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26 ARTICLES PUBLISHED IN JoVE

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Medicine

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice
Sharif U. Ahmed 1, Murtuza Zair 1, Kui Chen 1, Matthew Iu 1, Feng He 1, Oyedele Adeyi 2, Sean P. Cleary 3, Anand Ghanekar 1,3
1Toronto General Research Institute, University Health Network, 2Department of Pathology, University Health Network, 3Division of General Surgery, University Health Network

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

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JoVE Core

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose
Venkatesh Krishnan 1, Robert Clark 1, Marina Chekmareva 2, Amy Johnson 1, Sophia George 3, Patricia Shaw 4, Victoria Seewaldt 4,5, Carrie Rinker-Schaeffer 1
1Section of Urology, Department of Surgery, The University of Chicago, 2Department of Pathology, Robert Wood Johnson Medical School, 3Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, 4Department of Laboratory Medicine and Pathobiology, Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University of Toronto, University Health Network, 5Departments of Medicine, Pharmacology, and Cancer Biology, Duke University Medical Center

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

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Immunology and Infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection
Constance Finney 1, Lena Serghides 2
1Department of Biological Sciences, University of Calgary, 2Toronto General Research Institute, University Health Network

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

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Bioengineering

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance
Yannan N. Dou 1, Robert A. Weersink 2,5, Warren D. Foltz 5,6, Jinzi Zheng 5,6, Naz Chaudary 7, David A. Jaffray 2,3,4,5,6, Christine Allen 1
1Leslie Dan Faculty of Pharmacy, University of Toronto, 2Department of Radiation Oncology, University of Toronto, 3Medical Biophysics, University of Toronto, 4Institute of Biomaterials & Biomedical Engineering, University of Toronto, 5Techna Institute and Radiation Medicine Program, Princess Margaret Cancer Center, University Health Network, 6STTARR Innovation Center, University Health Network, 7Ontario Cancer Institute, University Health Network

AN MRI-compatible custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

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Medicine

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder
Katharine Dunlop 1, Pauline Gaprielian 6, Daniel Blumberger 5,7, Zafiris J. Daskalakis 5,7, Sidney H. Kennedy 2,3,5, Peter Giacobbe 2,3,5, Jonathan Downar 2,3,4,5
1Institute of Medical Sciences, University of Toronto, 2MRI-Guided rTMS Clinic, University Health Network, 3Department of Psychiatry, University Health Network, 4Toronto Western Research Institute, University Health Network, 5Department of Psychiatry, University of Toronto, 6Faculty of Arts and Science, University of Toronto, 7Temerty Centre for Therapeutic Brain Intervention, Centre for Addiction and Mental Health

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

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Immunology and Infection

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow
Yonghong Chen *1, Azusa Maeda *1,2, Jiachuan Bu 1, Ralph DaCosta 1,2,3
1Princess Margaret Cancer Centre, 2Department of Medical Biophysics, University of Toronto, 3Techna Institute, University Health Network

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

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Medicine

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors
Shawn Stapleton 1,2,3, Daniel Mirmilshteyn 2, Jinzi Zheng 3,4, Christine Allen 2,4,5, David A. Jaffray 1,2,3,4,5,6
1Department of Medical Biophysics, University of Toronto, 2Leslie Dan Faculty of Pharmacy, University of Toronto, 3STTARR Innovation Centre, Princess Margaret Cancer Centre, 4Institute of Biomaterials and Biomedical Engineering, University of Toronto, 5Techna Institute, University Health Network, 6Radiation Medicine Program, Princess Margaret Cancer Centre

The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

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Immunology and Infection

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies
Andrzej Chruscinski 1, Flora Y. Y. Huang 1, Antigona Ulndreaj 2, Conan Chua 1, Michael Fehlings 3, Vivek Rao 4, Heather J. Ross 1, Gary A. Levy 1
1Multi-Organ Transplant Program, University Health Network, 2Institute of Medical Science, University of Toronto, 3Divison of Neurosurgery, University Health Network, 4Division of Cardiac Surgery, University Health Network

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

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Immunology and Infection

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses
Jeeyoon Jennifer Ahn *1, Thirumahal Selvanantham *1, Monan Angela Zhang *1, Thierry Mallevaey 1, Shannon E. Dunn 1,2,3
1Department of Immunology, University of Toronto, 2Toronto General Research Institute, University Health Network, 3Women's College Research Institute

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-γ (IFN-γ) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

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Immunology and Infection

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain
Tingxi Guo 1,2, Toshiki Ochi *2, Munehide Nakatsugawa *2, Yuki Kagoya 2, Mark Anczurowski 1,2, Chung-Hsi Wang 1,2, Muhammed A. Rahman 2, Kayoko Saso 2, Marcus O. Butler 1,2, Naoto Hirano 1,2
1Department of Immunology, University of Toronto, 2Princess Margaret Cancer Centre, University Health Network

