Children's Hospital of Philadelphia View Institution's Website 25 articles published in JoVE Medicine Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor Rene Machietto1, Nicholas Giacobbe1, Jessica Perazzelli2, Ted J. Hofmann2, Allison Barz Leahy2,3, Stephan A. Grupp1,2,3, Yongping Wang1,3,4, Stephan Kadauke1,3,4 1Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, 3Perelman School of Medicine at the University of Pennsylvania, 4 This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial. Neuroscience Maintenance of a Lateral Fluid Percussion Injury Device Anthony M. Farrugia2, Samuelle A. S. Delcy2,3, Brian N. Johnson1, Akiva S. Cohen1,2 1 Proper care and maintenance are essential for a lateral fluid percussion injury (LFPI) device to function reliably. Here, we demonstrate how to properly clean, fill, and assemble an LFPI device, and ensure that it is adequately maintained for optimal results. Cancer Research Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice Kyra Harvey1, Peter J. Madsen2,3, Tiffany Smith2, Crystal Griffin1,2, Luke Patterson1,2, Nicholas A. Vitanza4,5, Phillip B. Storm2,3, Adam C. Resnick2,3, Jessica B. Foster1,2 1Center for Data Driven Discovery in Biomedicine, Children’s Hospital of Philadelphia, 3Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, 5Department of Pediatrics, Seattle Children’s Hospital, University of Washington Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors. Biochemistry Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection Jonathan T. Specker1, Alexander B. Smith2, Orlaith Keenan2, Joseph P. Zackular2,3, Boone M. Prentice1 1Department of Chemistry, University of Florida, 2 A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry. Biology Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping Chao Song1,2, Arianna Valeri1, Fanxin Long1 1 This protocol describes an easy-to-use method to examine substrate oxidation by tracking 14CO2 production in vitro. Biology Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System Md Nasimuzzaman1,2, Sophia Villaveces1, Johannes C. M. van der Loo1,2,3, Sivani Alla1 1Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration. Behavior Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease Manuela Lavorato*1, Neal D. Mathew*1, Nina Shah1, Eiko Nakamaru-Ogiso1,2, Marni J. Falk1,2 1 This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function. Bioengineering Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization Vaishali V. Inamdar1, Emmett G. Fitzpatrick1, Ivan S. Alferiev1,2, Robert J. Levy1,2, Stanley J. Stachelek*1,2, Ilia Fishbein*1,2 1 Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility. Medicine International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure Jing Liu1,2, Dalibor Kurepa3, Francesco Feletti4,5, Almudena Alonso-Ojembarrena6, Jovan Lovrenski7, Roberto Copetti8, Erich Sorantin9, Javier Rodriguez-Fanjul10, Karishma Katti3, Andrea Aliverti4, Huayan Zhang11,12, Misun Hwang13, Tsu F. Yeh14, Cai-Bao Hu15, Xing Feng16, Ru-Xin Qiu1,2, Jing-Han Chi17, Li-Li Shang18, Guo-Rong Lyu19, Shao-Zheng He20, Yan-Fen Chai21, Zhan-Jun Qiu22, Hai-Ying Cao2,23, Yue-Qiao Gao1,2, Xiao-Ling Ren1,2, Guo Guo1,24, Li Zhang1,2, Ying Liu1,2, Wei Fu1,2, Zu-Lin Lu1,2, Hong-Lei Li1,2 1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The National Neonatal Lung Ultrasound Training Base, 3 Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax. Genetics Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells Fabian L. Cardenas-Diaz*1, Jean Ann Maguire*1, Paul Gadue1,2,3, Deborah L. French1,2,3 1 This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells. Medicine Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus Jing Liu1,2, Roberto Copetti3, Erich Sorantin4, Jovan Lovrenski5, Javier Rodriguez-Fanjul6, Dalibor Kurepa7, Xing Feng8, Luigi Cattaross9, Huayan Zhang10,11, Misun Hwang12, Tsu F. Yeh13,14, Yisrael Lipener7, Abhay Lodha15, Jia-Qin Wang16, Hai-Ying Cao2,17, Cai-Bao Hu2,18, Guo-Rong Lyu19, Xin-Ru Qiu1,2, Li-Qun Jia20, Xiao-Man Wang20, Xiao-Ling Ren1,2, Jiu-Ye Guo1,2, Yue-Qiao Gao1,2, Jian-Jun Li1,2, Ying Liu1,2, Wei Fu1,2, Yan