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1. Compressive stress triggers fibroblasts spreading over cancer cells to generate carcinoma in situ organization

Author

Fabien Bertillot, Laetitia Andrique, Carlos Ureña Martin, Olivier Zajac, Ludmilla de Plater, Michael M. Norton, Aurélien Richard, Kevin Alessandri, Basile G. Gurchenkov, Florian Fage, Atef Asnacios, Christophe Lamaze, Moumita Das, Jean- Léon Maître, Pierre Nassoy, and Danijela Matic Vignjevic

Subjects
Biology (General), QH301-705.5
Abstract

Abstract At the early stage of tumor progression, fibroblasts are located at the outer edges of the tumor, forming an encasing layer around it. In this work, we have developed a 3D in vitro model where fibroblasts’ layout resembles the structure seen in carcinoma in situ. We use a microfluidic encapsulation technology to co-culture fibroblasts and cancer cells within hollow, permeable, and elastic alginate shells. We find that in the absence of spatial constraint, fibroblasts and cancer cells do not mix but segregate into distinct aggregates composed of individual cell types. However, upon confinement, fibroblasts enwrap cancer cell spheroid. Using a combination of biophysical methods and live imaging, we find that buildup of compressive stress is required to induce fibroblasts spreading over the aggregates of tumor cells. We propose that compressive stress generated by the tumor growth might be a mechanism that prompts fibroblasts to form a capsule around the tumor.

Published
2024
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2. Quantitative cell-based model predicts mechanical stress response of growing tumor spheroids over various growth conditions and cell lines.

Author

Paul Van Liedekerke, Johannes Neitsch, Tim Johann, Kevin Alessandri, Pierre Nassoy, and Dirk Drasdo

Subjects
Biology (General), QH301-705.5
Abstract

Model simulations indicate that the response of growing cell populations on mechanical stress follows the same functional relationship and is predictable over different cell lines and growth conditions despite experimental response curves look largely different. We develop a hybrid model strategy in which cells are represented by coarse-grained individual units calibrated with a high resolution cell model and parameterized by measurable biophysical and cell-biological parameters. Cell cycle progression in our model is controlled by volumetric strain, the latter being derived from a bio-mechanical relation between applied pressure and cell compressibility. After parameter calibration from experiments with mouse colon carcinoma cells growing against the resistance of an elastic alginate capsule, the model adequately predicts the growth curve in i) soft and rigid capsules, ii) in different experimental conditions where the mechanical stress is generated by osmosis via a high molecular weight dextran solution, and iii) for other cell types with different growth kinetics from the growth kinetics in absence of external stress. Our model simulation results suggest a generic, even quantitatively same, growth response of cell populations upon externally applied mechanical stress, as it can be quantitatively predicted using the same growth progression function.

Published
2019
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3. Engineering 3D micro-compartments for highly efficient and scale-independent expansion of human pluripotent stem cells in bioreactors

Author

Philippe J.R. Cohen, Elisa Luquet, Justine Pletenka, Andrea Leonard, Elise Warter, Basile Gurchenkov, Jessica Carrere, Clément Rieu, Jerome Hardouin, Fabien Moncaubeig, Michael Lanero, Eddy Quelennec, Helene Wurtz, Emilie Jamet, Maelle Demarco, Celine Banal, Paul Van Liedekerke, Pierre Nassoy, Maxime Feyeux, Nathalie Lefort, and Kevin Alessandri

Subjects
Biomaterials, Mechanics of Materials, Biophysics, Ceramics and Composites, Bioengineering
Published
2023
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4. A novel 3D culture model recapitulates primary FL B cell features and promotes their survival

