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Chapter 7 - Methods for Analysis of Apical Lumen Trafficking Using Micropatterned 3D Systems

Chapter 7 - Methods for Analysis of Apical Lumen Trafficking Using Micropatterned 3D Systems | CYTOO Newsletter | Scoop.it

Epithelial organs are made of interconnected branched networks of tubules, with a central lumen lined by a monolayer of epithelial cells. Certain epithelial cell lines can be converted into organotypic cultures by the addition of extracellular matrix components. When cultured in these conditions, epithelial cells reorient the axis of polarity, reorganize the membrane surfaces, and transport apical proteins to form the lumen in a process that recapitulates essential aspects of de novo apical membrane formation during epithelial organ morphogenesis. Micropatterns are a simple technique that allows cell culture in a controlled adhesive environment with extremely high precision, close to the nanometer scale. We have recently developed a method to culture MDCK cysts on micropatterns of different sizes and composition. Using this method we found that changes in micropattern shape and size can be used to modify cell contractility to understand its contribution to apical membrane formation. When imaging cysts on micropatterns the main advantage is that apical-directed vesicle trafficking is visualized in the x–y plane, which presents higher resolution on confocal microscopes. Thus, the use of micropatterns is an efficient setup to analyze polarized secretion with unprecedented higher resolution in both time and space.

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CYTOO’s 2D+/3D Cell Culture Platform in the spotlight with Manuel Théry presenting at the ASCB 2013 Annual Meeting

CYTOO will be at the American Society for Cell Biology Annual Meeting in New Orleans, LA, USA, to present its 2D+ and 3D Cell Culture Platform. This innovative solution opens fresh perspectives for cell based assay development, through more physiologically relevant cellular models.

On December 17, from 1:45 pm to 3:45 pm, Manuel Théry, PhD (iRTSV, CEA) will present a showcase entitled Enabling cell physiology through 2D+/3D cell culture on micropatterns with Sébastien Degot, Senior R&D Scientist (CYTOO).

CYTOO is also participating to the Thermo Scientific workshop, held on Sunday, December 15, 2013 from 11:30 to 1:30, to present the Advantages of CYTOO's 2D+/3D Cell Culture Platform for the imaging and analysis of complex cell models such as myogenesis: assessment using the Thermo Scientific Cellomics CellInsight.

 

 

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2nd prize for her poster on #EMT on #micropatterns at TEMTIA meeting... Congratulations Mithila Burute!

2nd prize for her poster on #EMT on #micropatterns at TEMTIA meeting... Congratulations Mithila Burute! | CYTOO Newsletter | Scoop.it
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Figure: Centrosome positioning on epithelial MCF10A cells.

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MEHTRICS Micropatterns networks

2D+ unlimited with micropatterns networks... Cells seeded on such networks can spread on one or more patterns and adopt a stable and normalized shape. After mitosis and migration to the neighboring pattern, cells readopt similar morphologies providing all the benefits of our 2D+ technology over time!

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Comprendre et contrôler la division des cellules souches

Comprendre et contrôler la division des cellules souches | CYTOO Newsletter | Scoop.it
Des chercheurs du CEA, du CNRS, de l’Université Grenoble Alpes, de l’Unité de Thérapie Cellulaire de l’hôpital Saint-Louis (AP-HP) et de l’UMR940 Inserm-Université Paris Diderot de l’IUH (Institut Universitaire d’Hématologie), montrent que la division des cellules souches est conditionnée par des contraintes physiques. Toute modification spatiale du microenvironnement de ces cellules peut ainsi influencer la façon dont elles assurent le renouvellement des tissus. Les chercheurs ont utilisé des techniques de traitement de surface issues de la micro-électronique pour obtenir ce résultat : cette méthode apparaît prometteuse pour maîtriser les capacités régénératives des cellules souches et orienter le devenir de leur descendance. Ces résultats sont publiés dans Cell Reports du 17 octobre.

