University of Manchester View Institution's Website 35 articles published in JoVE Bioengineering Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting Jessica J. Senior*1, Richard J. A. Moakes*2, Megan E. Cooke3, Samuel R. Moxon4, Alan M. Smith1, Liam M. Grover2 1Department of Pharmacy, University of Huddersfield, 2School of Chemical Engineering, University of Birmingham, 3Chemical and Biological Engineering, University of Colorado Boulder, 4School of Biological Sciences, University of Manchester Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described. Medicine High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening Yu-Fen Chang1, Wan-Chi Su1, Chih-Chuan Su1, Min-Wen Chung1, Jin Chang2, You-Yi Li3, Yi-Ju Kao3, Wen-Pin Chen3, Matthew J. Daniels4,5,6 1LumiSTAR Biotechnology, Inc., 2NEXEL Co., Ltd., 3Institute of Pharmacology, College of Medicine, National Taiwan University, 4Manchester Heart Centre, Manchester Royal Infirmary, Manchester University NHS Foundation Trust, 5Division of Cardiovascular Sciences, Manchester Academic Health Sciences Centre, University of Manchester, 6Division of Cell Matrix Biology and Regenerative Medicine, University of Manchester The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated. Bioengineering A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects Prasad Sawadkar1,3, Jason Wong2, Vivek Mudera1 1Division of Surgery and Interventional Science, University College London, 2Blond McIndoe Laboratories, Division of Cell Matrix and Regenerative Medicine, MAHSC, University of Manchester, 3Department of Surgical Research, Northwick Park Institute for Medical Research, Northwick Park Hospital In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue. Cancer Research Characterization and Functional Prediction of Bacteria in Ovarian Tissues Lanbo Zhao*1, Weichu Zhao*2, Qi Wang*1, Dongxin Liang1, Yu Liu3, Guoxing Fu4, Lu Han1, Yiran Wang1, Chao Sun1, Qing Wang1, Qing Song1, Qiling Li1, Qinrui Lu1 1Department of Pathology, First Affiliated Hospital of Xi’an Jiaotong University, 4Omega Bioservices Inc Immunohistochemistry staining and 16S ribosomal RNA gene (16S rRNA gene) sequencing were performed in order to discover and distinguish bacteria in cancerous and noncancerous ovarian tissues in situ. The compositional and functional differences of the bacteria were predicted by using BugBase and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Bioengineering Finite Element Modelling of a Cellular Electric Microenvironment Miruna Verdes1, Catherine Disney1, Chinnawich Phamornnak1, Lee Margetts2, Sarah Cartmell1,3 1Department of Materials, Faculty of Science and Engineering, The University of Manchester, 2Department of Mechanical, Aerospace and Civil Engineering, Faculty of Science and Engineering, The University of Manchester, 3The Henry Royce Institute, Royce Hub Building, The University of Manchester This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation. Medicine Slicing and Culturing Pig Hearts under Physiological Conditions Qinghui Ou*1, Riham R.E. Abouleisa*1, Xian-Liang Tang1, Hamzah R. Juhardeen1, Moustafa H. Meki2, Jessica M. Miller2, Guruprasad Giridharan2, Ayman El-Baz2, Roberto Bolli1, Tamer M.A. Mohamed1,3,4,5,6 1Institute of Molecular Cardiology, Department of Medicine, University of Louisville, 2Department of Bioengineering, University of Louisville, 3Diabetes and Obesity Center, Department of Medicine, University of Louisville, 4Department of Pharmacology and Toxicology, University of Louisville, 5Institute of Cardiovascular Sciences, University of Manchester, 6Faculty of Pharmacy, Zagazig University This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model. Engineering Design, Instrumentation and Usage Protocols for Distributed In Situ Thermal Hot Spots Monitoring in Electric Coils using FBG Sensor Multiplexing Anees Mohammed1, Siniša Durović1 1School of Electrical and Electronic Engineering, University of Manchester This paper presents a protocol that enables instrumentation of random wound electric coils with fiber Bragg grating (FBG) thermal sensors for the purpose of distributed condition monitoring of internal thermal hot spots. Genetics Measuring Microbial Mutation Rates with the Fluctuation Assay Rok Krašovec1, Huw Richards2,3, Guillaume Gomez3, Danna R. Gifford1, Adrien Mazoyer4, Christopher G. Knight3 1School of Biological Sciences, The University of Manchester, 2School of Science, University of Waikato, 3School of Natural Sciences, The University of Manchester, 4Department of Mathematics, Université du Québec à Montréal Here, a protocol is presented to perform a fluctuation assay and estimate microbial mutation rate using phenotypic markers. This protocol will enable researchers to assay mutations in diverse microbes and environments, determining how genotype and ecological context affect spontaneous mutation rates. Neuroscience Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins Cristina Melero*1, Aljona Kolmogorova*1, Paul Atherton1, Brian Derby2, Adam Reid3,4, Karin Jansen5, Christoph Ballestrem1 1Faculty of Biology, Medicine and Health. Division of Cell Matrix, Biology and Regenerative Medicine, Wellcome Trust Centre for Cell-Matrix Research, The University of Manchester, 2School of Materials, The University of Manchester, 3Blond McIndoe Laboratories, School of Biological Sciences, Faculty of Biology, Medicine and Health, Division of Cell Matrix Biology and Regenerative