Author

Valérie Le Morvan, Pierre Nassoy, Patricia Legoix, Céline Pangault, Simon Latour, Pierre Soubeyran, Laurence Bresson-Bepoldin, Frédéric Mourcin, Kevin Alessandri, Elise Dessauge, Martina Prochazkova-Carlotti, Julien Saint-Vanne, Lea Broca-Brisson, Nelson Hélaine, Karin Tarte, Jean-Philippe Merlio, Sylvain Baulande, Laurent Deleurme, Isabelle Mahouche, Laetitia Andrique, Céline Monvoisin, Gaëlle Recher, Christelle Dussert, Claire Lamaison, Marine Seffals, Microenvironment, Cell Differentiation, Immunology and Cancer (MICMAC), Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Etablissement français du sang [Rennes] (EFS Bretagne), University of Toronto, Laboratoire Photonique, Numérique et Nanosciences (LP2N), Université de Bordeaux (UB)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), Actions for OnCogenesis understanding and Target Identification in ONcology (ACTION), Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Bordeaux (UB), Université de Rennes (UR), Institut Curie [Paris], Plateforme de sequencage ICGEX, Bordeaux Research In Translational Oncology [Bordeaux] (BaRITOn), Université de Bordeaux (UB)-CHU Bordeaux [Bordeaux]-Institut National de la Santé et de la Recherche Médicale (INSERM), UNICANCER, CHU Bordeaux [Bordeaux], This work was supported by INSERM, University of Bordeaux, Ligue Régionalecontre le Cancer (comités de Gironde, Charentes, Pyrénées, Landes), the FondationARC pour la Recherche sur le Cancer (Grant PGA1 RF20170205386), the InstitutNational du cancer (INCA AAP PNP-19-009), Emergence GSO Cancéropole, andSIRIC BRIO. S.L. is supported by Ligue Nationale contre le Cancer and C.L. is arecipient of a doctoral fellowship from the FHU CAMIn and LBB (Léa Broca-Brisson)of a master fellowship from CNRS GDR Imabio. J.S.V. is funded by the InfrastructureeCellFrance (ANR-11-INBS-005). The 3D capsules were obtained from the VoxCellOrganoïds Plateform, a service of the CNRS-INSERM and Bordeaux University -UMS TBMCore 3427. We thank Stéphanie Durand-Panteix for her technical supportwith transduction experiments. We thank Atika Zouine and Vincent Pitard fortechnical assistance at the Flow cytometry facility, CNRS UMS 3427, INSERM US005, Univ. Bordeaux, F-33000 Bordeaux, France. We acknowledge the BordeauxImaging Center, a service unit of the CNRS-INSERM and Bordeaux University,member of the national infrastructure France BioImaging supported by the FrenchResearch Agency (ANR-10-INBS-04). Cell sorting was performed at the Biosit FlowCytometry and Cell Sorting Facility CytomeTRI (UMS 6480 Biosit, Rennes, France).High-throughput sequencing has been performed by the ICGex NGS platform of theInstitut Curie supported by the grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from the ANR, by the ITMO-CANCER,and by the SiRIC-Curie program (SiRIC Grant INCa-DGOS-4654)., The authors are indebted to the Centre de Ressources Biologiques (CRB)-Santé (BB-0033-00056) of Rennes hospital for the processing of biological samples., ANR-11-INBS-0005,ECELLFRANCE,Développement d'une Plateforme Nationale pour la médecine régénératrice(2011), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-EQPX-0003,ICGex,Equipement de biologie intégrative du cancer pour une médecine personnalisée(2010), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Plateforme de Séquençage ADN haut débit [Institut Curie] (NGS - ICGex), H2P2 - Histo Pathologie Hight Precision (H2P2), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Université de Rennes 1 - Faculté de Médecine (UR1 Médecine), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Bordeaux Segalen - Bordeaux 2-Institut Bergonié [Bordeaux], UNICANCER-UNICANCER, Université de Rennes (UNIV-RENNES), Chard-Hutchinson, Xavier, Infrastructures - Développement d'une Plateforme Nationale pour la médecine régénératrice - - ECELLFRANCE2011 - ANR-11-INBS-0005 - INBS - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, and Equipements d'excellence - Equipement de biologie intégrative du cancer pour une médecine personnalisée - - ICGex2010 - ANR-10-EQPX-0003 - EQPX - VALID

