Universite de Lyon View Institution's Website 12 articles published in JoVE Biology A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts Kawtar Arizkane1,2,3, Kevin Geistlich1,2,3,4, Laurine Moindrot1,2,3, Emma Risson1,2,3, Sandrine Jeanpierre1,2,3,4, Léa Barral1,2,3, Anaëlle Bobard1,2,3, Giulio Menegazzi5, Thibault Voeltzel1,2,3, Véronique Maguer-Satta1,2,3, Sylvain Lefort1,2,3 1Centre de Recherche en Cancérologie de Lyon, 2Centre de Recherche en Cancérologie de Lyon, 3Université de Lyon, 4Centre Léon Bérard, 5Department of Experimental and Clinical Biomedical Sciences Mario Serio, Università degli Studi di Firenze Here, we describe an easy-to-implement, standardized, microphysiological system that reflects the complexity of the human bone marrow's in vivo structure, providing a pertinent model to finely study a broad range of normal and pathological events. Biology Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds Josselin Rigaill*1,2, Estelle Audoux*1, Killian Rodriguez1, Aurélien Peyron1, Philippe Berthelot1,3, Jérôme Josse4,5, Frédéric Laurent4,5, Robin Caire1, Paul O. Verhoeven1,2 1CIRI, Centre International de Recherche en Infectiologie, GIMAP team, University of Lyon, INSERM U1111, CNRS, UMR5308, ENS Lyon, UCBL1, University of St-Etienne, France, 2Department of Infectious Agents and Hygiene, University Hospital of St-Etienne, St-Etienne, France, 3Department of Infectious Diseases, University Hospital of St-Etienne, St-Etienne, France, 4CIRI, Centre International de Recherche en Infectiologie, Staphylococcal Pathogenesis team, University of Lyon, INSERM U1111, CNRS UMR5308, ENS Lyon, UCBL1, University of Lyon, Lyon, France, 5Department of Bacteriology, Institute for Infectious Agents, Hospices Civils de Lyon, Lyon, France This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds. Biology Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models Alejandro Lopez Gonzalez*1, Léa Luciana*1, Clémentine Le Nevé2, Julie Valantin2, Laura Francols2, Nicolas Gadot2, Christophe Vanbelle3, Laurianne Davignon4, Laura Broutier1 1Childhood Cancer & Cell Death (C3), Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon (CRCL), Lyon 69373, 2Plateforme Anatomopathologie Recherche, Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon (CRCL), 3Plateforme d’Imagerie cellulaire, Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon (CRCL), 4PerkinElmer S.A.S. Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 µm to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions. Cancer Research A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells Maria Virginia Giolito1,2, Léo Claret1,2, Carla Frau1, Michelina Plateroti1,2 1Département de la recherche, Centre de Recherche en Cancérologie de Lyon, INSERM U1052, CNRS UMR5286, Université de Lyon, Université Lyon 1, Centre Léon Bérard, 2UMR-S1113 - IRFAC INSERM, Université de Strasbourg This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy. Developmental Biology Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues Simone Bovio1,2, Yuchen Long2, Françoise Monéger2 1SFR Biosciences, Université de Lyon, 2Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCBL, INRA, CNRS Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure. Chemistry Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis Lukasz Bozycki1, Magdalena Komiazyk1, Saida Mebarek2,3,4,5,6, Rene Buchet2,3,4,5,6, Slawomir Pikula1, Agnieszka Strzelecka-Kiliszek1 1Laboratory of Lipid Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 2Université de Lyon, 3Université Lyon 1, 4 We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX). Biology Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions Christian T. Herbst1,2, Vit Hampala1, Maxime Garcia2,3, Riccardo Hofer2, Jan G. Svec1 1Voice Research Lab, Department of Biophysics, Faculty of Science, Palacky University Olomouc, 2Laboratory of Bio-Acoustics, Dept. of Cognitive Biology, University of Vienna, 3ENES Lab, NEURO-PSI,CNRS UMR 9197, Université Lyon/Saint-Etienne, France This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals. Biology Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes Stephanie Chanon1, Christine Durand1, Aurelie Vieille-Marchiset1, Maud Robert2, Charna Dibner3, Chantal Simon1, Etienne Lefai1 1CarMeN Laboratory, INSERM U1060, INRA 1397, University of Lyon, 2Department of digestive and bariatric surgery, Obesity Integrated Center, University Hospital of Edouard Herriot, Hospices Civils de Lyon, Lyon 1 University, 3Division of Endocrinology, Diabetes, Hypertension and Nutrition, Department of Clinical Medicine, Faculty of Medicine, University of Geneva In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose. Medicine Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics Marion Gilormini1, Anne-Sophie Wozny1,2, Priscillia Battiston-Montagne1, Dominique Ardail1,2, Gersende Alphonse1,2, Claire Rodriguez-Lafrasse1,2 1UCBL1, UMR/CNRS 5822, Laboratoire de Radiobiologie Cellulaire Moléculaire, Université de Lyon, 2Hospices-Civils-de-Lyon, Centre Hospitalier Lyon-Sud Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC). Biology Isolation and Culture of Mouse Primary Pancreatic Acinar Cells Johann Gout1,2,3,4,5, Roxane M. Pommier1,2,3,4,5, David F. Vincent1,2,3,4,5, Bastien Kaniewski1,2,3,4,5, Sylvie Martel1,2,3,4,5, Ulrich Valcourt*1,2,3,4,5, Laurent Bartholin*1,2,3,4,5 1INSERM U1052, Centre de Recherche en Cancérologie de Lyon, 2CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, 3Université de Lyon, 4Université Lyon 1, 5Centre Léon Bérard In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells. Bioengineering Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon Benoît Drogue1, Philippe Thomas2, Laurent Balvay3, Claire Prigent-Combaret1, Corinne Dorel4 1UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Université de Lyon, 2Département Biosciences, INSA de Lyon, Université de Lyon, 3INSERM U758, Ecole Normale Supérieure de Lyon, Université de Lyon, 4Laboratoire de Génie Civil et Ingénierie Environnementale, INSA de Lyon, Université de Lyon The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained. Bioengineering Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells Sébastien Degot1, Muriel Auzan1, Violaine Chapuis1, Anne Béghin2, Amélie Chadeyras1, Constantin Nelep1, Maria Luisa Calvo-Muñoz1, Joanne Young1, François Chatelain1, Alexandra Fuchs1 1CYTOO Cell Architects, Grenoble, France, 2Centre Commun de Quantimétrie, Faculté de Médecine Rockefeller, Lyon, France Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.
