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Children’s Hospital of Philadelphia

6 ARTICLES PUBLISHED IN JoVE

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. Prentice¹

¹Department of Chemistry, University of Florida, ²Division of Protective Immunity, Children’s Hospital of Philadelphia, ³Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

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

¹Division of Neurology, Children’s Hospital of Philadelphia, ²Department of Anesthesiology, Perelman School of Medicine at the University of Pennsylvania, ³Pharmacology Graduate Group, Perelman School of Medicine at the University of Pennsylvania

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

¹Division of Oncology, Children’s Hospital of Philadelphia, ²Center for Data Driven Discovery in Biomedicine, Children’s Hospital of Philadelphia, ³Division of Neurosurgery, Children’s Hospital of Philadelphia, ⁴Ben 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.

Medicine

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Rene Machietto¹, Nicholas Giacobbe¹, Jessica Perazzelli², Ted J. Hofmann², Allison Barz Leahy^2,3, Stephan A. Grupp^1,2,3, Yongping Wang^1,3,4, Stephan Kadauke^1,3,4

¹Cell and Gene Therapy Laboratory, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, ²Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, ³Perelman School of Medicine at the University of Pennsylvania, ⁴Division of Transfusion Medicine, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia

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.

Cancer Research

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

Kyra Harvey^1,2,3, Katherine Labella^1,2,3, Angela Liou^1,2,3, Stephanie Brosius^1,2,3, Thomas De Raedt^1,2,3

¹Division of Oncology, Children’s Hospital of Philadelphia, ²Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, ³School of Medicine, University of Pennsylvania

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

Developmental Biology

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Rong Qiu¹, Chayanne Petit¹, Christopher S. Thom^1,2

¹Division of Neonatology, Children’s Hospital of Philadelphia, ²Department of Pediatrics, University of Pennsylvania Perelman School of Medicine

We describe a protocol for extracting high-quality nuclei from cryopreserved induced pluripotent stem cell-derived stromal/endothelial and blood cell types to support single-nucleus next-generation sequencing analyses. Producing high-quality, intact nuclei is imperative for multiomics experiments but can be a barrier to entry in the field for some laboratories.