Subjects
Stromal cell, Lymphoma, B-Cell, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], [SDV]Life Sciences [q-bio], Follicular lymphoma, neoplasms, lymphoma, [SDV.CAN]Life Sciences [q-bio]/Cancer, doxorubicin, Extracellular matrix, 03 medical and health sciences, 0302 clinical medicine, alginates, [SDV.CAN] Life Sciences [q-bio]/Cancer, follicular lymphoma, immune system diseases, hemic and lymphatic diseases, b-lymphocytes, medicine, Tumor Microenvironment, antineoplastic agents, Humans, cancer, Doxorubicin, Cell encapsulation, B cell, 030304 developmental biology, Cell Proliferation, 0303 health sciences, Chemistry, Lymphoma, Non-Hodgkin, Hematology, medicine.disease, In vitro, Lymphoma, b-cell lymphomas, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, 030220 oncology & carcinogenesis, Cancer research, bcl-2 protein, medicine.drug
Abstract

Non-Hodgkin B-cell lymphomas (B-NHL) mainly develop within lymph nodes as aggregates of tumor cells densely packed with their surrounding microenvironment, creating a tumor niche specific to each lymphoma subtypes. In vitro preclinical models mimicking biomechanical forces, cellular microenvironment, and 3D organization of B-cell lymphomas remain scarce, while all these parameters are key determinants of lymphomagenesis and drug resistance. Using a microfluidic method based on cell encapsulation inside permeable, elastic, and hollow alginate microspheres, we developed a new tunable 3D model incorporating lymphoma B cells, extracellular matrix (ECM), and/or tonsil stromal cells (TSC). Under 3D confinement, lymphoma B cells were able to form cohesive spheroids resulting from overexpression of ECM components. Moreover, lymphoma B cells and TSC dynamically formed self-organized 3D spheroids favoring tumor cell growth. 3D culture induced resistance to the classical chemotherapeutic agent doxorubicin, but not to the BCL2 inhibitor ABT-199, identifying this approach as a relevant in vitro model to assess the activity of therapeutic agents in B-NHL. RNA-sequence analysis highlighted the synergy of 3D, ECM, and TSC in upregulating similar pathways in malignant B cells in vitro than those overexpressed in primary lymphoma B cells in situ. Finally, our 3D model including ECM and TSC allowed long-term in vitro survival of primary follicular lymphoma B cells. In conclusion, we propose a new high-throughput 3D model mimicking lymphoma tumor niche and making it possible to study the dynamic relationship between lymphoma B cells and their microenvironment and to screen new anti-cancer drugs.

Published
2021
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5. Stromal cells regulate malignant B-cell spatial organization, survival, and drug response in a new 3D model mimicking lymphoma tumor niche

Author

Gaëlle Recher, N. Helaine, Céline Monvoisin, Laurence Bresson-Bepoldin, E. Dessauge, Kevin Alessandri, Frédéric Mourcin, Isabelle Mahouche, Céline Pangault, C. Lamaison, L. Broca-Brisson, Sylvain Latour, V. Le Morvan, Marine Seffals, Pierre Nassoy, Philippe Soubeyran, C. Dussert, Karin Tarte, Microenvironment, Cell Differentiation, Immunology and Cancer (MICMAC), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Institut Bergonié [Bordeaux], UNICANCER, Actions for OnCogenesis understanding and Target Identification in ONcology (ACTION), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Bordeaux Segalen - Bordeaux 2-Institut Bergonié [Bordeaux], UNICANCER-UNICANCER, Laboratoire Photonique, Numérique et Nanosciences (LP2N), Université de Bordeaux (UB)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), H2P2 - Histo Pathologie Hight Precision (H2P2), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Plateforme de génétique moléculaire des cancers d'Aquitaine, CHU Pontchaillou [Rennes], Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Rennes (UR)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), and Recher, Gaelle

Subjects
0303 health sciences, Cell type, Stromal cell, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Spheroid, Biology, medicine.disease, In vitro, Lymphoma, Cell biology, Extracellular matrix, 03 medical and health sciences, Crosstalk (biology), 0302 clinical medicine, medicine.anatomical_structure, immune system diseases, 030220 oncology & carcinogenesis, hemic and lymphatic diseases, medicine, B cell, 030304 developmental biology
Abstract

