscopie sans lentille, en plaçant directement les cellules vivantes sur un capteur numérique en regard d'une illumination cohérente selon le principe de l'holographie en ligne. Cette technique permet l'observation d'une culture cellulaire sur un large champ de vue (24 mm² soit plusieurs dizaines de milliers de cellules), et ce à l'intérieur même de l'incubateur, autorisant de surcroît des acquisitions dynamiques couvrant des périodes allant de quelques jours à plusieurs semaines. À partir des images holographiques brutes acquises, nous pouvons remonter aux images refocalisées par reconstruction numérique jusqu'à une résolution de 2µm. Le traitement de ces images donne accès à des niveaux d'information quantifiables allant de la cellule unique à l'organisation inter-individus de la population. Avec des premières études sur des cultures standard de cellules sur substrat 2D, nous sommes aujourd'hui en mesure, avec notre dispositif et la force de l'imagerie holographique, d'explorer et d'étudier la vie cellulaire en 3D, nous rapprochant un peu plus de la réalité physiologique des phénomènes biologiques

Développement de microtechnologies et application à la culture cellulaire 3D pour étudier la formation d'acini prostatiques et la cancérogénèse

In all secretory epithelia from glandular tissues, there is a common structural and functional unit, the acinus. It is a well polarized and organized pluricellular structure that is spontaneously reconstructed in 3D culture, therefore closely mimics the real structure we find in vivo. For my purpose, acini are used as models for tumor initiation and cancer development. One of the objectives of Biomics laboratory is to identify the genetic and microenvironmental determinants of prostate acini morphogenesis and polarity. The strategy is based on High-Throughput (HT) RNA interference (RNAi)-based screening. To meet this objective, my project was to develop appropriate 3D cell models which closely mimic the cyst-like and duct-like structure of prostate. By optimizing conventional 3D culture in Matrigel, I could recapitulate prostate acini morphogenesis and showed that lumen formation is independent to the polarity, which appears later. However, the conventional 3D cell culture formats and analytical tools are not suited for HT Screening (HTS). They lack control over acini size, are label-dependant and therefore time-consuming and labor intensive. Also, classical microscopy offers a very limited field of view and hence does not allow observing a large amount of 3D structures for statistical analysis.Tout épithélium glandulaire sécrétoire est constitué d'une unité structurale et fonctionnelle commune, l'acinus. C'est une architecture sphérique pluricellulaire parfaitement différentiée et polarisée qui, reconstruite en culture 3D, mime l'organisation réelle du tissu. Etudier les déterminants environnementaux et génétiques qui gouvernent la transformation d'un acinus en sphéroïde s'apparentant à une tumeur est l'un des enjeux majeurs des modèles in vitro. Un des défis actuels est d'adapter ces modèles in vitro à des conditions de culture 3D qui soient compatibles avec la réalisation de cribles génétiques en 3D, basés par exemple sur l'ARN interférence (RNAi). Cependant, les formats standards de culture 3D et les méthodes analytiques ne sont pas compatibles aux cribles haut-débit. Ils ne permettent pas de contrôler la taille et la distribution des acini, sont dépendants d'immuno-marquages et les acquisitions sont longues. Par ailleurs, la microscopie confocale et vidéomicroscopie offrent un champ d'observation restreint qui ne permet pas d'observer un grand nombre de structures 3D en même temps, pour permettre une analyse statistique. Ainsi, dans le but i) de développer des modèles cellulaires appropriés en 3D, ii) d'adresser des questions fondamentales relatives au cancer de la prostate et iii) de réaliser des cribles RNAi dans un contexte plus pertinent que la culture 2D, j'ai développé des outils innovants au format microsystèmes adaptés à l'analyse haut-débit d'un grand nombre d'objets 3D. En optimisant les conditions de culture cellulaire 3D sur le modèle de la lignée cellulaire RWPE1, j'ai pu récapituler les étapes de formation des acini prostatiques et montrer que la formation du lumen est indépendante de la polarité et est gouvernée par deux mécanismes, « hollowing » et cavitation

Développement de microtechnologies et application à la culture cellulaire 3D pour étudier la formation d'acini prostatiques et la cancérogénèse

In all secretory epithelia from glandular tissues, there is a common structural and functional unit, the acinus. It is a well polarized and organized pluricellular structure that is spontaneously reconstructed in 3D culture, therefore closely mimics the real structure we find in vivo. For my purpose, acini are used as models for tumor initiation and cancer development. One of the objectives of Biomics laboratory is to identify the genetic and microenvironmental determinants of prostate acini morphogenesis and polarity. The strategy is based on High-Throughput (HT) RNA interference (RNAi)-based screening. To meet this objective, my project was to develop appropriate 3D cell models which closely mimic the cyst-like and duct-like structure of prostate. By optimizing conventional 3D culture in Matrigel, I could recapitulate prostate acini morphogenesis and showed that lumen formation is independent to the polarity, which appears later. However, the conventional 3D cell culture formats and analytical tools are not suited for HT Screening (HTS). They lack control over acini size, are label-dependant and therefore time-consuming and labor intensive. Also, classical microscopy offers a very limited field of view and hence does not allow observing a large amount of 3D structures for statistical analysis.Tout épithélium glandulaire sécrétoire est constitué d'une unité structurale et fonctionnelle commune, l'acinus. C'est une architecture sphérique pluricellulaire parfaitement différentiée et polarisée qui, reconstruite en culture 3D, mime l'organisation réelle du tissu. Etudier les déterminants environnementaux et génétiques qui gouvernent la transformation d'un acinus en sphéroïde s'apparentant à une tumeur est l'un des enjeux majeurs des modèles in vitro. Un des défis actuels est d'adapter ces modèles in vitro à des conditions de culture 3D qui soient compatibles avec la réalisation de cribles génétiques en 3D, basés par exemple sur l'ARN interférence (RNAi). Cependant, les formats standards de culture 3D et les méthodes analytiques ne sont pas compatibles aux cribles haut-débit. Ils ne permettent pas de contrôler la taille et la distribution des acini, sont dépendants d'immuno-marquages et les acquisitions sont longues. Par ailleurs, la microscopie confocale et vidéomicroscopie offrent un champ d'observation restreint qui ne permet pas d'observer un grand nombre de structures 3D en même temps, pour permettre une analyse statistique. Ainsi, dans le but i) de développer des modèles cellulaires appropriés en 3D, ii) d'adresser des questions fondamentales relatives au cancer de la prostate et iii) de réaliser des cribles RNAi dans un contexte plus pertinent que la culture 2D, j'ai développé des outils innovants au format microsystèmes adaptés à l'analyse haut-débit d'un grand nombre d'objets 3D. En optimisant les conditions de culture cellulaire 3D sur le modèle de la lignée cellulaire RWPE1, j'ai pu récapituler les étapes de formation des acini prostatiques et montrer que la formation du lumen est indépendante de la polarité et est gouvernée par deux mécanismes, « hollowing » et cavitation