 

 

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Human Bone Marrow Mesenchymal Stem Cells Regulate Biased DNA Segregation in Response to Cell Adhesion Asymmetry

Human Bone Marrow Mesenchymal Stem Cells Regulate Biased DNA Segregation in Response to Cell Adhesion Asymmetry | CYTOO Newsletter | Scoop.it

Biased DNA segregation is a mitotic event in which the chromatids carrying the original template DNA strands and those carrying the template copies are not segregated randomly into the two daughter cells. Biased segregation has been observed in several cell types, but not in human mesenchymal stem cells (hMSCs), and the factors affecting this bias have yet to be identified. Here, we have investigated cell adhesion geometries as a potential parameter by plating hMSCs from healthy donors on fibronectin-coated micropatterns. On symmetric micropatterns, the segregation of sister chromatids to the daughter cells appeared random. In contrast, on asymmetric micropatterns, the segregation was biased. This sensitivity to asymmetric extracellular cues was reproducible in cells from all donors but was not observed in human skin-derived fibroblasts or in a fibroblastic cell line used as controls. We conclude that the asymmetry of cell adhesion is a major factor in the regulation of biased DNA segregation in hMSCs.

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Patterning Pluripotent Stem Cells at a Single Cell Level

Studies of cell-extracellular matrix (ECM) interactions at a single cell level have drawn interest from scientists around the world. Subcellular ECM micropatterning techniques allow researchers to control cell shape, migration, and spindle orientation during mitosis potentially influencing the stem cell fate. Generally these studies have been limited to somatic cells rather than human pluripotent stem cells (hPSCs) which are capable of enormous differentiation potential. hPSCs require a defined ECM for attachment and express characteristic integrins mediating cell-substrate interactions. hPSCs also rely on cell-cell contacts for survival and to maintain self-renewal properties, but these circumstances also significantly limit hPSC observation at a single cell level. In addition, currently available methods for ECM micropatterning generally require a facility with trained personnel and intricate equipment to produce protein micropatterns. To overcome this problem, we have developed a protocol for vitronectin micropatterning using simple UV/ozone modification of polystyrene. Single hPSCs were able to attach and form characteristic stress fibers and focal adhesions similar to somatic cell types which demonstrate hPSC responsiveness to extracellular adhesive cues. Micropatterned hPSCs were able to be cultured for up to 48 hours while maintaining expression of pluripotency-associated transcription factor OCT4. Although further studies are necessary, the results of our investigation will potentially have a large impact on cell regenerative medicine and tissue engineering.

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Cells Architectures

Cells Architectures | CYTOO Newsletter | Scoop.it
CYTOO sponsors the "Cells Architectures" project, which stems from the collaboration between the biologists at the CEA and the artists of Group LAPS. Video-projection is used to overlay a cell's architecture on to a building's architecture to reveal similarities and differences.

 

http://cytoo.com/cytoo_events.php

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CYTOO’s 2D+ and 3D+ Cell Culture Platform at MipTec 2013

CYTOO’s 2D+ and 3D+ Cell Culture Platform at MipTec 2013 | CYTOO Newsletter | Scoop.it

CYTOO will attend the MipTec 2013 conference in Basel, Switzerland. The company will present the 2D+ and 3D+ Cell Culture Platforms, opening fresh perspectives for cell based assay development in drug discovery.

During MipTec, CYTOO will be Gold Sponsor for the Stem Cells in Biomedicine Scientific Forum, held on September 24. Sébastien Degot, Senior R&D Scientist, will then present the “Phenotypic profiling using probabilistic density maps generated from micropatterned cells”.

During the Thermo Scientific Industrial Symposium, held on September 26, he will also present the “advantages of CYTOO’s 2D+/3D+ Cell Culture Platform for the imaging and analysis of complex cell models: assessment using the Thermo Scientific Cellomics CellInsight”.

