The University of Tokyo 17 articles published in JoVE Medicine Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients Itsuma Nagao1,2, Meg Nakazawa1, Yoko M. Ambrosini1 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, 2Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health. Biology A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models Genri Numata1,2, Eiki Takimoto1,3 1Department of Cardiovascular Medicine, The University of Tokyo Hospital, 2Department of Therapeutic Strategy for Heart Failure, Graduate School of Medicine, The University of Tokyo, 3Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions The present protocol describes obtaining the pressure-volume relationship through transesophageal pacing, which serves as a valuable tool in evaluating diastolic function in mouse models of heart failure. Medicine Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats Shigeto Tsuji1, Shogo Shimada1, Minoru Ono1 1Department of Cardiac Surgery, The University of Tokyo This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting. Biology Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator Kaede Saito1,2, Yuta Hiraike1,2,3, Misato Oguchi2, Toshimasa Yamauchi2 1Division for Health Service Promotion, The University of Tokyo, 2Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, 3The University of Tokyo Excellent Young Researcher Program, The University of Tokyo This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility. Neuroscience Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons Noriko Koganezawa1, Reiko T. Roppongi2, Yuko Sekino3,4, Izuo Tsutsui3, Ayaka Higa5, Tomoaki Shirao1,5 1Department of Pharmacology, Graduate School of Medicine, Gunma University, 2Gunma University Initiative for Advanced Research, Gunma University, 3Department of Veterinary Pathophysiology and Animal Health, Graduate school of Agricultural and Life Sciences, The University of Tokyo, 4Institute for Drug Discovery Innovation, 5AlzMed, Inc. A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate. Developmental Biology CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models Manabu Ozawa*1, Chihiro Emori*2, Masahito Ikawa1,2 1Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, 2Research Institute for Microbial Diseases, Osaka University Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method. Neuroscience Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice Hisato Maruoka1, Ryosuke Kamei1, Shunsuke Mizutani1, Qingrui Liu1, Shigeo Okabe1 1Department of Cellular Neurobiology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Neuroscience In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window Satoshi Manita1, Eiji Shigetomi2,3, Haruhiko Bito4, Schuichi Koizumi2,3, Kazuo Kitamura1 1Department of Neurophysiology, Faculty of Medicine, University of Yamanashi, 2Department of Neuropharmacology, Faculty of Medicine, University of Yamanashi, 3Yamanashi GLIA center, Interdisciplinary Graduate School of Medicine, University of Yamanashi, 4Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse. Developmental Biology Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum Sosuke Fujita1, Erina Kuranaga1, Masayuki Miura2, Yu-ichiro Nakajima2 1Graduate School of Life Sciences, Tohoku University, 2Graduate School of Pharmaceutical Sciences, The University of Tokyo Here, we describe a protocol for visualizing stem-like proliferating cells in the jellyfish Cladonema. Whole-mount fluorescent in situ hybridization with a stem cell marker allows for the detection of stem-like cells, and 5-ethynyl-2'-deoxyuridine labeling enables the identification of proliferating cells. Together, actively proliferating stem-like cells can be detected. Medicine Whole-Kidney Three-Dimensional Staining with CUBIC Sho Hasegawa1, Masaomi Nangaku1 1Division of Nephrology and Endocrinology, The University of Tokyo The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries. Neuroscience In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus Ryosuke Kamei1, Shinji Urata1, Hisato Maruoka1, Shigeo Okabe1 1Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy. Biology Optical Clearing of Plant Tissues for Fluorescence Imaging Daisuke Kurihara1, Yoko Mizuta1,2, Shiori Nagahara1, Yoshikatsu Sato1,3, Tetsuya Higashiyama1,3,4 1Institute of Transformative Bio-Molecules (ITbM), Nagoya University, 2Institute for Advanced Research (IAR), Nagoya University, 3Division of Biological Science, Graduate School of Science, Nagoya University, 4Department of Biological Sciences, Graduate School of Science, The University of Tokyo Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning. Developmental Biology Electroporation-mediated RNA Interference Method in Odonata Genta Okude1,2, Takema Fukatsu1,2,3, Ryo Futahashi2 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 3Graduate School of Life and Environmental Sciences, University of Tsukuba We provide a detailed protocol for electroporation-mediated RNA interference in insects of the order Odonata (dragonflies and damselflies) using the blue-tailed damselfly (Ischnura senegalensis: Coenagironidae: Zygoptera) and the pied skimmer dragonfly (Pseudothemis zonata: Libellulidae: Anisoptera). Biology Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes) Muhammad Rahmad Royan1, Shinji Kanda2, Daichi Kayo3, Weiyi Song4, Wei Ge4, Finn-Arne Weltzien1, Romain Fontaine1 1Physiology Unit, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 2Laboratory of Physiology, Atmosphere and Ocean Research Institute, The University of Tokyo, 3Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 4Centre of Reproduction, Development and Aging (CRDA), Faculty of Health Sciences, University of Macau The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology. Neuroscience Three-Dimensional Motor Nerve Organoid Generation Tatsuya Osaki1,2, Siu Yu A. Chow1,2, Yui Nakanishi1,2, Joel Hernández1,3, Jiro Kawada4, Teruo Fujii1, Yoshiho Ikeuchi1,2 1Institute of Industrial Science, The University of Tokyo, 2Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 3Faculty of Science and Engineering, Tecnologico de Monterrey, 4Jiksak Bioengineering, Inc. This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip. Medicine Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System Kazuma Sekiba1,2, Motoyuki Otsuka1, Kazuhiko Koike1 1Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 2Research Fellow of Japan Society for the Promotion of Science Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions. Medicine Technical Aspects of the Mouse Aortocaval Fistula Kota Yamamoto1,2, Xin Li1,3, Chang Shu3, Tetsuro Miyata2, Alan Dardik1,4 1The Department of Surgery and the Interdepartmental Program in Vascular Biology and Therapeutics, Yale University, 2Department of Vascular Surgery, The University of Tokyo, 3Department of Vascular Surgery, Central South University, 4VA Connecticut Healthcare Systems The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.