Leiden University Medical Center 27 articles published in JoVE Immunology and Infection Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures Dennis K. Ninaber1, Anne M. van der Does1, Pieter S. Hiemstra1 1PulmoScience Laboratory, Department of Pulmonology, Leiden University Medical Center Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface. Developmental Biology Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos Marije Koning1,2, Ellen Lievers1,2, Thierry Jaffredo3, Cathelijne W. van den Berg1,2,4, Ton J. Rabelink1,2,4 1Department of Internal Medicine - Nephrology, Leiden University Medical Center, 2Einthoven Laboratory of Vascular and Regenerative Medicine, Leiden University Medical Center, 3IBPS, CNRS UMR7622, Developmental Biology Laboratory, Sorbonne Université, 4The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden University Medical Center Here, we present a detailed protocol for the transplantation of kidney organoids in the celomic cavity of chicken embryos. This method induces vascularization and enhanced maturation of the organoids within 8 days and can be used to study these processes in an efficient manner. Medicine Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing Yang Ge1,2, Lieke van Roon1, H. Sophia Chen1,2, Ruben Methorst1, Martin Paton2, Marco C. DeRuiter1, Szymon M. Kielbasa3, Monique R. M. Jongbloed1,2 1Department of Anatomy & Embryology, Leiden University Medical Center, 2Department of Cardiology, Leiden University Medical Center, 3Department of Medical Statistics and Bioinformatics, Leiden University Medical Center This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding. Biology Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro Natalija Bogunovic*1,2,3, Karlijn B. Rombouts*1,2, Kak Khee Yeung1,2 1Department of Vascular Surgery, Amsterdam UMC, Vrije Universiteit Amsterdam, 2Department of Physiology, Amsterdam Cardiovascular Sciences, Amsterdam UMC, Vrije Universiteit Amsterdam, 3Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells. Biology In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing Georgios Galaris1, Jérémy H. Thalgott1, Eliott Teston1, Franck P.G. Lebrin1,2,3 1Einthoven Laboratory for Experimental Vascular Medicine, Department of Internal Medicine (Nephrology), Leiden University Medical Center, 2Institute Physics for Medicine Paris, INSERM U1273, ESPCI Paris, CNRS FRE 2031, 3MEMOLIFE Laboratory of Excellence and PSL Research University This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal. Medicine Intraoperative Assessment of Resection Margins in Oral Cavity Cancer: This is the Way Yassine Aaboubout1,2, Elisa M. Barroso1,3,4, Mahesh Algoe1, Patricia C. Ewing-Graham1, Ivo ten Hove3,6, Hetty Mast3, José A. Hardillo2, Aniel Sewnaik2, Dominiek A. Monserez2, Stijn Keereweer2, Brend P. Jonker3, Cornelia G. F. van Lanschot2, Roeland W. H. Smits2, Maria R. Nunes Soares1,4, Lars Ottevanger1, Sanne E. Matlung1, Paul A. Seegers5, Vera van Dis1, Robert M. Verdijk1, Eppo B. Wolvius3, Peter J. Caspers4, Tom C. Bakker Schut4, Robert J. Baatenburg de Jong2, Gerwin J. Puppels4, Senada Koljenović1 1Department of Pathology, Erasmus MC University Medical Center, 2Department of Otorhinolaryngology and Head and Neck Surgery, Erasmus MC University Medical Center, 3Department of Oral and Maxillofacial Surgery, Erasmus MC University Medical Center, 4Department of Dermatology, Erasmus MC University Medical Center, 5PALGA foundation, The nationwide network and registry of histo- and cytopathology, 6Department of Oral and Maxillofacial Surgery, Leiden University Medical Center The goal of this protocol is to provide a clear overview of specimen-driven intraoperative assessment of resection margins. It is encouraged to implement this protocol to improve patient care at other institutes. Biology TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing Jin Ma1,2, Gerard van der Zon1,2, Gonzalo Sanchez-Duffhues1, Peter ten Dijke1,2 1Dept. Cell Chemical Biology, Leiden University Medical Center, 2Oncode Institute, Leiden University Medical Center We describe methods to investigate TGF-β2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-β2-induced EndMT. Medicine Location, Dissection, and Analysis of the Murine Stellate Ganglion Katharina Scherschel1,2,3, Hanna Bräuninger2,4, Klara Glufke2, Christiane Jungen2,4,5, Nikolaj Klöcker3, Christian Meyer1,2,3 1Division of Cardiology, EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium, 2DZHK (German Centre for Cardiovascular Research), 3Institute of Neural and Sensory Physiology, Medical Faculty, Heinrich Heine University Düsseldorf, 4Clinic for Cardiology, University Heart & Vascular Centre, University Hospital