 

 

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Chapter 7 - Studying Mitochondria and Microtubule Localization and Dynamics in Standardized Cell Shapes

Chapter 7 - Studying Mitochondria and Microtubule Localization and Dynamics in Standardized Cell Shapes | CYTOO Newsletter | Scoop.it

Mammalian cells show a large diversity in shape and are both shape-changing and mobile when cultured on conventional uniform substrates. The use of micropatterning techniques limits the number of variable parameters, by imposing shape and standardized adhesive areas on the cells, which facilitates analysis. By changing size or shape of the micropattern, for example, forcing a polar axis on the cell, it is possible to study how these parameters impact organelle organization, distribution, and dynamics inside the cell. To study the mitochondrial network, which is composed of dynamic tubular organelles dependent on the microtubule cytoskeleton for its distribution, it is important to be able to distinguish between distinct mitochondria. Here, we present a practical method with which we spread the cells on large patterns created with deep UV technique, which not only makes the cells uniform in size and shape as well as immobile, and therefore easier to compare and analyze, but also expands the mitochondrial network and allows for an easier tracking of appropriately labeled individual mitochondria.

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A Novel Organelle Map Framework for High-Content Cell Morphology Analysis in High Throughput

A screening procedure was developed that takes advantage of the cellular normalization by micropatterning and a novel quantitative organelle mapping approach that allows unbiased and automated cell morphology comparison using black-box statistical testing. Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape and distribution of intracellular compartments avoiding strong cell-to-cell variation that is a major limitation of classical culture conditions. To detect changes in cell morphology induced by compound treatment, fluorescently labeled intracellular structures from several tens of micropatterned cells were transformed into probabilistic density maps. Then, the similarity or difference between two given density maps was quantified using statistical testing that evaluates differences directly from the data without additional analysis or any subjective decision. The versatility of this organelle mapping approach for different magnifications and its performance for different cell shapes has been assessed. Density-based analysis detected changes in cell morphology due to compound treatment in a small-scale proof-of-principle screen demonstrating its compatibility with high-throughput screening. This novel tool for high-content and high-throughput cellular phenotyping can potentially be used for a wide range of applications from drug screening to careful characterization of cellular processes.

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Micropatterned substrates coated with neuronal adhesion molecules for high-content study of synapse formation

Studying the roles of different proteins and the mechanisms involved in synaptogenesis is hindered by the complexity and heterogeneity of synapse types, and by the spatial and temporal unpredictability of spontaneous synapse formation. Here we demonstrate a robust and high-content method to induce selectively presynaptic or postsynaptic structures at controlled locations. Neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with various synaptogenic adhesion molecules. When plated on neurexin-1β-coated micropatterns, neurons expressing neuroligin-1 exhibit specific dendritic organization and selective recruitment of the postsynaptic scaffolding molecule PSD-95. Furthermore, functional AMPA receptors are trapped at neurexin-1β dots, as revealed by live-imaging experiments. In contrast, neurons plated on SynCAM1-coated substrates exhibit strongly patterned axons and selectively assemble functional presynapses. N-cadherin coating, however, is not able to elicit synapses, indicating the specificity of our system. This method opens the way to both fundamental and therapeutic studies of various synaptic systems.

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Cortical Dynein and Asymmetric Membrane Elongation Coordinately Position the Spindle in Anaphase

Mitotic spindle position defines the cell-cleavage site during cytokinesis. However, the mechanisms that control spindle positioning to generate equal-sized daughter cells remain poorly understood. Here, we demonstrate that two mechanisms act coordinately to center the spindle during anaphase in symmetrically dividing human cells. First, the spindle is positioned directly by the microtubule-based motor dynein, which we demonstrate is targeted to the cell cortex by two distinct pathways: a Gαi/LGN/NuMA-dependent pathway and a 4.1G/R and NuMA-dependent, anaphase-specific pathway. Second, we find that asymmetric plasma membrane elongation occurs in response to spindle mispositioning to alter the cellular boundaries relative to the spindle. Asymmetric membrane elongation is promoted by chromosome-derived Ran-GTP signals that locally reduce Anillin at the growing cell cortex. In asymmetrically elongating cells, dynein-dependent spindle anchoring at the stationary cell cortex ensures proper spindle positioning. Our results reveal the anaphase-specific spindle centering systems that achieve equal-sized cell division.