Non-Hodgkin B-cell lymphomas (B-NHL) mainly develop within lymph nodes as densely packed aggregates of tumor cells and their surrounding microenvironment, creating a tumor niche specific to each lymphoma subtypes. Until now, in vitro preclinical models mimicking biomechanical forces, cellular microenvironment, and 3D organization of B lymphomas remain scarce while all these parameters constitute key determinants of lymphomagenesis and drug resistance. Using a microfluidic method based on the encapsulation of cells inside permeable, elastic, and hollow alginate microspheres, we developed a new tunable 3D-model incorporating extracellular matrix and/or stromal cells. Lymphoma B cells and stromal cells dynamically formed self-organized 3D spheroids, thus initiating a coevolution of these two cell types, reflecting their bidirectional crosstalk, and recapitulating the heterogeneity of B-NHL subtypes. In addition, this approach makes it suitable to assess in a relevant in vitro model the activity of new therapeutic agents in B-NHL.

Published
2020

6. Buckling of an Epithelium Growing under Spherical Confinement

Author

Jochen Guck, Aurélien Roux, Carles Blanch-Mercader, Ilaria Di Meglio, Kevin Alessandri, Aziza Merzouki, Pierre Nassoy, Anastasiya Trushko, Karsten Kruse, Bastien Chopard, Shada Abuhattum, Biochemistry Department - University of Geneva, University of Geneva [Switzerland], Department of Computer Science - University of Geneva, Biotechnology Center - Technische Universität Dresden, Biotechnology Center- Technische Universität Dresden, Laboratoire Photonique, Numérique et Nanosciences (LP2N), Université de Bordeaux (UB)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), and Department of Computer Science- University of Geneva

Subjects
[PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], ddc:500.2, Biology, Models, Biological, Epithelium, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, 0302 clinical medicine, Elastic Modulus, Monolayer, Cell Adhesion, Morphogenesis, medicine, Humans, Computer Simulation, Epithelium mechanics, ddc:025.063, Molecular Biology, Elastic modulus, ComputingMilieux_MISCELLANEOUS, Cell Proliferation, 030304 developmental biology, 3D cell culture, 0303 health sciences, Epithelial monolayer, Cell Biology, Mechanics, Biomechanical Phenomena, Stress field, medicine.anatomical_structure, Buckling, Homogeneous, Alginate capsules, ddc:540, Material properties, 030217 neurology & neurosurgery, Developmental Biology
Abstract

Summary Many organs are formed through folding of an epithelium. This change in shape is usually attributed to tissue heterogeneities, for example, local apical contraction. In contrast, compressive stresses have been proposed to fold a homogeneous epithelium by buckling. While buckling is an appealing mechanism, demonstrating that it underlies folding requires measurement of the stress field and the material properties of the tissue, which are currently inaccessible in vivo. Here, we show that monolayers of identical cells proliferating on the inner surface of elastic spherical shells can spontaneously fold. By measuring the elastic deformation of the shell, we infer the forces acting within the monolayer and its elastic modulus. Using analytical and numerical theories linking forces to shape, we find that buckling quantitatively accounts for the shape changes of our monolayers. Our study shows that forces arising from epithelial growth in three-dimensional confinement are sufficient to drive folding by buckling.

Published
2020
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7. Up to 151.106 cumulated fold expansion of encapsulated hips cells in bioreactor over 28 days, and comparison with 2D culture and standard spheroid culture

Author

E. Quelennec, L. Remichius, Maxime Feyeux, A. Leonard, F. Moncaubeig, E. Luquet, J. Pletenka, Nathalie Lefort, Kevin Alessandri, E. Warter, and Cohen Pj

Subjects
Cancer Research, Transplantation, Oncology, Fold (higher-order function), Chemistry, Immunology, Bioreactor, Spheroid, Immunology and Allergy, Cell Biology, Molecular biology, Genetics (clinical)
Published
2021
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8. Quantitative agent-based modeling reveals mechanical stress response of growing tumor spheroids is predictable over various growth conditions and cell lines

