Children's Hospital of Philadelphia

24 articles published in JoVE

• Neuroscience

  
  Maintenance of a Lateral Fluid Percussion Injury Device Anthony M. Farrugia², Samuelle A. S. Delcy^2,3, Brian N. Johnson¹, Akiva S. Cohen^{1,2 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. Madsen^2,3, Tiffany Smith², Crystal Griffin^1,2, Luke Patterson^1,2, Nicholas A. Vitanza^4,5, Phillip B. Storm^2,3, Adam C. Resnick^2,3, Jessica B. Foster^{1,2 1}Center 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. Zackular^2,3, Boone M. Prentice^{1 1}Department 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 Song^1,2, Arianna Valeri¹, Fanxin Long^{1 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 Nasimuzzaman^1,2, Sophia Villaveces¹, Johannes C. M. van der Loo^1,2,3, Sivani Alla^{1 1}Raymond 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-Ogiso^1,2, Marni J. Falk^{1,2 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. Alferiev^1,2, Robert J. Levy^1,2, 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 Liu^1,2, Dalibor Kurepa³, Francesco Feletti^4,5, Almudena Alonso-Ojembarrena⁶, Jovan Lovrenski⁷, Roberto Copetti⁸, Erich Sorantin⁹, Javier Rodriguez-Fanjul¹⁰, Karishma Katti³, Andrea Aliverti⁴, Huayan Zhang^11,12, Misun Hwang¹³, Tsu F. Yeh¹⁴, Cai-Bao Hu¹⁵, Xing Feng¹⁶, Ru-Xin Qiu^1,2, Jing-Han Chi¹⁷, Li-Li Shang¹⁸, Guo-Rong Lyu¹⁹, Shao-Zheng He²⁰, Yan-Fen Chai²¹, Zhan-Jun Qiu²², Hai-Ying Cao^2,23, Yue-Qiao Gao^1,2, Xiao-Ling Ren^1,2, Guo Guo^1,24, Li Zhang^1,2, Ying Liu^1,2, Wei Fu^1,2, Zu-Lin Lu^1,2, Hong-Lei Li^{1,2 1}Department 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 Gadue^1,2,3, Deborah L. French^{1,2,3 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 Liu^1,2, Roberto Copetti³, Erich Sorantin⁴, Jovan Lovrenski⁵, Javier Rodriguez-Fanjul⁶, Dalibor Kurepa⁷, Xing Feng⁸, Luigi Cattaross⁹, Huayan Zhang^10,11, Misun Hwang¹², Tsu F. Yeh^13,14, Yisrael Lipener⁷, Abhay Lodha¹⁵, Jia-Qin Wang¹⁶, Hai-Ying Cao^2,17, Cai-Bao Hu^2,18, Guo-Rong Lyu¹⁹, Xin-Ru Qiu^1,2, Li-Qun Jia²⁰, Xiao-Man Wang²⁰, Xiao-Ling Ren^1,2, Jiu-Ye Guo^1,2, Yue-Qiao Gao^1,2, Jian-Jun Li^1,2, Ying Liu^1,2, Wei Fu^1,2, Yan Wang²¹, Zu-Lin Lu^1,2, Hua-Wei Wang⁸, Li-Li Shang^{22 1}Department 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 Tomasula^{1 1}Dairy 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. Peranteau^{1 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 Fritsch^1,2, Jean-Francois Pierre Gout^3,4, Marc Vermulst^{1 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 Song^{1,2 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 Nasimuzzaman^1,2, Johannes C.M. van der Loo^1,2,3, Punam Malik^{1,2 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 Ramakrishnan^{1 1}Clinical 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. Slee^1,2, Ivan S. Alferiev^1,2, Robert J. Levy^1,2, Stanley J. Stachelek^{1,2 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. Alferiev^{1 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. Wells^{2 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. Grant^{1,2 1}Division 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. Smith^1,2, Brian N. Johnson¹, Jaclynn A. Elkind¹, Jill M. See¹, Guoxiang Xiong¹, Akiva S. Cohen^{1,3 1}Division 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 Mostoslavsky^{1 1}Center 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. Wells^{2 1}Division 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. Falk^1,2, Meera Rao*¹, Julian Ostrovsky*¹, Evgueni Daikhin¹, Ilana Nissim¹, Marc Yudkoff^{1,2 1}Department 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.