Agency for Science, Technology and Research (Singapore) View Institution's Website 11 articles published in JoVE Biochemistry Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow Alan Tin Lun Lam1, Premkumar Jayaraman2, Abigail Becker3, Ryan Lim2, Kim Leng Teo1, Jacqueline Ng2, Steve Oh1 1Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A STAR), 2Thermo Fisher Scientific, 3Thermo Fisher Scientific Here, we present a protocol to harvest adherent cells from multi-layered flasks in a closed semi-automated manner using a counterflow centrifugation system. This protocol can be applied for harvesting both adherent and suspension cells from other cell expansion platforms with few modifications to the existing steps. Medicine Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases Ivan Seah*1,2, Zengping Liu*1,3,4, Daniel Soo Lin Wong1, Wendy Wong2, Graham E. Holder1,2,5, Veluchamy Amutha Barathi1,4,6, Gopal Lingam1,2,4, Xinyi Su1,2,3,4, Boris V. Stanzel1,7,8 1Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Ophthalmology, National University Hospital, Singapore, 3Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 4Singapore Eye Research Institute (SERI), 5UCL Institute of Ophthalmology, 6Academic Clinical Program in Ophthalmology, Duke-NUS Medical School, 7Macula Center Saar, Eye Clinic Sulzbach, Knappschaft Hospital Saar, 8Department of Ophthalmology, University of Bonn The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation. Bioengineering Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies Kuin T. Pang*1,2, Mean Ghim*1, Mehwish Arshad1, Xiaomeng Wang2,3,4, Peter D. Weinberg1 1Department of Bioengineering, Imperial College London, 2Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 3Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, 4Singapore Eye Research Institute, The Academia This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model. Bioengineering In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding Chen Fan1, Lay Keng Priscilla Lim1, Zihao Wu1, Bhavya Sharma1, Shi Qi Gan1,2, Kun Liang1, Zee Upton1,3, David Leavesley1 1Skin Research Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 2School of Chemical and Life Sciences, Singapore Polytechnic, 3Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR) This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue. Cancer Research Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells Yuri Frosi, Siti Radhiah Ramlan, Christopher J. Brown 1p53 Laboratory, Neuros/Immunos, A*STAR (Agency for Science, Technology and Research) Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats. Behavior Fully Automated Leg Tracking in Freely Moving Insects using Feature Learning Leg Segmentation and Tracking (FLLIT) Animesh Banerjee1, Shuang Wu2, Li Cheng3, Sherry Shiying Aw1 1Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 2Bioinformatics Institute, Agency for Science, Technology and Research, 3Department of Electrical and Computer Engineering, University of Alberta We describe detailed protocols for using FLLIT, a fully automated machine learning method for leg claw movement tracking in freely moving Drosophila melanogaster and other insects. These protocols can be used to quantitatively measure subtle walking gait movements in wild type flies, mutant flies and fly models of neurodegeneration. Genetics Genome Editing in Mammalian Cell Lines using CRISPR-Cas Kaiwen Ivy Liu1, Norfala-Aliah Binte Sutrisnoh1, Yuanming Wang1,2, Meng How Tan1,2 1Genome Institute of Singapore, Agency for Science Technology and Research, 2School of Chemical and Biomedical Engineering, Nanyang Technological University CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency. Bioengineering An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components Sau Yin Chin1,2, Yukkee Cheung Poh2, Anne-Céline Kohler2, Samuel K. Sia2 1Molecular Engineering Laboratory, Biomedical Sciences Institute, Agency for Science Technology and Research, 2Department of Biomedical Engineering, Columbia University An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation. Bioengineering Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum Abhijeet Patra1, Tao Ding2, Minghui Hong3, Arthur Mark Richards2, Ten It Wong4, Xiaodong Zhou4, Chester Lee Drum2 1NUS Nanoscience and Nanotechnology Initiative, National University of Singapore, 2Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, 3Department of Electrical and Computer Engineering, National University of Singapore, 4Institute of Materials Research Engineering, A*STAR (Agency for Science, Technology and Research) This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff ∼10-400 pg/mL, depending on the assay). Immunology and Infection Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes Wen Jing Sim1, Frano Malinarich1, Anna-Marie Fairhurst2, John Edward Connolly1,3 1Translational Immunology, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 2Singapore Immunology Network, 3Institute of Biomedical Studies, Baylor University Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes. Biology Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation Yufen Goh*1, Melissa J. Fullwood*1,2,3, Huay Mei Poh1, Su Qin Peh1, Chin Thing Ong1, Jingyao Zhang1, Xiaoan Ruan1, Yijun Ruan1,3 1Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, 2A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, 3Department of Biochemistry, National University of Singapore, Singapore Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.