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15 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology
Xiaofeng Cui *1,2,3, Guifang Gao *2,4, Tomo Yonezawa 5,6, Guohao Dai 1
1Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 2Stemorgan Inc., 3Institute of Advanced Study, Technical University of Munich, 4Institute of Virology, School of Medicine, Wuhan University, 5Department of Molecular and Experimental Medicine, The Scripps Research Institute, 6Research Institute for Biomedical Sciences, Tokyo University of Science

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

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Deriving De Novo Atomic Models by Cryo Electron Microscopy
Peng Ge 1, Nicole Poweleit 1,2, Robert P Gunsalus 2,3, Hong Zhou 1,2
1Electron Imaging Center for Nanomachines (EICN), California NanoSystems Institute, University of California, 2Immunology and Molecular Genetics, University of California, 3UCLA Institute of Genomics and Proteomics

Cryo electron microscopy (cryoEM) can be employed to derive de novo atomic models of macromolecular complexes in solution. The steps involved in high resolution cryoEM of biological molecules, from image recording, to data processing, to atomic modeling based on the resulting cryoEM density map, are illustrated.

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Biology

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)
Jing Men 1,2, Jason Jerwick 2,3, Penghe Wu 1,2, Mingming Chen 3,4, Aneesh Alex 2,3, Yutao Ma 4, Rudolph E. Tanzi 5, Airong Li 5, Chao Zhou 1,2,3
1Bioengineering Program, Lehigh University, 2Center for Photonics and Nanoelectronics, Lehigh University, 3Department of Electrical and Computer Engineering, Lehigh University, 4State Key Laboratory of Software Engineering, Wuhan University, 5Genetics and Aging Research Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

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Biology

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy
Jingwen Yang 1, Yunjun Yan 1, Xiaowei Xiang 1, Yuchao Xu 1, Naiming Zhou 2, Tianming Wang 1
1National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College, Zhejiang Ocean University, 2Institute of Biochemistry, College of Life Sciences, Zhejiang University

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

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Bioengineering

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin
Kun Lv 1,2, Song Gao 1,2
1The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), School & Hospital of Stomatology, Wuhan University, 2Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone's outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

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Cancer Research

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues
Yuan Wen 1, Xie Song-ping 2, Liu Pan 3, Liu Xiao-yan 1, Pan Shan 4, Yin Qian 4, Sun Meng 5, Huang Xiao-xing 6, Xiao Rui-jing 4, Xiong Jie 4, Zhang Qiu-ping 4, Shao Liang 1
1Department of Hematology, Zhongnan Hospital of Wuhan University, 2Department of Thoracic Surgery, Renmin Hospital of Wuhan University, 3Department of Human Anatomy, Histology and Embryology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, 4Department of Immunology, School of Basic Medical Sciences, Wuhan University, 5Department of Gastroenterology, Central Hospital of Wuhan, 6Department of Transfusion, Zhongnan Hospital of Wuhan University

ZW10 interacting protein (ZWINT) participates in the mitotic spindle checkpoint and the pathogenesis of carcinoma. Here, we introduce a methodology of the immunostaining of ZWINT in human lung cancer tissues, followed by the digital scanning of whole slides and image analysis. This methodology can provide high-quality digital images and reliable results.

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Developmental Biology

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone
Jingwen Yang *1,2, Haichun Pan *2, Yuji Mishina 2
1The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, 2Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

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Genetics

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions
Lina Yu *1, Wenxiu He *1, Jie Xie *1, Rui Guo *1, Juan Ni 2, Xia Zhang 3, Quishi Xu 1, Caifeng Wang 1, Qiuling Yue 1, Fangfang Li 1, Mengcheng Luo 4, Bo Sun 3, Lan Ye 1, Ke Zheng 1
1State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 2The Affiliated Hospital of Hangzhou Normal University, 3School of Life Science and Technology, ShanghaiTech University, 4Department of Tissue and Embryology, School of Basic Medical Sciences, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely applicable for studying protein-nucleic acid interactions.

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Bioengineering

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks
Mingjun Xie 1,2, Kang Yu 1,2, Yuan Sun 1,2, Lei Shao 1,2, Jing Nie 1,2, Qing Gao 1,2, Jingjiang Qiu 3, Jianzhong Fu 1,2, Zichen Chen 1,2, Yong He 1,2
1State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, 2Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School of Mechanical Engineering, Zhejiang University, 3School of Mechanics and Safety Engineering, Zhengzhou University

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

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Biology

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells
Tianming Wang 1, Xu Chen 1, Ke Xu 1, Bing Zhang 1, Dexiang Huang 1, Jingwen Yang 1
1National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College, Zhejiang Ocean University

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

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Engineering

Interactive and Visualized Online Experimentation System for Engineering Education and Research
Zhongcheng Lei 1, Hong Zhou 1, Shengwang Ye 1, Wenshan Hu 1, Guo-Ping Liu 1, Zijie Wei 1
1Department of Artificial Intelligence and Automation, Wuhan University

This work describes an online experimentation system that provides visualized experiments, including the visualization of theories, concepts, and formulas, visualizing the experimental process with three-dimensional (3-D) virtual test rigs, and visualizing the control and monitoring system using widgets such as charts and cameras.

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Engineering

A Virtual Simulation Experiment of Mechanics: Material Deformation and Failure Based on Scanning Electron Microscopy
You Li 1, Zhongcheng Lei 2, Yong He 1, Ze Liu 1
1School of Civil Engineering, Wuhan University, 2Department of Artificial Intelligence and Automation, Wuhan University

This work presents a three-dimensional virtual simulation experiment for material deformation and failure that provides visualized experimental processes. Through a set of experiments, users can become familiar with the equipment and learn the operations in an immersive and interactive learning environment.

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Engineering

Image Recognition and Parameter Analysis of Concrete Vibration State Based on Support Vector Machine
Shuai Wang 1,2, Aixun Wang 2, Xudong Fu 1, Keyang Wu 2, Tong Lu 2
1Wuhan University, 2Wuhan Construction Engineering Group Co. Ltd.

The protocol described in this paper utilizes the directional gradient histogram technique to extract the characteristics of concrete image samples under various vibration states. It employs a support vector machine for machine learning, resulting in an image recognition method with minimal training sample requirements and low computer performance demands.

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Medicine

A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine
Shixu Li 1,2, Yu Xiao 3, Yan Tang 1,2, Yongxin Zhang 1,2, Yingge Ma 1,2, Ling Wang *1,2, Lin Ye *1,2
1Shenzhen Eye Hospital, Jinan University, 2Shenzhen Eye Institute, 3Shenzhen Research Institute, Wuhan University

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry eye.

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Engineering

Online Virtual Reality Networked Control Laboratory Applied in Control Engineering Education
Guoyao Zhang 1, Zhongcheng Lei 1, Wenshan Hu 1, Hong Zhou 1
1Department of Artificial Intelligence and Automation, Wuhan University

This study describes a WebVR-based online virtual reality (VR) laboratory system that provides users with immersive and interactive experimentation capabilities supported by VR devices. The proposed system not only helps to enhance the realism of user participation in online experiments but is also applicable to a wide range of online laboratory frameworks.

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