University Medical Center Utrecht View Institution's Website 27 articles published in JoVE Biology Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles Rachel M. E. Cahalane*1,2, Mandy E. Turner*1, Cassandra L. Clift1, Mark C. Blaser1, Gabrielle Bogut1, Sydney Levy1, Taku Kasai1, Tom A. P. Driedonks3,4, Esther N. M. Nolte-‘t Hoen4, Masanori Aikawa1,5, Sasha A. Singh1,5, Elena Aikawa1,5 1Center for Excellence in Vascular Biology, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School A detailed protocol is provided for cleaning and re-using polycarbonate ultracentrifuge tubes to perform extracellular vesicle isolation suitable for proteomics experiments. Medicine Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model Laura Charlès1,2,3, Thomas Agius*4,5, Irina Filz von Reiterdank*1,2,3,6, Janna Hagedorn1,2,3, Yanis Berkane1,2,3,7, Hyshem H. Lancia1,2,3, Basak E. Uygun2,3,8, Korkut Uygun2,3,8, Curtis L. Cetrulo Jr.1,2,3,9, Mark A. Randolph1,2,3,9, Alexandre G. Lellouch1,2,3,8 1Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, 2Harvard Medical School, 3 Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia. Medicine Reduction of Radiation Exposure during Endovascular Treatment of Peripheral Arterial Disease Combining Fiber Optic RealShape Technology and Intravascular Ultrasound Constantijn E. V. B. Hazenberg1, Suzan C. A. Wulms2, Jurre Klaassen1, Joost A. van Herwaarden1 1Department of Vascular Surgery, University Medical Center Utrecht, 2Technical Medicine, University of Twente Described here is a stepwise method of combining Fiber Optic RealShape technology and intravascular ultrasound to show the potential of merging both techniques, in view of the reduction of radiation exposure and improvement of navigation tasks and treatment success during an endovascular procedure for the treatment of peripheral arterial disease. Bioengineering Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids Renee G. C. Maas1, Tess Beekink*1, Nino Chirico*1, Christian J. B. Snijders Blok1, Inge Dokter1, Vasco Sampaio-Pinto1, Alain van Mil1, Pieter A. Doevendans1, Jan W. Buikema2, Joost P. G. Sluijter*1, Francesca Stillitano*1 1Utrecht Regenerative Medicine Center, Circulatory Health Laboratory, University Utrecht, Department of Cardiology, University Medical Center Utrecht, 2Amsterdam Cardiovascular Sciences, Department of Physiology, Amsterdam University Medical Center Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation. Biology Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids Marie Bannier-Hélaouët1,2, Maarten H. Geurts1,2, Jeroen Korving1, Harry Begthel1, Hans Clevers1,2 1Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), University Medical Center Utrecht, 2Oncode Institute, Hubrecht Institute This protocol describes how to establish, maintain, genetically modify, differentiate, functionally characterize, and transplant lacrimal gland organoids derived from primary mouse and human tissue. Immunology and Infection The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model Yanis Berkane*1,2,3,5, Austin Alana Shamlou*1,2,4,5, Jose Reyes1,2, Hyshem H. Lancia1,2,5, Irina Filz von Reiterdank1,2,5,6,7, Nicolas Bertheuil3, Basak E. Uygun2,5,6, Korkut Uygun1,2,5,6, William G. Austen Jr.2,4, Curtis L. Cetrulo Jr.1,2,4,5, Mark A. Randolph1,2,4,5, Alexandre G. Lellouch1,2,5,6,8 1Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, 2Harvard Medical School, 3Department of Plastic, Reconstructive and Aesthetic Surgery, Rennes University Hospital Center (CHU de Rennes), University of Rennes 1, 4Plastic Surgery Research Laboratory, Massachusetts General Hospital, 5 This protocol describes harvesting, suturing, and monitoring fasciocutaneous flaps in rats that allow for good visualization and manipulation of blood flow through the superficial inferior epigastric vessels by means of clamping and ligating the femoral vessels. This is critical for studies involving ischemic preconditioning. Medicine A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids Jorik M. van Rijn1,2,3, Marliek van Hoesel1,2, Sabine Middendorp1,2 1Division of Pediatrics, Department of Pediatric Gastroenterology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht University, 2Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, 3Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation. Behavior Brain Infarct Segmentation and Registration on MRI or CT for Lesion-symptom Mapping J. Matthijs