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University Medical Center Utrecht

25 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays
Ewoud R.E. Schmidt 1, Francesca Morello 1, R. Jeroen Pasterkamp 1
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.

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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 Koppen 1, Marianne C. Verhaar 1, Lennart G. Bongartz 1, Jaap A. Joles 1
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.

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Immunology and Infection

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy
Bas G.J. Surewaard 1, Jos A.G. van Strijp 1, Reindert Nijland 1
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.

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Medicine

Myocardial Infarction and Functional Outcome Assessment in Pigs
Stefan Koudstaal 1,2, Sanne J. Jansen of Lorkeers 1, Johannes M.I.H. Gho 1, Gerardus P.J van Hout 1, Marlijn S. Jansen 1, Paul F. Gründeman 1, Gerard Pasterkamp 1,2, Pieter A. Doevendans 1,2, 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.

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Bioengineering

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing
Sybren L. N. Maas 1,2, Jeroen De Vrij 1,2, Marike L. D. Broekman 1,2
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.

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Biology

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses
Muriel Mari 1, Janice Griffith 1, Fulvio Reggiori 1
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.

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Medicine

Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model
David Stecher 1, Glenn Bronkers 2, Jappe O.T. Noest 2, Cornelis A.F. Tulleken 3, Imo E. Hoefer 4, Lex A. van Herwerden 1, Gerard Pasterkamp 4, Marc P. Buijsrogge 1
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.

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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. Tseng 2, Maartje M. C. Bastings 1, Stefan Koudstaal 2, Pierfrancesco Agostoni 2, Steven A. J. Chamuleau 2, Patricia Y. W. Dankers 1
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.

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Medicine

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images
Susanne J. van Veluw 1, Geert Jan Biessels 1, Peter R. Luijten 2, Jaco J. M. Zwanenburg 2
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.

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Immunology and Infection

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori
Sina Bartfeld 1, Hans Clevers 1
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.

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Medicine

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction
Guilielmus H.J.M. Ellenbroek 1, Gerardus P.J. van Hout 1,2, Leo Timmers 1,2, Pieter A. Doevendans 2,4, Gerard Pasterkamp 1,3, Imo E. Hoefer 1,3
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.

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Medicine

3D Whole-heart Myocardial Tissue Analysis
Hans Thijs Van den Broek 1, Leon De Jong 1,2, Pieter A. Doevendans 1, Steven A.J. Chamuleau 1, 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.

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Medicine

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients
Sylvia F. Boj 1, Annelotte M. Vonk 2, Marvin Statia 1, Jinyi Su 1, Johanna F. Dekkers 3, Robert R. G. Vries 1, Jeffrey M. Beekman 2, Hans Clevers 1,4
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.

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Biology

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow
Arjan D. Barendrecht *1, Johan J. F. Verhoef *2, Silvia Pignatelli 1, Gerard Pasterkamp 1, Harry F. G. Heijnen 1, Coen Maas 1
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.

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Medicine

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)
George M. Solomon 1, Inez Bronsveld 2, Kathryn Hayes 3, Michael Wilschanski 4, Paola Melotti 5, Steven M. Rowe 1, Isabelle Sermet-Gaudelus 6,7
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.

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Behavior

Brain Infarct Segmentation and Registration on MRI or CT for Lesion-symptom Mapping
J. Matthijs Biesbroek 1, Hugo J. Kuijf 2, Nick A. Weaver 1, Lei Zhao 3, Marco Duering 4, , Geert Jan Biessels 1
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.

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Medicine

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization
Jae Gyun Oh 1, Jaydev Dave 1, Changwon Kho 1, Francesca Stillitano 1
1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

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Cancer Research

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids
Marrit Putker *1, Rosemary Millen *2, René Overmeer *3, Else Driehuis 2,4, Maurice M. J. M. Zandvliet 5, Hans Clevers 2, Sylvia F. Boj 3, Qi-Xiang Li 6
1Crown Bioscience Netherlands B.V., 2Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht, 3Hubrecht Organoid Technology (HUB), 4Pathology dept, University Medical Center Utrecht, 5Department of Clinical Sciences - Companion Animals, Faculty of Veterinary Medicine, Utrecht University, 6Crown Bioscience Inc.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

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Bioengineering

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids
Renee G. C. Maas 1, Tess Beekink *1, Nino Chirico *1, Christian J. B. Snijders Blok 1, Inge Dokter 1, Vasco Sampaio-Pinto 1, Alain van Mil 1, Pieter A. Doevendans 1, Jan W. Buikema 2, 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.

