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10 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Polymeric Microneedle Array Fabrication by Photolithography
Himanshu Kathuria 1, Jaspreet Singh Kochhar 1, Michelle Hui Min Fong 1, Michinao Hashimoto 2, Ciprian Iliescu 3, Hanry Yu 3,4,5, Lifeng Kang 1
1Department of Pharmacy, National University of Singapore, 2Singapore University of Technology and Design, 3Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research (A*STAR), 4Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 5Mechanobiology Institute, National University of Singapore

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

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Bioengineering

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening
Yong Yu 1, Siu Yee New 1, Jiaxian Lin 2, Xiaodi Su 1, Yen Nee Tan 1
1Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), 2School of Chemical & Biomedical Engineering, Nanyang Technological University

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

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Medicine

A Step by Step Protocol for Subretinal Surgery in Rabbits
Sami Al-Nawaiseh 1, Fabian Thieltges 1, Zengping Liu 1,2, Claudine Strack 1, Ralf Brinken 1, Norbert Braun 3, Marc Wolschendorf 3, Arvydas Maminishkis 5, Nicole Eter 4, Boris V. Stanzel 1,6
1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology, National University of Singapore, 3Geuder AG, 4Department of Ophthalmology, University of Münster, 5Section on Epithelial and Retinal Physiology and Disease, National Eye Institute/National Institutes of Health, 6Surgical Retina Department, Singapore National Eye Centre

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

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Neuroscience

Enumeration of Neural Stem Cells Using Clonal Assays
Gunaseelan Narayanan 1, Yuan Hong Yu 1, Muly Tham 1, Hui Theng Gan 1, Srinivas Ramasamy 1, Shvetha Sankaran 1, Srivats Hariharan 1, Sohail Ahmed 1
1Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR)

Neural stem cells (NSCs) refer to cells which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

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Biochemistry

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
Emery Haley *1, Wooyoung Choi *1, Chen Fan 1, Weinan Sun 2,3, Juan Du 1, Wei Lü 1
1Van Andel Institute, 2Vollum Institute, 3Janelia Research Campus

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

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Bioengineering

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding
Chen Fan 1, Lay Keng Priscilla Lim 1, Zihao Wu 1, Bhavya Sharma 1, Shi Qi Gan 1,2, Kun Liang 1, Zee Upton 1,3, David Leavesley 1
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.

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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 Arshad 1, Xiaomeng Wang 2,3,4, Peter D. Weinberg 1
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.

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Biology

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control
Inn Chuan Ng *1, Li Zhang *2, Narelle Nichola Yi Ying Shen 3, Yun Ting Soong 4, Chan Way Ng 5, Phoebe Kang Sheing Koh 1, Yan Zhou 3, Hanry Yu 1,3,4,5,6
1Department of Physiology & The Institute for Digital Medicine (WisDM), National University of Singapore, 2College of Agriculture and Biology, Zhongkai University of Agriculture and Engineering, 3Mechanobiology Institute, National University of Singapore, 4Institute of Bioengineering and Nanotechnology, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering, 6CAMP, Singapore-MIT Alliance for Research and Technology

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

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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 Wong 1, Wendy Wong 2, Graham E. Holder 1,2,5, Veluchamy Amutha Barathi 1,4,6, Gopal Lingam 1,2,4, Xinyi Su 1,2,3,4, Boris V. Stanzel 1,7,8
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.

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Cancer Research

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells
Eva Dovjak 1, Mario Mairhofer 1, Claudia Wöß 1, Jingjing Qi 1, Dorothy N. Y. Fan 2, Clemens A. Schmitt 1,2,3, Yong Yu 1
1Johannes Kepler University Linz, 2Charité - University Medicine Berlin, 3Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

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