University of New South Wales

22 ARTICLES PUBLISHED IN JoVE

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

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.

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.

JoVE Core

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

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.

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.

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.

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.

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.

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.

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.

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

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Ayse S. Dereli¹, Evan J. Bailey¹, Natasha N. Kumar¹

¹Department of Pharmacology, School of Medical Sciences, University of New South Wales

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

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.

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.

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, ³Department of Respiratory Medicine, Sydney Children's Hospital, ⁴Department of Pathology, School of Medical Sciences, University of New South Wales Australia, ⁵Katharina Gaus Light Microscopy Facility, Mark Wainwright Analytical Centre, University of New South Wales

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.

Neuroscience

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Andrew P. Tosolini^1,2, David Villarroel-Campos^1,2,3, Giampietro Schiavo^1,2,3, James N. Sleigh^1,2,3

¹Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, ²UCL Queen Square Motor Neuron Disease Centre, University College London, ³UK Dementia Research Institute, University College London

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

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.

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.

Biology

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture

Jonathan Daly^1,2, Rebecca Hobbs¹, Nikolas Zuchowicz³, Mary Hagedorn^4,5, Justine K. O’Brien^1,2

¹Taronga Institute of Science and Learning, Taronga Conservation Society Australia, ²School of Biological, Earth and Environmental Sciences, University of New South Wales, ³Department of Mechanical Engineering, University of Minnesota, ⁴Hawaii Institute of Marine Biology, University of Hawaii, ⁵Smithsonian National Zoo and Conservation Biology Institute

This protocol describes a semi-automated pathway to improve the efficiency and capacity of processing and cryopreservation of sperm from threatened coral species, aiming to secure genetic diversity and support reef restoration efforts.

Biology

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures

Qitao Zhang^*1, Daisy Y. Shu^*2, Richard A. Bryan³, John Y. S. Han¹, Gillian A. Gulette¹, Kin Lo³, Leo A. Kim⁴, Jason M. L. Miller^1,5

¹Kellogg Eye Center, University of Michigan, Ann Arbor, ²School of Optometry and Vision Science, University of New South Wales, ³Lucid Scientific, ⁴Schepens Eye Research Institute of Mass. Eye and Ear, Department of Ophthalmology, Harvard Medical School, ⁵Cellular and Molecular Biology Program, University of Michigan, Ann Arbor

We introduce a novel device for measuring oxygen consumption rates (OCR) in retinal pigment epithelial (RPE) cultures. The device can measure OCR for weeks at a time on RPE grown on standard cell culture plates with standard media while the plates are in a standard cell culture incubator.