Hamburg-Eppendorf, 5Department of Cardiology, Leiden University Medical Center Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes. Cancer Research Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells Jing Zhang1, Midory Thorikay1, Gerard van der Zon1, Maarten van Dinther1, Peter ten Dijke1 1Oncode Institute and Department of Cell Chemical Biology, Leiden University Medical Center We describe a systematic workflow to investigate TGF-β signaling and TGF-β-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining. Cancer Research Management of Respiratory Motion Artefacts in 18F-fluorodeoxyglucose Positron Emission Tomography using an Amplitude-Based Optimal Respiratory Gating Algorithm Willem Grootjans1, Peter Kok2, Jurrian Butter2, Erik Aarntzen2 1Department of Radiology, Leiden University Medical Centre, 2Department of Radiology and Nuclear Medicine, Radboud University Medical Centre Amplitude-based optimal respiratory gating (ORG) effectively removes respiratory-induced motion blurring from clinical 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) images. Correction of FDG-PET images for these respiratory motion artefacts improves image quality, diagnostic and quantitative accuracy. Removal of respiratory motion artefacts is important for adequate clinical management of patients using PET. Immunology and Infection Visualization and Quantification of High-Dimensional Cytometry Data using Cytofast and the Upstream Clustering Methods FlowSOM and Cytosplore Guillaume Beyrend1, Koen Stam2, Ferry Ossendorp1, Ramon Arens1 1Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2Department of Parasitology, Leiden University Medical Center Cytofast is a visualization tool used to analyze output from clustering. Cytofast can be used to compare two clustering methods: FlowSOM and Cytosplore. Cytofast can rapidly generate a quantitative and qualitative overview of mass cytometry data and highlight the main differences between different clustering algorithms. Bioengineering Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro Vincent van Duinen1,2, Wendy Stam1, Viola Borgdorff3, Arie Reijerkerk3, Valeria Orlova4, Paul Vulto5, Thomas Hankemeier2, Anton Jan van Zonneveld1 1Department of Internal Medicine (Nephrology) and the Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, 2Division of Systems Biomedicine and Pharmacology, Department of Analytical BioSciences and Metabolomics, Leiden University, 3Ncardia, 4Department of Anatomy and Embryology, Leiden University Medical Center, 5Mimetas This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner. Medicine CO2-Lasertonsillotomy Under Local Anesthesia in Adults Justin E.R.E. Wong Chung1,2, Noud van Helmond3, Rozemarie van Geet1, Peter Paul G. van Benthem2, Henk M. Blom1,2 1Department of Otolaryngology, HagaZiekenhuis, 2Department of Otolaryngology, Leiden University Medical Center, 3Department of Anesthesiology, Cooper Medical School of Rowan University, Cooper University Hospital CO2-lasertonsillotomy under local anesthesia is an interesting alternative treatment method for tonsillectomy under general anesthesia for tonsil-related complaints in adults. This report presents a step-by-step protocol detailing the execution of CO2-lasertonsillotomy under local anesthesia. Immunology and Infection A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation Eline J. Arends*1, Laura S. van Dam*1, Tineke Kraaij1, Sylvia W.A. Kamerling1, Ton J. Rabelink1, Cees van Kooten1, Y.K. Onno Teng1 1Department of Nephrology, Leiden University Medical Center This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies. Developmental Biology Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs) Oleh V. Halaidych1, Francijna van den Hil1, Christine L. Mummery1,2, Valeria V. Orlova1 1Department of Anatomy and Embryology, Leiden University Medical Center, 2Department of Applied Stem Cell Technologies, University of Twente This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow. Developmental Biology The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens Esther Dronkers1, Asja T. Moerkamp1, Tessa van Herwaarden1, Marie-José Goumans1, Anke M. Smits1 1Department of Molecular Cell Biology, Leiden University Medical Center The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics. Medicine A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells Daniëlle G. Leuning1, Ellen Lievers1, Marlies E.J. Reinders1, Cees van Kooten1, Marten A. Engelse1, Ton J. Rabelink1 1Department of Internal Medicine, Leiden University Medical Centre Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy. Cancer Research Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples Simone Punt1, Robert J. Baatenburg de