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCRα or TCRβ of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

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Developmental Biology

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors
Emily C. McGaugh 1,2,3, M. Cristina Nostro 1,2,3
1Toronto General Research Institute, University Health Network, 2McEwen Centre for Regenerative Medicine, University Health Network, 3Department of Physiology, University of Toronto

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

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Immunology and Infection

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry
Stephen C. Juvet 1, Sajad Moshkelgosha 2, Sharon Sanderson 3, Joanna Hester 4, Kathryn J. Wood 4, Andrew Bushell 4
1Division of Respirology, Departments of Medicine and Immunology, Toronto Lung Transplant Program, Multiorgan Transplant Program, Toronto General Research Institute, University of Toronto and University Health Network, 2Latner Thoracic Surgery Laboratories, Toronto General Research Institute, University Health Network, 3National Institutes of Health Research, Oxford Biomedical Research Centre, Translational Immunology Laboratory, NDORMS, Kennedy Institute of Rheumatology, University of Oxford, 4Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

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Developmental Biology

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation
Xuetao Sun 1, Sara S. Nunes 1,2,3,4
1Toronto General Research Institute, University Health Network, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Heart & Stroke/Richard Lewar Centre of Excellence, University of Toronto, 4Laboratory of Medicine and Pathobiology, University of Toronto

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

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JoVE Journal

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid
Shaun Pacheco 1, Shan-Yu Fung 2, Mingyao Liu 1,3
1Latner Thoracic Surgery Research Laboratories, University Health Network, 2Department of Pediatrics, British Columbia Children's Hospital & University of British Columbia, 3Institute of Medical Science, University of Toronto

This protocol describes a clinically-applicable means of dissolving hydrophobic compounds in an aqueous environment using combinations of self-assembling peptide and amino acid solutions. Our method resolves a major limitation of hydrophobic therapeutics, which lack safe, efficient means of solubility and delivery methods into clinical settings.

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Cancer Research

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice
Rui Xue Zhang 1, Tian Zhang 1, King Chen 1, Ji Cheng 1, Paris Lai 1, Andrew M. Rauth 2, K. Sandy Pang 1, Xiao Yu Wu 1
1Department of Pharmaceutical Sciences, University of Toronto, 2Departments of Medical Biophysics and Radiation Oncology, University of Toronto, Ontario Cancer Institute, University Health Network

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

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Bioengineering

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles
Hisato Konoeda 1, Hong Yang 2, Chengliang Yang 1, Annette Gower 1, Chun Xu 1, Wei Zhang 1, Mingyao Liu 1,3
1Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Respiratory Medicine Research Laboratory, Institute of Translation Medicine, Shanghai General Hospital, Shanghai Jiaotong University, 3Institute of Medical Science, Faculty of Medicine, University of Toronto

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

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Medicine

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation
Roberto V. P. Ribeiro 1,2, Juglans S. Alvarez 1, Frank Yu 1, Mitchell B. Adamson 1,2, Naoto Fukunaga 1, Cyril Serrick 3, Ved Bissoondath 1, Massimiliano Meineri 4,5, Mitesh V. Badiwala 1,6, Vivek Rao 1,2,6
1Division of Cardiovascular Surgery, Peter Munk Cardiac Center, Toronto General Hospital, University Health Network, 2Institute of Medical Science, University of Toronto, 3Perfusion and Anesthesia Services, Toronto General Hospital, University Health Network, 4Department of Anesthesia and Pain Management, Toronto General Hospital, University Health Network, 5Department of Anesthesia, University of Toronto, 6Department of Surgery, Faculty of Medicine, University of Toronto

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

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Cancer Research

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells
Lauren Philp 1,2, Harley Chan 3, Marjan Rouzbahman 4, Ariana Rostami 5, Lili Ding 6, Scott V. Bratman 5,6, Margarete K. Akens 5,7,8, Jonathan C. Irish 7,9,11, Marcus Q. Bernardini 10, Gang Zheng 5,11
1Institute of Medical Science, University of Toronto, 2Department of Obstetrics and Gynecology, University of Toronto, 3Guided Therapeutics Laboratory, TECHNA Institute, University Health Network, 4Department of Pathology, Princess Margaret Cancer Center, University Health Network, 5Department of Medical Biophysics, University of Toronto, 6Princess Margaret Cancer Center, University Health Network, 7Techna Institute, University Health Network, 8Department of Surgery, University of Toronto, 9Department of Otolaryngology, Head and Neck Surgery, University of Toronto, 10Division of Gynecologic Oncology, University of Toronto /Princess Margaret Cancer Center, University Health Network, 11Princess Margaret Cancer Center, University Health Network

