Cheung Kenneth C P, Marelli-Berg Federica M
William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom.
Bio Protoc. 2018 Jun 20;8(12):e2886. doi: 10.21769/BioProtoc.2886.
The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. Murine endothelial cell culture is an important tool for cardiovascular disease research. This protocol demonstrates a quick, efficient method for the isolation of microvascular endothelial cells from murine tissues without any special equipment. To isolate endothelial cells, the lung or heart were mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained was then incubated with an anti-CD31, anti-CD105 antibody and with biotinylated isolectin B-4. The endothelial cells were harvested using magnetic bead separation with rat anti-mouse Ig- and streptavidin-conjugated microbeads. Endothelial cells were expanded and collected for subsequent analyses. The morphological and phenotypic features of these cultures remained stable over 10 passages in culture. There was no overgrowth of contaminating cells of non-endothelial origin at any stage.
血管内皮对于正常的血管稳态至关重要。其功能障碍参与各种心血管疾病。小鼠内皮细胞培养是心血管疾病研究的重要工具。本方案展示了一种无需任何特殊设备即可从鼠组织中快速、高效分离微血管内皮细胞的方法。为了分离内皮细胞，将肺或心脏机械切碎并用胶原酶和胰蛋白酶进行酶消化。然后将获得的单细胞悬液与抗CD31、抗CD105抗体以及生物素化的异凝集素B-4一起孵育。使用与大鼠抗小鼠Ig和链霉亲和素偶联的磁珠通过磁珠分离收获内皮细胞。内皮细胞进行传代扩增并收集用于后续分析。这些培养物的形态和表型特征在传代培养10代过程中保持稳定。在任何阶段均未出现非内皮来源污染细胞的过度生长。