Medicine, The University of Manchester, Manchester Academic Health Science Centre, 4Department of Plastic Surgery & Burns, Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, 5Department of Pathology, UMC Utrecht Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding. Biology Fabrication of Amyloid-β-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease Bushra Almari1, David Brough2, Michael Harte1, Annalisa Tirella1 1Division of Pharmacy and Optometry, School of Health Studies, Faculty of Biology, Medicine and Health, University of Manchester, 2Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-β over time and can be used to study the effects of secreted amyloid-β in in vitro and in vivo models. Chemistry Synthesis and Characterization of Amphiphilic Gold Nanoparticles Zekiye P. Guven*1, Paulo H. Jacob Silva*1, Zhi Luo1, Urszula B. Cendrowska1, Matteo Gasbarri1, Samuel T. Jones1,2, Francesco Stellacci1,3 1Institute of Materials, École Polytechnique Fédérale de Lausanne, 2School of Materials, University of Manchester, 3Interfaculty Institute of Bioengineering, École Polytechnique Fédérale de Lausanne Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented. Neuroscience Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke Siobhan Crilly1, Alexandra Njegic2, Adrian R. Parry-Jones2,3, Stuart M. Allan1,3, Paul R. Kasher1,3 1Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Manchester Academic Health Science Centre, University of Manchester, 2Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, 3Lydia Becker Institute of Immunology and Inflammation, University of Manchester Here we present a protocol to quantify brain injury, locomotor deficits and neuroinflammation following bleeding in the brain in zebrafish larvae, in the context of human intracerebral hemorrhage (ICH). Cancer Research A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells Gray Kueberuwa1, Weiming Zheng1, Milena Kalaitsidou1, David E. Gilham1,2, Robert E. Hawkins1 1Manchester Cancer Research Centre Building, Department Cancer Sciences, University of Manchester, 2Celyad Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning. Immunology and Infection Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α Alba Llibre*1,2, Vincent Bondet*1,2, Mathieu P. Rodero3, David Hunt4, Yanick J. Crow3,5, Darragh Duffy1,2 1Immunobiology of Dendritic Cells, Institut Pasteur, 2INSERM U1223, 3Laboratory of Neurogenetics and Neuroinflammation, INSERM UMR1163, Institut Imagine, 4MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, 5Manchester Centre for Genomic Medicine, University of Manchester Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-α subtypes in human samples. Bioengineering Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies I-Ning Lee*1,2, Joseph Hosford*1, Shuai Wang3, John A. Hunt4, Judith M. Curran2, William P. Heath3, Lu Shin Wong1 1Manchester Institute of Biotechnology & School of Chemistry, University of Manchester, 2School of Engineering, University of Liverpool, 3School of Electrical and Electronic Engineering, University of Manchester, 4School of Science and Technology, Nottingham Trent University Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies. Bioengineering Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement Jun Liu1, John Wilson2, Claire Davis1, Anthony Peyton2 1Advanced Steel Research Centre, Warwick Manufacturing Group, University of Warwick, 2School of Electrical and Electronic Engineering, University of Manchester This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements. Chemistry Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation Monika S. Walczak1, Perla Morales-Gil1,2, Turia Belashehr1, Kiran Kousar1, Paulina Arellanes Lozada1,3, Robert Lindsay1 1Corrosion and Protection Centre, School of Materials, The University of Manchester, 2Laboratorio de Caracterización de Materiales Sintéticos y Naturales, Instituto Mexicano del Petróleo, 3Departamento de Metalurgia y Materiales, Instituto Politécnico Nacional A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented. Engineering Experimental Procedure for Warm Spinning of Cast Aluminum Components Matthew J. Roy1, Daan M. Maijer2 1School of Mechanical, Aerospace, and Civil Engineering, University of Manchester, 2Materials Engineering, University of British Columbia An experimental protocol for instrumented warm rotary forming of cast aluminum alloys employing a bespoke industrially scaled apparatus is presented. Experimental considerations including thermal and mechanical effects are discussed, as well as similitude with full-scale processing of automotive wheels. Cancer Research Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures Eamonn Morrison1,2, Patty Wai1,2, Andri Leonidou1,2, Philip Bland1,2, Saira Khalique1,2, Gillian Farnie3, Frances Daley1, Barrie Peck1,2, Rachael Natrajan1,2 1The Breast Cancer Now Toby Robins Research Centre, Division of Breast Cancer, The Institute of Cancer Research, 2Division of Molecular Pathology, The Institute of Cancer Research, 3Institute of Cancer Sciences, University of Manchester Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo. Biology Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling Zai Hao Zhao1, Chun Li Jin1, Ji Hyun Jang1, Yu Na Wu1, Sung Joon Kim1, Hong Hua Jin2, Lan Cui2, Yin Hua Zhang1,2,3 1Department of Physiology & Biomedical Sciences, Ischemic/hypoxic Disease Institute, Seoul National University College of Medicine, 2Yan