Isolation of endothelial cells from murine tissue

The isolation and long-term culture of murine endothelial cells (ECs) has o ften proven a difficult task. In this paper we describe a quick, efficient protocol for the isolation of microvascular endothelial cells from murine t issues. Murine lung or heart are mechanically minced and enzymatically dige sted with collagenase and trypsin. The single cell suspension obtained is t hen incubated with an anti-CD31 antibody, anti-CD105 antibody and with biot inylated isolectin B-4. Pure EC populations are finally obtained by magneti c bead separation using rat anti-mouse Ig- and streptavidin-conjugated micr obeads. EC cultures are subsequently expanded and characterised. The surfac e molecule expression by the primary cultures of murine EC obtained from lu ng and heart tissue is analysed and compared to that of a murine endothelio ma and of primary cultures of murine renal tubular epithelial cells. The ph enotype and morphology of these cultures remain stable over 10-15 passages in culture, and no overgrowth of contaminating cells of non-endothelial ori gin is observed at any stage. (C) 2000 Elsevier Science B.V. All rights res erved.