Agency for Science, Technology and Research (A*STAR)

10 ARTICLES PUBLISHED IN JoVE

Bioengineering

Polymeric Microneedle Array Fabrication by Photolithography

Himanshu Kathuria¹, Jaspreet Singh Kochhar¹, Michelle Hui Min Fong¹, Michinao Hashimoto², Ciprian Iliescu³, Hanry Yu^3,4,5, Lifeng Kang¹

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.

Bioengineering

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Yong Yu¹, Siu Yee New¹, Jiaxian Lin², Xiaodi Su¹, Yen Nee Tan¹

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.

Medicine

A Step by Step Protocol for Subretinal Surgery in Rabbits

Sami Al-Nawaiseh¹, Fabian Thieltges¹, Zengping Liu^1,2, Claudine Strack¹, Ralf Brinken¹, Norbert Braun³, Marc Wolschendorf³, Arvydas Maminishkis⁵, Nicole Eter⁴, 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.

Neuroscience

Enumeration of Neural Stem Cells Using Clonal Assays

Gunaseelan Narayanan¹, Yuan Hong Yu¹, Muly Tham¹, Hui Theng Gan¹, Srinivas Ramasamy¹, Shvetha Sankaran¹, Srivats Hariharan¹, Sohail Ahmed¹

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.

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 Fan¹, Weinan Sun^2,3, Juan Du¹, Wei Lü¹

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.

Bioengineering

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

Chen Fan¹, Lay Keng Priscilla Lim¹, Zihao Wu¹, Bhavya Sharma¹, Shi Qi Gan^1,2, Kun Liang¹, Zee Upton^1,3, 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.

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 Wang^2,3,4, 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.

Biology

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Inn Chuan Ng^*1, Li Zhang^*2, Narelle Nichola Yi Ying Shen³, Yun Ting Soong⁴, Chan Way Ng⁵, Phoebe Kang Sheing Koh¹, Yan Zhou³, 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.

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. 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.

Cancer Research

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Eva Dovjak¹, Mario Mairhofer¹, Claudia Wöß¹, Jingjing Qi¹, Dorothy N. Y. Fan², Clemens A. Schmitt^1,2,3, Yong Yu¹

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.