The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital

10 ARTICLES PUBLISHED IN JoVE

Developmental Biology

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

Zhaofeng Jia^1,2, Yujie Liang³, Bin Ma^4,5, Xiao Xu^2,6, Jianyi Xiong², Li Duan², Daping Wang^1,2

¹Guangzhou Medical University, ²Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopeadic Engineering, Department of Orthopedics, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), ³Department of Chemistry, The Chinese University of Hong Kong, ⁴School of Biomedical Engineering, Shanghai Jiao Tong University, ⁵Renji Hospital Clinical Stem Cell Research Center, Shanghai Jiao Tong University School of Medicine, ⁶Shantou University Medical College

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

Neuroscience

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Xiao Xu¹, Yinwei You^1,2, Long Zhang¹

¹Department of Entomology, China Agricultural University, ²Bio-tech Research Center, Shandong Academy of Agricultural Sciences

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Biology

A Method for Islet Transplantation to the Omentum in Mouse

Ying Lu^*1, Shangyou Zou^*1, Suzanne Bertera^*2, Rita Bottino², David K.C. Cooper³, Zhengzhao Liu¹, Yi Huang¹, Chengjun Wang¹, Chungu Hong¹, Tian He¹, Hancheng Zhang¹, Qin Huo⁴, Hongxing Fu⁵, Zhiming Cai¹, Lisha Mou¹

¹Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen University Health Science Center, ²Institute for Cellular Therapeutics, Allegheny-Singer Research Institute, ³Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, ⁴College of Life Science and Oceanography, Shenzhen University, ⁵School of Pharmcy, Wenzhou Medical College

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Biology

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Zhaofeng Jia^1,2,3, Yujie Liang^4,5, Xingfu Li^2,3, Xiao Xu^1,2,3, Jianyi Xiong^2,3, Daping Wang^1,2,3, Li Duan^2,3

¹Postgraduate institution, Guangzhou Medical University, ²Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, ³Shenzhen Key Laboratory of Tissue Engineering, Shenzhen Laboratory of Digital Orthopaedic Engineering, Shenzhen Second People's Hospital (The First Hospital Affiliated to Shenzhen University), ⁴Department of Chemistry, Chinese University of Hong Kong, ⁵Shenzhen Kangning Hospital, Shenzhen Mental Health Center

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Biology

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Yanli Zhao^1,2,3, Chengjiang Zhao⁴, David K.C. Cooper⁵, Ying Lu², Kewang Luo⁶, Huiyun Wang³, Pengfei Chen³, Changchun Zeng³, Shaodong Luan¹, Lisha Mou², Hanchao Gao^1,2,3

¹Department of Nephrology, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, ²Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, ³Department of Medical Laboratory, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, ⁴Department of Endocrinology, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, ⁵Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, ⁶People's Hospital of Longhua

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

JoVE Journal

Thermocapillary Convection Space Experiment on the SJ-10 Recoverable Satellite

Li Duan^*1,2, Yongli Yin^*3, Jia Wang^*1, Qi Kang^1,2, Di Wu¹, Huan Jiang¹, Pu Zhang¹, Liang Hu¹

¹National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, ²School of Engineering Sciences, University of Chinese Academy of Sciences, ³China Astronaut Research and Training Center

A protocol for the space payload design, the space experiment on thermocapillary convection, and analyses of experimental data and images are presented in this paper.

Medicine

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Yuanzheng Peng^*1, Zhicheng Zou^*1, Jiao Chen¹, Hancheng Zhang¹, Ying Lu¹, Rito Bittino², Hongxing Fu³, David K. C. Cooper⁴, Shan Lin¹, Mengtao Cao¹, Yifan Dai⁵, Zhiming Cai¹, Lisha Mou¹

¹Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Health Science Center, ²Institute for Cellular Therapeutics, Allegheny-Singer Research Institute, ³School of Pharmaceutical Sciences, Wenzhou Medical University, ⁴Xenotransplantation Program/Department of Surgery, University of Alabama at Birmingham, ⁵Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Biology

Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model

Yangyang Deng^*1,2, Ming Zhou^*1,3, Ying Lu¹, Jiao Chen¹, Zuhui Pu⁴, Dongjing Yu^1,5, Yifan Dai⁶, Yongqiang Zhan⁷, Lisha Mou¹

In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Using species-specific primers, cpsDNA was quantified by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models.

Biology

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Li Duan^1,2, Xingfu Li^1,2, Xiao Xu^1,2, Limei Xu^1,2, Daping Wang^1,2, Kan Ouyang^1,2, Yujie Liang^1,3

¹Department of Orthopedics, The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital, ²Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, Shenzhen Second People's Hospital, ³Department of Child and Adolescent Psychiatry, Shenzhen Kangning Hospital, Shenzhen Mental Health Center, Shenzhen Key Laboratory for Psychological Healthcare & Shenzhen Institute of Mental Health

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Biology

Adult Pig Islet Isolation

Ying Lu^*1,2, Zuhui Pu^*3, Jiao Chen², Jing Deng², Ying Deng^2,4, Shufang Zhu², Chunliang Xu⁵, Fuwen Yao^1,2, Zijing Wu^1,2, Yong Ni¹, Yongqiang Zhan¹, Jun Cheng¹, Naiyang Zhan¹, Wenlong Huang⁶, Zhiming Cai², Rita Bottino⁷, Lisha Mou²

¹Department of Hepatopancreatobiliary Surgery, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, ²Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Institute of Translational Medicine, First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, ³Imaging Department, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, ⁴Faculty of Science, University of Waterloo, ⁵School of Life Science, The Chinese University of Hong Kong, ⁶General Surgery, The First Affiliated Hospital of Shantou University Medical College, ⁷Director Islet Programs, Imagine Pharma

This detailed protocol covers the methodological steps of adult pig islet isolation from the digestion phase via purification to the final functional assessment of the islets. This outline can be used as a guideline for adult pig islet isolation in research institutions.