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A review of microfabrication and hydrogel engineering for micro-organs on chips

A review of microfabrication and hydrogel engineering for micro-organs on chips | CYTOO Newsletter | Scoop.it

This review highlights recent trends towards the development of in vitro multicellular systems with definite architectures, or “organs on chips”. First, the chemical composition and mechanical properties of the scaffold have to be consistent with the anatomical environment in vivo. In this perspective, the flourishing interest in hydrogels as cellular substrates has highlighted the main parameters directing cell differentiation that need to be recapitulated in artificial matrix. Another scaffold requirement is to act as a template to guide tissue morphogenesis. Therefore specific microfabrication techniques are required to spatially pattern the environment at microscale. 2D patterning is particularly efficient for organizing planar polarized cell types such as endothelial cells or neurons. However, most organs are characterized by specific sub units organized in three dimensions at the cellular level. The reproduction of such 3D patterns in vitro is necessary for cells to fully differentiate, assemble and coordinate to form a coherent micro-tissue. These physiological microstructures are often integrated in microfluidic devices whose controlled environments provide the cell culture with more life-like conditions than traditional cell culture methods. Such systems have a wide range of applications, for fundamental research, as tools to accelerate drug development and testing, and finally, for regenerative medicine.

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Tour de France de LaBiotech: Cytoo, Grenoble

Tour de France de LaBiotech: Cytoo, Grenoble | CYTOO Newsletter | Scoop.it

 

CYTOO est une société spécialisée dans les systèmes et outils pour les sciences de la vie qui a la particularité d’offrir une solution révolutionnaire permettant une fiabilité, une précision et une quantification puissante des cultures cellulaires...

 

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Tour de France of LaBiotech : the unique documentary film

 

 

CYTOO 's insight:

Now available with English subtitles!

 

Watch Mathieu Raul's interview at 38:26...

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The Bin/Amphiphysin/Rvs (BAR) Domain Protein Endophilin B2 Interacts with Plectin and Controls Perinuclear Cytoskeletal Architecture

Proteins of the Bin/amphiphysin/Rvs (BAR) domain superfamily are essential in controlling the shape and dynamics of intracellular membranes. Here, we present evidence for the unconventional function of a member of the endophilin family of BAR and Src homology 3 domain-containing proteins, namely endophilin B2, in the perinuclear organization of intermediate filaments. Using mass spectrometry analysis based on capturing endophilin B2 partners in in situ pre-established complexes in cells, we unravel the interaction of endophilin B2 with plectin 1, a variant of the cytoskeleton linker protein plectin as well as with vimentin. Endophilin B2 directly binds the N-terminal region of plectin 1 via Src homology 3-mediated interaction and vimentin indirectly via plectin-mediated interaction. The relevance of these interactions is strengthened by the selective and drastic reorganization of vimentin around nuclei upon overexpression of endophilin B2 and by the extensive colocalization of both proteins in a meshwork of perinuclear filamentous structures. By generating mutants of the endophilin B2 BAR domain, we show that this phenotype requires the BAR-mediated membrane binding activity of endophilin B2. Plectin 1 or endophilin B2 knockdown using RNA interference disturbed the perinuclear organization of vimentin. Altogether, these data suggest that the endophilin B2-plectin 1 complex functions as a membrane-anchoring device organizing and stabilizing the perinuclear network of vimentin filaments. Finally, we present evidence for the involvement of endophilin B2 and plectin 1 in nuclear positioning in individual cells. This points to the potential importance of the endophilin B2-plectin complex in the biological functions depending on nuclear migration and positioning.

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Tour de France de LaBiotech : le documentaire exclusif

Le Tour de France de LaBiotech est une expérience unique et inédite à la rencontre des entreprises de biotechnologies françaises.

CYTOO 's insight:

Interview de Mathieu Raul vous présentant CYTOO à 38:26

Encore merci à Philip et Joachim pour leur venue à Grenoble et bravo pour ce projet!

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CYTOO Inc. is looking for a high-performance Sales Representative!

