Isolation and epithelial co-culture of mouse renal peritubular endothelial cells

BMC Cell Biology

Methodology Article

Steve I Alexander¹  Yuan Min Wang¹  Ya Wang²  Yiping Wang²  Guoping Zheng²  Tania Tsatralis²  Qi Cao²  Ye Zhao²  David C Harris²  Hong Zhao³  Yun Zhang⁴ 

[1] Centre for Kidney Research, Children’s Hospital at Westmead, Sydney, NSW, Australia;Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia;Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia;Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, PR China;Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia;Experimental Centre of Science and Research, The First Clinical Hospital of Shanxi Medical University, Taiyuan, PR China;

关键词: Peritubular endothelial cells;    Tubular epithelial cells;    CD146;    Co-culture;    Vascular endothelial growth factor;   

DOI  :  10.1186/s12860-014-0040-6

 received in 2014-03-04, accepted in 2014-10-16,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundEndothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.ResultsFlow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.ConclusionIn this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

【 授权许可】

Unknown   
© Zhao et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.