Jong2, Ekaterina S. Jordanova1,3 1Department of Pathology, Leiden University Medical Center, 2Department of Otorhinolaryngology and Head and Neck Surgery, Erasmus University Medical Center, 3Center for Gynecological Oncology Amsterdam, VU Medical Center Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer. Developmental Biology Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs) Luca Sala1, Dorien Ward-van Oostwaard1, Leon G. J. Tertoolen1, Christine L Mummery1,2, Milena Bellin1 1Department of Anatomy and Embryology, Leiden University Medical Center, 2Department of Applied Stem Cell Technologies, University of Twente Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals. Cancer Research Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish Jiang Ren*1, Sijia Liu*1, Chao Cui1, Peter ten Dijke1 1Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively. Medicine Percutaneous Hepatic Perfusion (PHP) with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver Eleonora M. de Leede1, Mark C. Burgmans2, Christian H. Martini3, Fred G. J. Tijl4, Arian R. van Erkel2, Jaap Vuyk3, Ellen Kapiteijn5, Cornelis Verhoef6, Cornelis J. H. van de Velde1, Alexander L. Vahrmeijer1 1Department of Surgery, Leiden University Medical Centre, 2Department of Radiology, Leiden University Medical Centre, 3Department of Anesthesiology, Leiden University Medical Centre, 4Department of Extracorporeal Circulation, Leiden University Medical Centre, 5Department of Medical Oncology, Leiden University Medical Centre, 6Department of Surgery, Erasmus MC Cancer Institute In this manuscript, we describe percutaneous isolated hepatic perfusion with simultaneous chemofiltration as treatment for unresectable liver metastases. This procedure is performed under general anaesthesia in the angiosuite by an experienced team, consisting of an interventional radiologist, a clinical perfusionist and anaesthesiologist. Medicine A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail Chun Yu Wong1,2,3, Margreet R. de Vries2,3, Yang Wang4, Joost R. van der Vorst3, Alexander L. Vahrmeijer3, Anton-Jan van Zonneveld1,2, Jaap F. Hamming3, Prabir Roy-Chaudhury4, Ton J. Rabelink1,2, Paul H. A. Quax2,3, Joris I. Rotmans1,2 1Department of Nephrology, Leiden University Medical Center, 2Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 3Department of Surgery, Leiden University Medical Center, 4Division of Nephrology, University of Cincinnati Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions. Bioengineering Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System Boudewijn P.T. Kruithof1, Samuel C. Lieber2, Marianna Kruithof-de Julio3, Vincian Gaussin4, Marie José Goumans1 1Department of Molecular Cell Biology, Leiden University Medical Center, 2Department of Engineering Technology, New Jersey Institute of Technology, 3Department of Urology, Leiden University Medical Center, 4Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve. Medicine Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions Sofia Karkampouna1, Peter Kloen2, Miryam C. Obdeijn3, Scott M. Riester4, Andre J. van Wijnen4,5, Marianna Kruithof-de Julio1,6 1Department of Molecular Cell Biology, Cancer Genomics Centre and Centre for Biomedical Genetics, Leiden University Medical Center, 2Department of Orthopedic Surgery, Academic Medical Center, 3Department of Plastic, Reconstructive and Hand Surgery, Academic Medical Center, 4Department of Orthopedic Surgery, Mayo Clinic, 5Department of Biochemistry and Molecular Biology, Mayo Clinic, 6Department of Dermatology, Leiden University Medical Center Dupuytren’s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue. Medicine Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth. Begüm Kocatürk1, Henri H. Versteeg1 1Department of Hemostasis and Thrombosis, Leiden University Medical Center Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology. Medicine State of the Art Cranial Ultrasound Imaging in Neonates Ginette M. Ecury-Goossen1, Fleur A. Camfferman2, Lara M. Leijser3,4, Paul Govaert1,5, Jeroen Dudink1,2 1Department of Pediatrics, Division of Neonatology, Erasmus MC-Sophia Children's Hospital, 2Department of Radiology, Erasmus MC-Sophia Children's Hospital, 3Department of Pediatrics, Division of Neonatology, UZ Brussel, 4Department of Pediatrics, Division of Neonatology, Leiden University Medical Center, 5Department of Pediatrics, Division of Neonatology, Isala Hospital, 6 Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique. Behavior Assessing Functional Performance in the Mdx Mouse Model Annemieke Aartsma-Rus1, Maaike van Putten1 1Department of Human Genetics, Leiden University Medical Center The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.