Diversity of occupants’ activity impact on indoor air

The problem of indoor air quality (IAQ) has attracted a great deal of attention recently. One of interesting aspects of this problem is the characterization of human impact on IAQ. This work presents a method which allows to determine the diversity of building occupants influence on indoor air. The method is based on the supervised clustering of the indoor air measurement data in domains of various indoor air parameters. The introduced approach was applied to examine the exemplary data set. The diversity of impact was studied in the domain of air parameters which characterize thermal conditions indoors and in the domain of chemical parameters of indoor air. The obtained results showed that the diversity of impact was greatly related to the fact that occupants’ activities occurred in combinations, rather than individually. It was also demonstrated that the diversity of impact was domain dependent. Most of examined activities showed greater diversity of influence in the domain of parameters which characterize thermal conditions. This shows that the activity-specific impacts would be rather associated with the domain of chemical parameters of indoor air. The proposed method allows to obtain information which can be useful in the diagnostics of IAQ and for the control of ventilation

Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development

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High Accuracy Weigh-In-Motion Systems for Direct Enforcement

In many countries, work is being conducted to introduce Weigh-In-Motion (WIM) systems intended for continuous and automatic control of gross vehicle weight. Such systems are also called WIM systems for direct enforcement (e-WIM). The achievement of introducing e-WIM systems is conditional on ensuring constant, known, and high-accuracy dynamic weighing of vehicles. WIM systems weigh moving vehicles, and on this basis, they estimate static parameters, i.e., static axle load and gross vehicle weight. The design and principle of operation of WIM systems result in their high sensitivity to many disturbing factors, including climatic factors. As a result, weighing accuracy fluctuates during system operation, even in the short term. The article presents practical aspects related to the identification of factors disturbing measurement in WIM systems as well as methods of controlling, improving and stabilizing the accuracy of weighing results. Achieving constant high accuracy in weighing vehicles in WIM systems is a prerequisite for their use in the direct enforcement mode. The research results presented in this paper are a step towards this goal

Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression

International audienceThe surrounding microenvironment limits tumour expansion, imposing a compressive stress on the tumour, but little is known how pressure propagates inside the tumour. Here we present non-destructive cell-like microsensors to locally quantify mechanical stress distribution in three-dimensional tissue. Our sensors are polyacrylamide microbeads of well-defined elasticity, size and surface coating to enable internalization within the cellular environment. By isotropically compressing multicellular spheroids (MCS), which are spherical aggregates of cells mimicking a tumour, we show that the pressure is transmitted in a non-trivial manner inside the MCS, with a pressure rise towards the core. This observed pressure profile is explained by the anisotropic arrangement of cells and our results suggest that such anisotropy alone is sufficient to explain the pressure rise inside MCS composed of a single cell type. Furthermore, such pressure distribution suggests a direct link between increased mechanical stress and previously observed lack of proliferation within the spheroids core

Deciphering Cell Intrinsic Properties: A Key Issue for Robust Organoid Production

International audienceWe highlight the disposition of various cell types to self-organize into complexorgan-like structures without necessarily the support of any stromal cells,provided they are placed into permissive 3D culture conditions. The goal ofgenerating organoids reproducibly and efficiently has been hampered by poorunderstanding of the exact nature of the intrinsic cell properties at the origin oforganoid generation, and of the signaling pathways governing their differen-tiation. Using microtechnologies like microfluidics to engineer organoidswould create opportunities for single-cell genomics and high-throughputfunctional genomics to exhaustively characterize cell intrinsic properties. Amore complete understanding of the development of organoids wouldenhance their relevance as models to study organ morphology, function,and disease and would open new avenues in drug development and regener-ative medicine

Effect of an osmotic stress on multicellular aggregates

International audienceThere is increasing evidence that multicellular structures respond to mechanical cues, such as the confinement and compression exerted by the surrounding environment. In order to understand the response of tissues to stress, we investigate the effect of an isotropic stress on different biological systems. The stress is generated using the osmotic pressure induced by a biocompatible polymer. We compare the response of multicellular spheroids, individual cells and matrigel to the same osmotic perturbation. Our findings indicate that the osmotic pressure occasioned by polymers acts on these systems like an isotropic mechanical stress. When submitted to this pressure, the volume of multicellular spheroids decreases much more than one could expect from the behavior of individual cells