Wang21, Zu-Lin Lu1,2, Hua-Wei Wang8, Li-Li Shang22 1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Division of Neonatology, Children's Hospital of Philadelphia, 12Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making. Bioengineering Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota Jenni Firrman1, LinShu Liu1, Pieter Van den Abbeele2, Ceylan Tanes3, Kyle Bittinger3, Peggy Tomasula1 1Dairy and Functional Foods Research Unit, Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 2ProDigest, 3 Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract. Developmental Biology Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model Nicholas J. Ahn1, John D. Stratigis1, Barbara E. Coons1, Alan W. Flake1, Hyun-Duck Nah-Cederquist2, William H. Peranteau1 1 We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment. Genetics Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms Clark Fritsch1,2, Jean-Francois Pierre Gout3,4, Marc Vermulst1 1 This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms. Medicine A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System Dan Li*1, Feng Li*1, Pavithran Guttipatti1, Yuanquan Song1,2 1 Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration. Genetics Production and Purification of Baculovirus for Gene Therapy Application Md Nasimuzzaman1,2, Johannes C.M. van der Loo1,2,3, Punam Malik1,2 1 In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application. Biology Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors Jesse J. Hubbard1, Spencer K. Sullivan2, Jason A. Mills3, Brian J. Hayes1, Beverly J. Torok-Storb1, Aravind Ramakrishnan1 1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Department of Pathology, The Children's Hospital of Philadelphia Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors. Bioengineering The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits Joshua B. Slee1,2, Ivan S. Alferiev1,2, Robert J. Levy1,2, Stanley J. Stachelek1,2 1 Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. Medicine Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers Ilia Fishbein1, Scott P. Forbes1, Richard F. Adamo1, Michael Chorny1, Robert J. Levy1, Ivan S. Alferiev1 1 These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries. Biology Isolation of Neonatal Extrahepatic Cholangiocytes Sara Karjoo1, Rebecca G. Wells2 1 A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology. Biology Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq) Sandra Deliard1, Jianhua Zhao1, Qianghua Xia1, Struan F.A. Grant1,2 1Division of Human Genetics, Children's Hospital of Philadelphia Research Institute, 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example. Neuroscience Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury Colin J. Smith1,2, Brian N. Johnson1, Jaclynn A. Elkind1, Jill M. See1, Guoxiang Xiong1, Akiva S. Cohen1,3 1Division of Neurology, Children's Hospital of Philadelphia, 2Neuroscience Graduate Group, Perelman School of Medicine at the University of Pennsylvania, 3Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions. Biology Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector Andreia Gianotti Sommer1, Sarah S. Rozelle1, Spencer Sullivan2, Jason A. Mills3, Seon-Mi Park1, Brenden W. Smith1, Amulya M. Iyer1, Deborah L. French3, Darrell N. Kotton1, Paul Gadue3, George J. Murphy1, Gustavo Mostoslavsky1 1Center for Regenerative Medicine (CReM), Boston University School of Medicine, 2Department of Hematology, Children's Hospital of Philadelphia, 3Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community. Biology Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion Jessica W. Wen1, Abby L. Olsen2, Maryna Perepelyuk2, Rebecca G. Wells2 1Division of Gastroenterology, Hepatology & Nutrition, Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Medicine, University of Pennsylvania A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture. Biology Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans Marni J. Falk1,2, Meera Rao*1, Julian Ostrovsky*1, Evgueni Daikhin1, Ilana Nissim1, Marc Yudkoff1,2 1Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.