Biesbroek1, Hugo J. Kuijf2, Nick A. Weaver1, Lei Zhao3, Marco Duering4, Meta VCI Map Consortium, Geert Jan Biessels1 1Department of Neurology and Neurosurgery, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, 2Image Sciences Institute, University Medical Center Utrecht, 3BrainNow Research Institute, 4Institute for Stroke and Dementia Research, University Hospital, LMU Munich Provided here is a practical tutorial for an open-access, standardized image processing pipeline for the purpose of lesion-symptom mapping. A step-by-step walkthrough is provided for each processing step, from manual infarct segmentation on CT/MRI to subsequent registration to standard space, along with practical recommendations and illustrations with exemplary cases. Bioengineering Fabrication of Decellularized Cartilage-derived Matrix Scaffolds Kim E.M. Benders1, Margo L. Terpstra1, Riccardo Levato1, Jos Malda1,2 1Department of Orthopedics, University Medical Center Utrecht, The Netherlands, 2Department of Veterinary Medicine, Equine Surgery, University of Utrecht, The Netherlands Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings. Medicine Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD) George M. Solomon1, Inez Bronsveld2, Kathryn Hayes3, Michael Wilschanski4, Paola Melotti5, Steven M. Rowe1, Isabelle Sermet-Gaudelus6,7 1Department of Medicine and the Gregory Fleming James Cystic Fibrosis Center, University of Alabama at Birmingham, 2Department of Pulmonology and Tuberculosis, University Medical Center Utrecht, 3Center for Experimental Medicine, Queens University, Northern Ireland, 4Hadassah Hebrew University Medical Center, Jerusalem, 5Centro Fibrosi Cistica, Azienda Ospedaliera Universitaria Integrata, 6Service de Pneumologie et Allergologie Pédiatriques and Center de Ressources et de Compétence de la Mucoviscidose, Hôpital Necker Enfants Malades, 7INSERM U 1151, Institut Necker Enfants Malades Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure. Medicine Development of Recombinant Proteins to Treat Chronic Pain Judith Prado1, Jelena Popov-Celeketic2, Cristine Steen-Louws1, Ramin Raoof1, Eric Hack1, Niels Eijkelkamp1,3 1Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, 2Department of Rheumatology & Clinical Immunology, University Medical Center Utrecht, Utrecht University, 3Laboratory of Neuroimmunology and Developmental Origins of Disease, University Medical Center Utrecht, Utrecht University In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain. Biology Live-cell Imaging of Platelet Degranulation and Secretion Under Flow Arjan D. Barendrecht*1, Johan J. F. Verhoef*2, Silvia Pignatelli1, Gerard Pasterkamp1, Harry F. G. Heijnen1, Coen Maas1 1Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, 2Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis. Bioengineering An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage Vahid Arbabi*1,2, Behdad Pouran*1,2, Amir. A. Zadpoor1, Harrie Weinans1,2,3 1Department of Biomechanical Engineering, Faculty of Mechanical, Maritime, and Materials Engineering, Delft University of Technology (TU Delft), 2Department of Orthopedics, UMC Utrecht, 3Department of Rheumatology, UMC Utrecht We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods. Medicine 3D Whole-heart Myocardial Tissue Analysis Hans Thijs Van den Broek1, Leon De Jong1,2, Pieter A. Doevendans1, Steven A.J. Chamuleau1, Frebus J. Van Slochteren*1, René Van Es*1 1Department of Cardiology, Division Heart and Lungs, University Medical Center Utrecht, 2MIRA Institute, University Twente This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction. Medicine Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients Sylvia F. Boj1, Annelotte M. Vonk2, Marvin Statia1, Jinyi Su1, Johanna F. Dekkers3, Robert R. G. Vries1, Jeffrey M. Beekman2, Hans Clevers1,4 1Foundation Hubrecht Organoid Technology, 2Department of Pediatric Pulmonology, Regenerative Medicine Centre Utrecht, Wilhelmina Children's Hospital, University Medical Centre Utrecht, 3Department of Stem Cells and Cancer, Walter and Eliza Hall Institute of Medical Research, 4Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators. Medicine Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction Guilielmus H.J.M. Ellenbroek1, Gerardus P.J. van Hout1,2, Leo Timmers1,2, Pieter A. Doevendans2,4, Gerard Pasterkamp1,3, Imo E. Hoefer1,3 1Department of Experimental Cardiology, University Medical Center Utrecht, 2Department