CYTOO Inc. is looking for a high-performance Sales Representative! | CYTOO Newsletter | Scoop.it

http://cytoo.com/cytoo_career.php

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Advancements in high content analysis and application to toxicology

Advancements in high content analysis and application to toxicology | CYTOO Newsletter | Scoop.it

On November 4th, at 10:30, during the In Vitro Toxicology Society meeting, Joanne Young (Senior Reserch Scientist) will present the "Advancements in high content analysis and application to toxicology".

 

 

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CYTOO is looking for a Cell Applications Specialist

CYTOO is looking for a Cell Applications Specialist | CYTOO Newsletter | Scoop.it

CYTOO is looking for an enthusiastic and creative individual to contribute to its R&D and Services activities. You will be a key player in the current R&D and Services projects, capable of working in a challenging environment ensuring design, execution, analysis and reporting of experiments for completion of on time deliverables.

 

 

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The adhesive properties of tail primary cells tell a tale about mother's diet

The adhesive properties of tail primary cells tell a tale about mother's diet | CYTOO Newsletter | Scoop.it

Here is the new CYTOO Story!

Thank you Bertrand Kaeffer for writing "The adhesive properties of tail Primary cells tell a tale about mother's diet"!

Read the full text: http://www.cytoo.com/documents/CYTOO-Story-cell-adhesion.pdf

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Mechanisms of spindle positioning: cortical force generators in the limelight

Mechanisms of spindle positioning: cortical force generators in the limelight | CYTOO Newsletter | Scoop.it

Correct positioning of the spindle governs placement of the cytokinesis furrow and thus plays a crucial role in the partitioning of fate determinants and the disposition of daughter cells in a tissue. Converging evidence indicates that spindle positioning is often dictated by interactions between the plus-end of astral microtubules that emanate from the spindle poles and an evolutionary conserved cortical machinery that serves to pull on them. At the heart of this machinery lies a ternary complex (LIN-5/GPR-1/2/Gα in Caenorhabditis elegans and NuMA/LGN/Gαi in Homo sapiens) that promotes the presence of the motor protein dynein at the cell cortex. In this review, we discuss how the above components contribute to spindle positioning and how the underlying mechanisms are precisely regulated to ensure the proper execution of this crucial process in metazoan organisms

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A Mechanical Checkpoint Controls Multicellular Growth through YAP/TAZ Regulation by Actin-Processing Factors

A Mechanical Checkpoint Controls Multicellular Growth through YAP/TAZ Regulation by Actin-Processing Factors | CYTOO Newsletter | Scoop.it

Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.

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Tour de France 2013 Sup'Biotech : Sous le soleil de Marseille

Tour de France 2013 Sup'Biotech : Sous le soleil de Marseille | CYTOO Newsletter | Scoop.it

Déjà plus d'une semaine que Philip et Joachim sont sur les routes de France pour faire connaître au grand public les biotechnologies. Comme vous pouvez le voir dans la vidéo ci-dessous, les deux étudiants de Sup'Biotech ont déjà beaucoup de souvenirs en tête ! De Paris à Dijon, en passant par l'Allemagne et le sud de la France, ils ne chôment pas ! Au programme, vélo et visite de grandes entreprises du secteur comme Aldevron, Oncodesign ou Imaxio. Pour conclure cette belle première semaine, Philip et Joachim ont réussi à joindre l'utile à l'agréable en faisant une pause au bord de la piscine chez une de leurs amies ! Le vélo ça crève. Ensuite, nous l'avions vu, les deux étudiants étaient vite repartis sur les routes pour se rendre à Lyon. Mais cette fois-ci, les voilà à Grenoble pour visiter Cytoo, entreprise spécialisée dans la culture cellulaire. Mathieu Raul, le directeur du développement explique qu'ils sont désormais capables de faire prendre à la cellule la forme qu'ils souhaitent et ainsi augmenter l'efficacité des criblages sur ces cellules. A l'étage du dessus, se trouve PX' Therapeutics, qui propose un accompagnement de la recherche à toutes les entreprises de biotechnologies ne disposant pas des compétences en interne. Les pépites de Grenoble sont nombreuses !

CYTOO 's insight:

Merci à LaBiotech pour ce Tour de France et un petit crochet par Grenoble pour découvrir CYTOO et les micropatterns!

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