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University of New South Wales

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30 articles published in JoVE

Medicine

Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models Brett A. Fong¹, James D. Crowley¹, William R. Walsh¹, Matthew H. Pelletier¹ ¹Surgical and Orthopaedic Research Laboratories (SORL), Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales (UNSW) Sydney The present protocol describes the maceration and cleaning of cadaveric bone with a vacuum-sealed, hot water bath immersion technique. This is a low-cost, safe, and effective method to produce anatomical specimens for surgical planning and medical education as an alternative to three-dimensional (3D) printed models.
Bioengineering

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs Gagan Jalandhra¹, Sara Romanazzo², Stephanie Nemec¹, Iman Roohani³, Kristopher A. Kilian^1,2 ¹School of Materials Science and Engineering, University of New South Wales, ²School of Chemistry, Australian Centre for NanoMedicine, University of New South Wales, ³School of Biomedical Engineering, University of Sydney This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.
Chemistry

3D Printing and In Situ Surface Modification via Type I Photoinitiated Reversible Addition-Fragmentation Chain Transfer Polymerization Nathaniel Corrigan¹, Cyrille Boyer¹ ¹Cluster for Advanced Macromolecular Design, and Australian Centre for Nanomedicine, School of Chemical Engineering, University of New South Wales The present protocol describes the digital light processing-based 3D printing of polymeric materials using type I photoinitiated reversible addition-fragmentation chain transfer polymerization and the subsequent in situ material post-functionalization via surface-mediated polymerization. Photoinduced 3D printing provides materials with independently tailored and spatially controlled bulk and interfacial properties.
Medicine

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency Katelin M. Allan^1,2, Sharon L. Wong^1,2, Laura K. Fawcett^1,2,3, Alexander Capraro^1,2, Adam Jaffe^1,2,3, Cristan Herbert⁴, Elvis Pandzic⁵, Shafagh A. Waters^1,2,3 ¹School of Women's and Children's Health, Faculty of Medicine and Health, University of New South Wales, ²Molecular and Integrative Cystic Fibrosis Research Centre (miCF RC), Faculty of Medicine and Health, University of New South Wales, ³ This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.
Medicine

Laparoscopic Repair of Para-Esophageal Hernia Using Absorbable Biosynthetic Mesh My Pham^1,2, Ruben Cohen-Hallaleh¹, Christophe R. Berney^1,2 ¹Department of General Surgery, Bankstown-Lidcombe Hospital, ²Medicine Faculty, University of New South Wales Presented here is a protocol of para-esophageal hernia repair. Use of absorbable biosynthetic mesh avoids the risk of erosion through the esophagus whilst reinforcing the repair. Glue fixation is preferred to avoid the risk of trauma such as bleeding or cardiac tamponade, which are associated with stitches or tacks.
Neuroscience

Objectively Assessing Sports Concussion Utilizing Visual Evoked Potentials Daryl H. Fong¹, Adrian J. Cohen², Dylan E. Mahony², Neil G. Simon⁴, Joseph E. Herrera³, Rebecca B. Baron³, David Putrino³ ¹School of Aerospace, Mechanical and Mechatronic Engineering, Faculty of Engineering and Information Technologies, University of Sydney, ²HeadsafeIP, ³Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, ⁴St Vincent's Clinical School, Faculty of Medicine, University of New South Wales A portable system capable of measuring steady-state visual-evoked potentials was developed and trialed on 65 amateur rugby players over 18 weeks to investigate SSVEP as a potential electrophysiological biomarker for concussion. Players' baselines were measured pre-season, with retesting for reliability, concussion, and recovery assessment being conducted within controlled time-periods, respectively.
Cancer Research

An Orthotopic Resectional Mouse Model of Pancreatic Cancer Tony C. Y. Pang^1,2,4,5, Zhihong Xu^1,2, Alpha Raj Mekapogu^1,2, Srinivasa Pothula^1,2, Therese M. Becker³, David Goldstein¹, Romano C. Pirola^1,2, Jeremy S. Wilson^1,2, Minoti V. Apte^1,2 ¹Pancreatic Research Group, South Western Sydney Clinical School, University of New South Wales, ²Ingham Institute for Applied Medical Research, ³Centre for Circulating Tumour Cell Diagnostics and Research, Ingham Institute for Applied Medical Research, ⁴Surgical Innovations Unit, Westmead Hospital, ⁵Westmead Clinical School, University of Sydney In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.
Immunology and Infection