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

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Immunology and Infection

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils
Jeffrey W. Chadwick 1,2, Michael Glogauer 1,2
1Department of Dental Oncology and Maxillofacial Prosthetics, Princess Margaret Cancer Centre, University Health Network, 2Faculty of Dentistry, University of Toronto

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

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Medicine

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation
Xu-Chun Chen *1, Manmeet Sekhon *1,2, Xue-Zhong Ma *1, Justin Manuel 1, Sai Chung 1,2, Eddie He 1, Agata Bartczak 1, Sandra Fischer 3, Cornelia Thoeni 3, Graziano Oldani 1, Catia T. Perciani 1, Sonya MacParland 1,2,3, Ian McGilvray 1
1Multi-Organ Transplant Program, University Health Network, 2Department of Immunology, University of Toronto, 3Department of Laboratory Medicine and Pathobiology, University of Toronto

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver's common hepatic artery to the recipient liver's proper hepatic artery.

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Bioengineering

Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment
Meng-Fen Tsai 1,3, Andrea Bandini 3, Rosalie H. Wang 2,3,5, José Zariffa 1,3,4,5
1Institute of Biomedical Engineering, University of Toronto, 2Department of Occupational Science and Occupational Therapy, University of Toronto, 3KITE, Toronto Rehabilitation Institute, University Health Network, 4Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, 5Rehabilitation Sciences Institute, University of Toronto

A protocol is proposed to capture natural hand function of individuals with hand impairments during their daily routines using an egocentric camera. The goal of the protocol is to ensure that the recordings are representative of an individual's typical hand use during activities of daily living at home.

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Neuroscience

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis
Darren M. O'Hara 1,2, Minesh Kapadia 1,2, Susan Ping 1,2, Suneil K. Kalia 1,2,3, Lorraine V. Kalia 1,2,4,5,6
1Krembil Research Institute, Toronto Western Hospital, University Health Network, 2Department of Laboratory Medicine and Pathobiology, University of Toronto, 3Department of Surgery, Division of Neurosurgery, University of Toronto, 4Department of Medicine, Division of Neurology, University of Toronto, 5Department of Medicine, Division of Neurology, Edmond J. Safra Program in Parkinson's Disease and the Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, University Health Network, 6Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

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Immunology and Infection

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System
Marieke de Korte 1,2, Armand Keating 1,2,4, Chen Wang 1,3
1Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, 2Krembil Research Institute, Toronto Western Hospital, University Health Network, 3Pathology and Lab Medicine, Hematopathology, Mount Sinai Hospital, Sinai Health Systems, 4Medical Oncology and Hematology, Princess Margaret Cancer Centre

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

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Medicine

Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound
Catherine A. Bellissimo 1, Idunn S. Morris 2,3,4, Jenna Wong 1, Ewan C. Goligher 1,2,3,5
1Toronto General Hospital Research Institute, 2Interdepartmental Division of Critical Care Medicine, University of Toronto, 3Department of Physiology, Faculty of Medicine, University of Toronto, 4Deparatment of Intensive Care Medicine, Nepean Hospital, 5Division of Respirology, Department of Medicine, University Health Network

Diaphragm thickness and function can be assessed in healthy individuals and critically ill patients using point-of-care ultrasound. This technique offers an accurate, reproducible, feasible, and well-tolerated method for evaluating diaphragm structure and function.

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Medicine

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation
Yamato Suzuki 1,2, Stephen Juvet 1,2, Mingyao Liu 1,2, Shaf Keshavjee 1,2
1Latner Thoracic Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Temerty Faculty of Medicine, University of Toronto

The murine intrapulmonary tracheal transplantation (IPTT) model is valuable for studying obliterative airway disease (OAD) after lung transplantation. It offers insights into lung-specific immunological and angiogenic behavior in airway obliteration after allotransplantation with high reproducibility. Here, we describe the IPTT procedure and its expected results.

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Medicine

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation
Paolo Oliveira *1,3, Keiji Yamanashi *1, Aizhou Wang 1, Marcelo Cypel 1,2
1Toronto General Hospital Research Institute, University Health Network, 2Department of Surgery, University of Toronto, 3Departamento de Cardiopneumologia, Instituto do Coração, Faculdade de Medicina HCFMUSP, Universidade de Sao Paulo

Here, we describe the necessary steps for establishing a rat EVLP model and show the inflammatory profile associated with the perfused lungs. The aim is to propagate knowledge and experiences about the rat EVLP model, enabling the integral understanding of the biological responses associated with that revolutionary technique.

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