title: Isolation and Culture Expansion of Tumor-specific Endothelial Cells.
abstract: Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5(cre):ZsGreen(l/s/l) reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.
authors.affiliation: Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill.
authors.forename: Lin
authors.initials: L
authors.lastname: Xiao
authors.affiliation: Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill.
authors.forename: James V
authors.initials: JV
authors.lastname: McCann
authors.affiliation: Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill; McAllister Heart Institute, University of North Carolina at Chapel Hill; acdudley@med.unc.edu.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Dudley
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D042783:Endothelial Cells / Q000473:pathology*; D005260:Female; D005347:Fibroblasts / Q000473:pathology; D005818:Genetic Engineering / Q000379:methods; D018189:Immunomagnetic Separation / Q000379:methods*; D008325:Mammary Neoplasms, Experimental / Q000098:blood supply* / Q000473:pathology*; D051379:Mice; D009389:Neovascularization, Pathologic / Q000473:pathology