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP) Jordan F. Hastings¹, Jeremy Z.R. Han¹, Robert F. Shearer^1,2, Sean P. Kennedy^1,3, Mary Iconomou^1,4, Darren N. Saunders⁵, David R. Croucher^1,6,7 ¹The Kinghorn Cancer Centre, Garvan Institute of Medical Research, ²Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, ³RCSI Molecular Medicine, Royal College of Surgeons in Ireland, ⁴Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, ⁵School of Medical Sciences, University of New South Wales, ⁶St Vincent's Hospital Clinical School, University of New South Wales, ⁷School of Medicine and Medical Science, University College Dublin This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.
Neuroscience

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing Brandon M. Wild¹, Renée Morris¹, Mihai Moldovan², Christian Krarup², Arun V. Krishnan³, Ria Arnold¹ ¹School of Medical Science, University of New South Wales, ²Department of Clinical Neurophysiology, Rigshospitalet and the Institute of Neuroscience and Pharmacology, University of Copenhagen, ³Prince of Wales Clinical School, University of New South Wales Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.
Immunology and Infection

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34⁺ Cells Jose Perdomo¹, Feng Yan¹, Halina H.L. Leung¹, Beng H. Chong^1,2 ¹Haematology Research Unit, St George and Sutherland Clinical School, University of New South Wales, ²Haematology Department, St George and Sutherland Hospitals A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34⁺ cells. A method for CD34⁺cell isolation and megakaryocyte differentiation is described here.
Developmental Biology

Generation of Genetically Modified Mice through the Microinjection of Oocytes Fabien Delerue¹, Lars M. Ittner¹ ¹Transgenic Animal Unit, Mark Wainwright Analytical Centre, University of New South Wales The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.
Engineering

Recombination Dynamics in Thin-film Photovoltaic Materials via Time-resolved Microwave Conductivity Joanna A. Guse^1,2, Timothy W. Jones³, Andrew Danos⁴, Dane R. McCamey^1,2 ¹ARC Centre of Excellence in Exciton Science, ²School of Physics, University of New South Wales, ³CSIRO, CSIRO Energy Centre, ⁴School of Chemistry, University of New South Wales A Time Resolved Microwave Conductivity technique for investigating direct and trap-mediated recombination dynamics and determining carrier mobilities of thin film semiconductors is presented here.
Bioengineering

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles Murray C. Killingsworth^1,2,3,4, Yuri V. Bobryshev^3,4,5 ¹South Western Sydney Clinical School, Faculty of Medicine, University of New South Wales Australia, ²School of Medicine, Western Sydney University, ³Correlative Microscopy Group, Ingham Institute for Applied Medical Research, ⁴Electron Microscopy Laboratory, Department of Anatomical Pathology, Sydney South West Pathology Service, New South Wales Health Pathology, ⁵School of Medical Sciences, Faculty of Medicine, University of New South Wales Australia A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.
Developmental Biology

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol Hananeh Fonoudi*^1,2,3,8, Hassan Ansari*^1,8, Saeed Abbasalizadeh¹, Gillian M Blue^6,7, Nasser Aghdami¹, David S Winlaw^6,7, Richard P Harvey^2,3,4, Alexis Bosman*^2,3, Hossein Baharvand*^1,5 ¹Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, ²Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, ³St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales, ⁴School of Biotechnology and Biomolecular Sciences, University of New South Wales, ⁵Department of Developmental Biology, University of Science and Culture, ⁶Heart Centre for Children, The Children´s Hospital at Westmead, ⁷Sydney Medical School, University of Sydney, ⁸Department of Developmental Biology, University of Science and Culture, Tehran, Iran Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.
Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry Alexander F. Mason¹, Pall Thordarson¹ ¹School of Chemistry, The Australian Centre for Nanomedicine and the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of New South Wales This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.
Chemistry

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst Jonathan Yeow^1,2,3, Jiangtao Xu^1,2,3, Cyrille Boyer^1,2,3 ¹Centre for Advanced Macromolecular Design (CAMD), The University of New South Wales, ²Australian Centre for NanoMedicine (ACN), The University of New South Wales, ³School of Chemical Engineering, The University of New South Wales This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.
Immunology and Infection

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data George Ashdown¹, Elvis Pandžić³, Andrew Cope², Paul Wiseman⁴, Dylan Owen¹ ¹Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, Division of Immunology, Infection and Inflammatory Disease, King's College London, ³ARC Centre for Advanced Molecular Imaging, Australian Centre for NanoMedicine, University of New South Wales Australia, ⁴Departments of Chemistry and Physic, McGill University To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.
Bioengineering

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology Su C. Chew^1,2, Scott A. Rice^2,3,4,5, Staffan Kjelleberg^2,3,4,6, Liang Yang^2,3 ¹Interdisciplinary Graduate School, Nanyang Technological University, ²Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, ³School of Biological Sciences, Nanyang Technological University, ⁴Centre for Marine Bio-Innovation, University of New South Wales, ⁵School of Biological, Earth and Environmental Sciences, University of New South Wales, ⁶School of Biotechnology and Biomolecular Sciences Sciences, University of New South Wales Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.
Neuroscience