Bian University Hospital, 3Institute of Cardiovascular Sciences, University of Manchester This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts. Engineering Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles Thomas J. A. Slater1, Edward A. Lewis1, Sarah J. Haigh1 1School of Materials, University of Manchester The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described. Neuroscience Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining Siwei Zhang*1,2, Jingjing Li*1,3, Robert Lea1, Enrique Amaya1 1The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, 2Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 3 This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections. Immunology and Infection Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network Nicolas Dutzan*1, Loreto Abusleme*1, Joanne E. Konkel2,3, Niki M. Moutsopoulos1 1Oral Immunity and Inflammation Unit, NIDCR, NIH, 2Manchester Immunology Group, Faculty of Life Sciences, University of Manchester, 3Manchester Collaborative Centre for Inflammation Research, University of Manchester We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva. Neuroscience Enrichment of Bruch's Membrane from Human Donor Eyes Selina McHarg1,2, Nicole Brace1,2, Paul N. Bishop1,2, Simon J. Clark1,2 1Centre for Ophthalmology & Vision Sciences, Institute of Human Development, University of Manchester, 2Centre for Advanced Discovery and Experimental Therapeutics, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre Identifying proteins specifically associated with Bruch’s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis. Bioengineering Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(ε-caprolactone) Electrospun Yarns Lucy A. Bosworth1, Sarah R. Rathbone1, Sarah H. Cartmell1 1School of Materials, The University of Manchester This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density. Neuroscience Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration Alba C. de Luca1, Alessandro Faroni2, Adam J. Reid2,3 1Centre for Neuroprosthesis, EPFL | STI | IMT/IBI | LSBI, 2Blond McIndoe Research Laboratories, Institute of Inflammation & Repair, The University of Manchester, 3University Hospital of South Manchester Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site. Medicine A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology Aamer Sandoo1,2, George D. Kitas2,3 1School of Sport, Health and Exercise Sciences, Bangor University, 2Department of Rheumatology, Dudley Group of Hospitals NHS Trust, Russells Hall Hospital, 3Arthritis Research UK Epidemiology Unit, University of Manchester The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis. Biology Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae Naomi Pollock1, Natasha Cant1, Tracy Rimington1, Robert C. Ford1 1Faculty of Life Sciences, University of Manchester Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies. Neuroscience A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions Antonio G. Zippo1, Sara Nencini1, Gian Carlo Caramenti2, Maurizio Valente1, Riccardo Storchi3, Gabriele E.M. Biella1 1Institute of Molecular Bioimaging and Physiology (IBFM), Department of Biomedicine, National Research Council, 2Institute of Biomedical Technologies (ITB), Department of Biomedicine, National Research Council, 3Faculty of Life Sciences, University of Manchester To elucidate the complex transition from Local Field Potentials (LFPs) to spikes a suitable stimulator for light mechanical peripheral stimuli was built. As an application, the spiking activities recorded from somatosensory cortex were analyzed by a multi-objective optimization strategy. The results demonstrated that the proposed stimulator was able to deliver tactile stimuli with millisecond and millimeter precisions. Biology In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors Açelya Yilmazer1, Irene de Lázaro1, Cyrill Bussy1, Kostas Kostarelos2 1Nanomedicine Lab, UCL School of Pharmacy, University College London, 2Nanomedicine Lab, Faculty of Medical & Human Sciences, University of Manchester This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency. Biology Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae Liam O'Ryan1, Tracy Rimington1, Natasha Cant1, Robert C. Ford1 1Faculty of Life Sciences, University of Manchester Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein. Medicine Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts Urszula M. Polanska*1, Ahmet Acar*1, Akira Orimo1,2 1CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, University of Manchester, 2Atopy Research Center, Juntendo University Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis. Medicine Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies Mitra Tavakoli*1, Rayaz A. Malik*1 1Division of Cardiovascular Medicine, University of Manchester Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity. Biology Preparation of Highly Coupled Rat Heart Mitochondria Irina Gostimskaya1, Alexander Galkin2 1Faculty of Life Sciences, University of Manchester, 2School of Biological Sciences, Queen's University Belfast We describe а protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis. Biology Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation Enrique Amaya1, Kristen Kroll2 1The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, 2Department of Developmental Biology, Washington University School of Medicine This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.