University Hospital of Geneva View Institution's Website 9 articles published in JoVE Biology Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy Thais Bascuas*1,2, Martina Kropp*1,2, Nina Harmening1,2, Brittany M. Wong1, Sandra Johnen3, Zsuzsanna Izsvák4, Gabriele Thumann1,2 1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Department of Ophthalmology, University Hospital RWTH Aachen, 4Max Delbrück Center for Molecular Medicine Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo analyses and in vivo studies transferable to humans. Medicine Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System Sandra Johnen1, Nina Harmening2,3, Corinne Marie4,5, Daniel Scherman4, Zsuzsanna Izsvák6, Zoltán Ivics7, Peter Walter1, Gabriele Thumann2,3 1Department of Ophthalmology, University Hospital RWTH Aachen, 2Experimental Ophthalmology, University of Geneva, 3Department of Ophthalmology, University Hospitals of Geneva, 4Université de Paris, CNRS, INSERM, UTCBS, Unité des technologies Chimiques et Biologiques pour la Santé, 5Chimie ParisTech, PSL Research University, 6Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 7Division of Medical Biotechnology, Paul-Ehrlich-Institute We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Medicine Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells Thais Bascuas1,2, Martina Kropp1,2, Nina Harmening1,2, Mohammed Asrih1, Zsuzsanna Izsvák3, Gabriele Thumann1,2 1Experimental Ophthalmology, University of Geneva, 2Department of Ophthalmology, University Hospitals of Geneva, 3Max Delbrück Center for Molecular Medicine We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration. Cancer Research Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma Archana Vijaya Kumar1,2, Carmen Donate2, Beat A. Imhof1, Thomas Matthes2 1Department of Pathology and Immunology, University of Geneva, 2Hematology Service, University Hospital, Geneva Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology. Genetics Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures Volodymyr Petrenko*1, Camille Saini*2, Laurent Perrin*1, Charna Dibner1 1Department of Medical Specialties, Division of Endocrinology, Diabetes, Hypertension and Nutrition, Diabetes Center, University of Geneva Medical School, Institute of Genetics and Genomics in Geneva (iGE3), 2Population Epidemiology Unit (UEP), Community Medicine, Geneva University Hospital Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion. Neuroscience Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays Stefan Hahnewald1,2, Marta Roccio1,2, Anne Tscherter3, Jürg Streit3, Ranjeeta Ambett1,2, Pascal Senn1,2,4 1Department of Otorhinolaryngology, Inselspital and University of Bern, 2Department of Clinical Research, Inselspital and University of Bern, 3Department of Physiology, University of Bern, 4Department of Clinical Neurosciences, Service ORL & HNS, University Hospital Geneva We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device. Bioengineering Manufacturing Devices and Instruments for Easier Rat Liver Transplantation Graziano Oldani1,2, Stephanie Lacotte3, Lorenzo Orci1, Philippe Morel4, Gilles Mentha1, Christian Toso1 1Transplantation Division, Department of Surgery, University of Geneva Hospitals, 2Department of Surgery, University of Pavia, 3Department of Surgery, University of Geneva, 4Division of Abdominal Surgery, Department of Surgery, University of Geneva Hospitals We describe the design of the “quick-linker” device for easier orthotopic rat liver transplantation. Medicine Orthotopic Liver Transplantation in Rats Graziano Oldani1,2, Stephanie Lacotte3, Philippe Morel4, Gilles Mentha1, Christian Toso1 1Transplantation Division, Department of Surgery, University of Geneva Hospitals, 2Department of Surgery, University of Pavia, 3Department of Surgery, University of Geneva, 4Division of Abdominal Surgery, Department of Surgery, University of Geneva Hospitals We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat. Bioengineering Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study Michael J. McClure1, Scott A. Sell1, David G. Simpson2, Beat H. Walpoth3, Gary L. Bowlin1 1Department of Biomedical Engineering, Virginia Commonwealth University, 2Department of Anatomy and Neurobiology, Virginia Commonwealth University, 3Department of Cardiovascular Surgery, University Hospital of Geneva The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.