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Medicine

Reduction of Radiation Exposure during Endovascular Treatment of Peripheral Arterial Disease Combining Fiber Optic RealShape Technology and Intravascular Ultrasound
Constantijn E. V. B. Hazenberg 1, Suzan C. A. Wulms 2, Jurre Klaassen 1, Joost A. van Herwaarden 1
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.

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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 Reyes 1,2, Hyshem H. Lancia 1,2,5, Irina Filz von Reiterdank 1,2,5,6,7, Nicolas Bertheuil 3, Basak E. Uygun 2,5,6, Korkut Uygun 1,2,5,6, William G. Austen Jr. 2,4, Curtis L. Cetrulo Jr. 1,2,4,5, Mark A. Randolph 1,2,4,5, Alexandre G. Lellouch 1,2,5,6,8
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, 5Shriners Children's Boston, 6Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, 7Department of Plastic Surgery and Hand Surgery, University Medical Center Utrecht, 8Department of Plastic, Reconstructive, and Aesthetic Surgery, Groupe Almaviva Santé, Clinique de l'Alma

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.

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Biology

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids
Marie Bannier-Hélaouët 1,2, Maarten H. Geurts 1,2, Jeroen Korving 1, Harry Begthel 1, Hans Clevers 1,2
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.

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Medicine

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model
Laura Charlès 1,2,3, Thomas Agius *4,5, Irina Filz von Reiterdank *1,2,3,6, Janna Hagedorn 1,2,3, Yanis Berkane 1,2,3,7, Hyshem H. Lancia 1,2,3, Basak E. Uygun 2,3,8, Korkut Uygun 2,3,8, Curtis L. Cetrulo Jr. 1,2,3,9, Mark A. Randolph 1,2,3,9, Alexandre G. Lellouch 1,2,3,8
1Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, 2Harvard Medical School, 3Shriners Children’s Boston, 4Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, 5Department of Vascular Surgery, Centre Hospitalier Universitaire Vaudois and University of Lausanne, 6Department of Plastic, Reconstructive and Hand Surgery, University Medical Center Utrecht, 7Department of Plastic, Reconstructive and Aesthetic Surgery, Rennes University Hospital Center (CHU de Rennes), Rennes 1 University, 8Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, 9Plastic Surgery Research Laboratory, Massachusetts General Hospital

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.

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Biology

Surgical Technique of the 3-Dimensional-printed Personalized Hip Implant for the Treatment of Canine Hip Dysplasia
Irin Kwananocha 1,2, Femke Verseijden 1, Seyed A Kamali 1, Joëll Magré 3, Koen Willemsen 3, Jacobine CM Schouten 4, Daniela Salvatori 4, Marianna A Tryfonidou 1, Björn P Meij 1
1Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 2Research and Academic Service, Faculty of Veterinary Medicine, Kasetsart University, 3Department of Orthopedics, University Medical Center Utrecht, 4Department of Clinical Sciences, Anatomy and Physiology, Faculty of Veterinary Medicine, Utrecht University

This work describes a novel surgical technique for extracapsular implantation of a personalized, 3-dimensional-printed, joint-preserving implant. This novel treatment aims to restore hip stability in young adult dogs suffering from hip dysplasia with laxity by uniquely reproducing the anatomical shape of the acetabular rim of the hip joint.

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Biology

Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles
Rachel M. E. Cahalane *1,2, Mandy E. Turner *1, Cassandra L. Clift 1, Mark C. Blaser 1, Gabrielle Bogut 1, Sydney Levy 1, Taku Kasai 1, Tom A. P. Driedonks 3,4, Esther N. M. Nolte-‘t Hoen 4, Masanori Aikawa 1,5, Sasha A. Singh 1,5, Elena Aikawa 1,5
1Center for Interdisciplinary Cardiovascular Sciences, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Mechanobiology and Medical Device Research Group (MMDRG), Biomedical Engineering, College of Science and Engineering, University of Galway, 3Department of CDL Research, University Medical Center Utrecht, 4Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, 5Center 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.

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