Author

Johannes Neitsch, Pierre Nassoy, Paul Van Liedekerke, Tim Johann, Dirk Drasdo, Kevin Alessandri, Modelling and Analysis for Medical and Biological Applications (MAMBA), Inria de Paris, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Jacques-Louis Lions (LJLL (UMR_7598)), Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Interdisciplinary Centre for Bioinformatics [Leipzig] (IZBI), Universität Leipzig [Leipzig], Laboratoire Photonique, Numérique et Nanosciences (LP2N), Université de Bordeaux (UB)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), SImulations en Médecine, BIOtechnologie et ToXicologie de systèmes multicellulaires (SIMBIOTX ), Inria Saclay - Ile de France, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), Universität Leipzig, and Centre National de la Recherche Scientifique (CNRS)-Institut d'Optique Graduate School (IOGS)-Université de Bordeaux (UB)

Subjects
0303 health sciences, education.field_of_study, Cell type, Strain (chemistry), Chemistry, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Population, Cell, [SDV.CAN]Life Sciences [q-bio]/Cancer, Growth curve (biology), [PHYS.MECA]Physics [physics]/Mechanics [physics], Osmosis, 01 natural sciences, 03 medical and health sciences, medicine.anatomical_structure, Cell culture, 0103 physical sciences, Compressibility, medicine, [PHYS.MECA.BIOM]Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph], 010306 general physics, education, Biological system, 030304 developmental biology
Abstract

Model simulations indicate that the response of growing cell populations on mechanical stress follows the same functional relationship and is predictable over different cell lines and growth conditions despite the response curves look largely different. We develop a hybrid model strategy in which cells are represented by coarse-grained individual units calibrated with a high resolution cell model and parameterized measurable biophysical and cell-biological parameters. Cell cycle progression in our model is controlled by volumetric strain, the latter being derived from a bio-mechanical relation between applied pressure and cell compressibility. After parameter calibration from experiments with mouse colon carcinoma cells growing against the resistance of an elastic alginate capsule, the model adequately predicts the growth curve in i) soft and rigid capsules, ii) in different experimental conditions where the mechanical stress is generated by osmosis via a high molecular weight dextran solution, and iii) for other cell types with varying doubling times. Our model simulation results suggest that the growth response of cell population upon externally applied mechanical stress is the same, as it can be quantitatively predicted using the same growth progression function.Author summaryThe effect of mechanical resistance on the growth of tumor cells remains today largely unquantified. We studied data from two different experimental setups that monitor the growth of tumor cells under mechanical compression. The existing data in the first experiment examined growing CT26 cells in an elastic permeable capsule. In the second experiment, growth of tumor cells under osmotic stress of the same cell line as well as other cell lines were studied. We have developed and agent-based model with measurable biophysical and cell-biological parameters that can simulate both experiments. Cell cycle progression in our model is a Hill function of cell volumetric strain, derived from a bio-mechanical relation between applied pressure and cell compressibility. After calibration of the model parameters within the data of the first experiment, we are able predict the growth rates in the second experiment. We show that that the growth response of cell populations upon externally applied mechanical stress in the two different experiments and over different cell lines can be predicted using the same growth progression function once the growth kinetics of the cell lines in abscence of mechanical stress is known.

Published
2019

9. Self-interference 3D super-resolution microscopy for deep tissue investigations

Author

Pierre Bon, Laurent Cognet, Maxime Feyeux, Brahim Lounis, Kevin Alessandri, Pierre Nassoy, Jeanne Linarès-Loyez, Laboratoire Photonique, Numérique et Nanosciences (LP2N), Université de Bordeaux (UB)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), lp2n-04,lp2n-12, Université Sciences et Technologies - Bordeaux 1-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS)-Université Sciences et Technologies - Bordeaux 1-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), and lp2n-04,lp2n-16

Subjects
0301 basic medicine, Point spread function, Pluripotent Stem Cells, Microscope, Materials science, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Induced Pluripotent Stem Cells, Biochemistry, law.invention, Protein filament, 03 medical and health sciences, law, Microscopy, Humans, Microscopy, Interference, Induced pluripotent stem cell, Molecular Biology, Nanoscopic scale, ComputingMilieux_MISCELLANEOUS, Super-resolution microscopy, Resolution (electron density), Cell Biology, Actins, Single Molecule Imaging, 030104 developmental biology, Biophysics, Octamer Transcription Factor-3, Biotechnology
Abstract

Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native cellular processes in intact tissues.