of Cardiology, University Medical Center Utrecht, 3Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, 4Interuniversity Cardiology Institutes of the Netherlands (ICIN) Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model. Medicine Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images Susanne J. van Veluw1, Geert Jan Biessels1, Peter R. Luijten2, Jaco J. M. Zwanenburg2 1Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht, 2Department of Radiology, University Medical Center Utrecht A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images. Immunology and Infection Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori Sina Bartfeld1, Hans Clevers1 1Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori. Bioengineering An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart A. C. H. Pape*1, Maarten H. Bakker*1, Cheyenne C. S. Tseng2, Maartje M. C. Bastings1, Stefan Koudstaal2, Pierfrancesco Agostoni2, Steven A. J. Chamuleau2, Patricia Y. W. Dankers1 1Institute for Complex Molecular Systems, Department of Biomedical Engineering, Laboratory of Chemical Biology, Eindhoven University of Technology, 2Department of Cardiology, Division Heart and Lungs, Interuniversity Cardiology Institute of the Netherlands (ICIN), University Medical Center Utrecht Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol–gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart. Medicine Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model David Stecher1, Glenn Bronkers2, Jappe O.T. Noest2, Cornelis A.F. Tulleken3, Imo E. Hoefer4, Lex A. van Herwerden1, Gerard Pasterkamp4, Marc P. Buijsrogge1 1Department of Cardiothoracic Surgery, University Medical Center Utrecht, 2Vascular Connect b.v., 3Department of Neurosurgery, University Medical Center Utrecht, 4Department of Experimental Cardiology, University Medical Center Utrecht This paper describes a novel nonocclusive coronary anastomotic connector in a porcine off-pump coronary artery bypass (OPCAB) model. This easy-to-use coronary connector has intrinsic potential to facilitate minimally invasive OPCAB surgery. Bioengineering Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing Sybren L. N. Maas1,2, Jeroen De Vrij1,2, Marike L. D. Broekman1,2 1Department of Neurosurgery, University Medical Center Utrecht, 2Brain Center Rudolf Magnus, University Medical Center Utrecht Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing. Biology Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses Muriel Mari1, Janice Griffith1, Fulvio Reggiori1 1Department of Cell Biology, University Medical Center Utrecht Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies. Medicine Myocardial Infarction and Functional Outcome Assessment in Pigs Stefan Koudstaal1,2, Sanne J. Jansen of Lorkeers1, Johannes M.I.H. Gho1, Gerardus P.J van Hout1, Marlijn S. Jansen1, Paul F. Gründeman1, Gerard Pasterkamp1,2, Pieter A. Doevendans1,2, Imo E. Hoefer*1, Steven A.J. Chamuleau*1 1Department of Cardiology, Division Heart and Lungs, University Medical Center Utrecht, 2Interuniversity Cardiology Institute of the Netherlands This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented. Immunology and Infection Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy Bas G.J. Surewaard1, Jos A.G. van Strijp1, Reindert Nijland1 1Medical Microbiology, University Medical Center Utrecht We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy. Medicine 5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat Arianne van Koppen1, Marianne C. Verhaar1, Lennart G. Bongartz1, Jaap A. Joles1 1Department of Nephrology & Hypertension, University Medical Center Utrecht A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions. Neuroscience Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays Ewoud R.E. Schmidt1, Francesca Morello1, R. Jeroen Pasterkamp1 1Department of Neuroscience & Pharmacology, Rudolf Magnus Institute for Neuroscience, University Medical Center Utrecht Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues. Biology Dendra2 Photoswitching through the Mammary Imaging Window Bojana Gligorijevic1,2, Dmitriy Kedrin1, Jeffrey E Segall1, John Condeelis1,2, Jacco van Rheenen1,2,3 1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine - Yeshiva University, 2Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine - Yeshiva University, 3Hubrecht Institute-KNAW and University Medical Center Utrecht Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.