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons Rahul Mohan*¹, Andrew P. Tosolini*¹, Renée Morris¹ ¹Translational Neuroscience Facility, School of Medical Sciences, University of New South Wales The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.
Engineering

Silicon Metal-oxide-semiconductor Quantum Dots for Single-electron Pumping Alessandro Rossi¹, Tuomo Tanttu², Fay E. Hudson¹, Yuxin Sun¹, Mikko Möttönen², Andrew S. Dzurak¹ ¹School of Electrical Engineering & Telecommunications, University of New South Wales, ²QCD Labs, COMP Centre of Excellence, Department of Applied Physics, Aalto University The fabrication process and experimental characterization techniques relevant to single-electron pumps based on silicon metal-oxide-semiconductor quantum dots are discussed.
Bioengineering

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification Martin D. Rees¹, Shane R. Thomas^1,2 ¹Centre for Vascular Research, University of New South Wales, ²School of Medical Sciences, University of New South Wales Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.
Engineering

In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries William R. Brant¹, Siegbert Schmid¹, Guodong Du², Helen E. A. Brand³, Wei Kong Pang^2,4,5, Vanessa K. Peterson⁴, Zaiping Guo^2,5, Neeraj Sharma⁶ ¹School of Chemistry, University of Sydney, ²Institute for Superconducting & Electronic Materials, University of Wollongong, ³Australian Synchrotron, ⁴Australian Nuclear Science and Technology Organisation, ⁵School of Mechanical, Materials, and Mechatronic Engineering, University of Wollongong, ⁶School of Chemistry, University of New South Wales We describe the design and construction of an electrochemical cell for the examination of electrode materials using in situ neutron powder diffraction (NPD). We briefly comment on alternate in situ NPD cell designs and discuss methods for the analysis of the corresponding in situ NPD data produced using this cell.
Bioengineering

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy Siti Hawa Ngalim¹, Astrid Magenau¹, Ying Zhu², Lotte Tønnesen¹, Zoe Fairjones¹, J. Justin Gooding², Till Böcking¹, Katharina Gaus¹ ¹Centre for Vascular Research and Australian Centre for Nanomedicine, The University of New South Wales, ²School of Chemistry and Australian Centre for Nanomedicine, The University of New South Wales A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.
Bioengineering

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration L. John R. Foster¹, Elizabeth Karsten¹ ¹Bio/Polymer Research Group, School of Biotechnology & Biomolecular Sciences, University of New South Wales The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.
Engineering

Polycrystalline Silicon Thin-film Solar cells with Plasmonic-enhanced Light-trapping Sergey Varlamov¹, Jing Rao¹, Thomas Soderstrom¹ ¹School of Photovoltaics, University of New South Wales Polycrystalline silicon thin-film solar cells on glass are fabricated by deposition of boron and phosphorous doped silicon layers followed by crystallisation, defect passivation and metallisation. Plasmonic light-trapping is introduced by forming Ag nanoparticles on the silicon cell surface capped with a diffused reflector resulting in ~45% photocurrent enhancement.
Neuroscience

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity Susanna B. Park^1,2, Cindy S-Y. Lin³, Matthew C. Kiernan^1,2 ¹Prince of Wales Clinical School, University of New South Wales, ²Neuroscience Research Australia, University of New South Wales, ³School of Medical Sciences, University of New South Wales This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.
Medicine

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats Paul J. Austin¹, Ann Wu¹, Gila Moalem-Taylor¹ ¹School of Medical Sciences, University of New South Wales Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).
Bioengineering

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages Kuldip Sidhu¹, Jaemin Kim¹, Methichit Chayosumrit², Sophia Dean¹, Perminder Sachdev³ ¹Stem Cell Lab, School of Psychiatry, Faculty of Medicine, The University of New South Wales, ²Siriraj Center of Excellence for Stem cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, ³Neuropsychiatric Institute, Prince of Wales Hospital We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.
Immunology and Infection

Production and Titering of Recombinant Adeno-associated Viral Vectors Christina McClure*¹, Katy L. H. Cole*¹, Peer Wulff¹, Matthias Klugmann², Andrew J. Murray^1,3 ¹School of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, ²Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, University of New South Wales, ³Department of Biochemistry and Molecular Biophysics, Columbia University Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.
Biology

DNA Methylation: Bisulphite Modification and Analysis Kate Patterson*¹, Laura Molloy*¹, Wenjia Qu¹, Susan Clark^1,2 ¹Epigenetics Group, Cancer Research Program, Garvan Institute of Medical Research, ²St Vincent's Clinical School, University of NSW The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

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