Published
2018
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10. Controlled production of sub-millimeter liquid core hydrogel capsules for parallelized 3D cell culture

Author

Hugo Doméjean, Nicolas Atrux-Tallau, Pierre Nassoy, Nicolas Bremond, Kevin Alessandri, Anette Funfak, Mathieu de la Motte Saint Pierre, and Jérôme Bibette

Subjects
0301 basic medicine, Materials science, Capillary action, Cell Survival, Polymers, Biomedical Engineering, Cell Culture Techniques, Bioengineering, Nanotechnology, 02 engineering and technology, Biochemistry, Viscoelasticity, 03 medical and health sciences, 3D cell culture, Mice, Cell Line, Tumor, Monolayer, Animals, Coalescence (physics), Viscosity, Drop (liquid), Spheroid, Hydrogels, General Chemistry, 021001 nanoscience & nanotechnology, Breakup, 030104 developmental biology, Chemical engineering, Hydrodynamics, 0210 nano-technology
Abstract

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.

Published
2016

11. C-terminal domain of the Uup ATP-binding cassette ATPase is an essential folding domain that binds to DNA

Author

Dorothée Murat, Elie Dassa, Monica Y. Burgos Zepeda, Kevin Alessandri, and Chahrazade El Amri

Subjects
DNA, Bacterial, Transposable element, Protein Folding, ATPase, genetic processes, Biophysics, ATP-binding cassette transporter, Biology, medicine.disease_cause, environment and public health, Biochemistry, Protein Structure, Secondary, Analytical Chemistry, chemistry.chemical_compound, Escherichia coli, medicine, Electrophoretic mobility shift assay, Molecular Biology, Base Sequence, Protein Stability, Escherichia coli Proteins, C-terminus, Genetic Complementation Test, fungi, Recombinant Proteins, Protein Structure, Tertiary, enzymes and coenzymes (carbohydrates), Cross-Linking Reagents, chemistry, health occupations, biology.protein, Thermodynamics, ATP-Binding Cassette Transporters, CTD, Protein Multimerization, DNA, Plasmids, Protein Binding
Abstract

The Uup protein belongs to a subfamily of soluble ATP-binding cassette (ABC) ATPases that have been implicated in several processes different from transmembrane transport of molecules, such as transposon precise excision. We have demonstrated previously that Escherichia coli Uup is able to bind DNA. DNA binding capacity is lowered in a truncated Uup protein lacking its C-terminal domain (CTD), suggesting a contribution of CTD to DNA binding. In the present study, we characterize the role of CTD in the function of Uup, on its overall stability and in DNA binding. To this end, we expressed and purified isolated CTD and we investigated the structural and functional role of this domain. The results underline that CTD is essential for the function of Uup, is stable and able to fold up autonomously. We compared the DNA binding activities of three versions of the protein (Uup, UupΔCTD and CTD) by an electrophoretic mobility shift assay. CTD is able to bind DNA although less efficiently than intact Uup and UupΔCTD. These observations suggest that CTD is an essential domain that contributes directly to the DNA binding ability of Uup.

Published
2010
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12. Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro

Author

Vasily Gurchenkov, Felix Rico, Jérôme Bibette, Hugo Doméjean, Tobias Kießling, Bibhu Ranjan Sarangi, Leslie Rolland, Simon Scheuring, Luc Fetler, Sara Geraldo, Danijela Matic Vignjevic, Bidisha Sinha, Pierre Nassoy, Anthony Simon, Kevin Alessandri, Nicolas Bremond, Anette Funfak, Christophe Lamaze, Physico-Chimie-Curie (PCC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Compartimentation et dynamique cellulaires (CDC), Soft Matter Physics Division, University of Leipzig, Universität Leipzig [Leipzig], BIO-AFM-LAB Bio Atomic Force Microscopy Laboratory (Bio-AFM-Lab), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Colloïdes et Matériaux Divisés (LCMD), Centre National de la Recherche Scientifique (CNRS)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), lp2n-04,lp2n-16, Laboratoire Photonique, Numérique et Nanosciences (LP2N), Université Sciences et Technologies - Bordeaux 1-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS)-Université Sciences et Technologies - Bordeaux 1-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Indian Institute of Science Education and Research Kolbata (IISER Kolkata), Soft Matter Physics Division [Leipzig, Allemagne], Universität Leipzig, Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Université de Bordeaux (UB)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), LP2N_G3, LP2N_A3, Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), and Aix-Marseille Université, U1006

