A method for obtaining and culturing large numbers of purified organ-derived murine endothelial cells

Fibroblast growth factor 1 (FGF-1)-coated collagen-gelatin sponges were affixed to various tissues to generate vascular beds, in which the vessels originated in the tissue to which the sponges were affixed. Organ-derived endothelium was obtained from vascularized sponges implanted in or on the skin, peritoneal wall, abdominal mesentery, epimysium, spleen, and liver. Collagenase digestion yielded single-cell suspensions that were analyzed by flow cytometry. Approximately 25% of the cells were positive for the endothelial cell (EC) markers MECA-32 and Sca-1 and for uptake of diIAcLDL. Similar results were obtained when sponges were implanted in several different mouse strains, although there was some evidence of heterogeneity in the degree of vascularization and EC recovery. Long-term cultures of high purity were obtained when the ECs were grown on mitomycin C-treated L929 feeder layers, in medium supplemented with cis-hydroxyproline and FGF-1. These cells have been utilized in preliminary studies of T cell-EC binding. Thus we have developed a generalized method for the recovery and culture of organ-derived murine endothelial cells. This technique should greatly improve the feasibility of studies of the interactions between murine endothelial and immune effector cells.