Subjects
Cell, Cell Count, 02 engineering and technology, Mechanotransduction, Cellular, Mice, Glucuronic Acid, Cell Movement, Tumor Microenvironment, Mechanotransduction, 0303 health sciences, Multidisciplinary, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Hexuronic Acids, Mechanics, Microfluidic Analytical Techniques, 021001 nanoscience & nanotechnology, Cell biology, Biomechanical Phenomena, medicine.anatomical_structure, tumor growth, tissue mechanics, Physical Sciences, embryonic structures, Disease Progression, GROWTH, 0210 nano-technology, Alginates, ALGINATE GEL BEADS, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], microfluidics, Capsules, Biology, 03 medical and health sciences, Cell Line, Tumor, Spheroids, Cellular, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Humans, Neoplasm Invasiveness, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, CANCER-CELLS, 030304 developmental biology, Cell Proliferation, mechanotransduction, Cell invasion, ENVIRONMENT, Spheroid, In vitro, Elasticity, MODEL, SIZE, Tumor progression, Cancer cell, Multicellular spheroid, HeLa Cells
Abstract

International audience; 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. tissue mechanics | microfluidics | tumor growth | mechanotransduction

Published
2013
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13. Light-sheet microscopy in thick media using scanned Bessel beams and two-photon fluorescence excitation

Author

Vasily Gurchenkov, Florian Fahrbach, Kevin Alessandri, Pierre Nassoy, Alexander Rohrbach, Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Max-Planck-Gesellschaft, Laboratory for Bio- and Nano-Photonics, University of Freiburg, University of Freiburg [Freiburg], Physico-Chimie-Curie (PCC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC), Compartimentation et dynamique cellulaires (CDC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), lp2n-04,lp2n-16, Laboratoire Photonique, Numérique et Nanosciences (LP2N), Université Sciences et Technologies - Bordeaux 1-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS)-Université Sciences et Technologies - Bordeaux 1-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), Centre for Biological Signalling Studies (bioss), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), and Université Sciences et Technologies - Bordeaux 1 (UB)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS)-Université Sciences et Technologies - Bordeaux 1 (UB)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Materials science, DEEP, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], PROPAGATION, 01 natural sciences, 010309 optics, 03 medical and health sciences, symbols.namesake, Optics, Two-photon excitation microscopy, 0103 physical sciences, Microscopy, FIELD, Penetration depth, Image resolution, 030304 developmental biology, [PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics], 0303 health sciences, business.industry, Scattering, Atomic and Molecular Physics, and Optics, SELECTIVE PLANE ILLUMINATION, Light sheet fluorescence microscopy, DEPTH, symbols, business, Excitation, Bessel function
Abstract

In this study we show that it is possible to successfully combine the benefits of light-sheet microscopy, self-reconstructing Bessel beams and two-photon fluorescence excitation to improve imaging in large, scattering media such as cancer cell clusters. We achieved a nearly two-fold increase in axial image resolution and 5–10 fold increase in contrast relative to linear excitation with Bessel beams. The light-sheet penetration depth could be increased by a factor of 3–5 relative to linear excitation with Gaussian beams. These finding arise from both experiments and computer simulations. In addition, we provide a theoretical description of how these results are composed. We investigated the change of image quality along the propagation direction of the illumination beams both for clusters of spheres and tumor multicellular spheroids. The results reveal that light-sheets generated by pulsed near-infrared Bessel beams and two photon excitation provide the best image resolution, contrast at both a minimum amount of artifacts and signal degradation along the propagation of the beam into the sample.

Published
2013
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