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 wer e affixed to various tissues to generate vascular beds, in which the v essels originated in the tissue to which the sponges were affixed. Org an-derived endothelium was obtained from vascularized sponges implante d in or on the skin, peritoneal wall, abdominal mesentery, epimysium, spleen, and liver. Collagenase digestion yielded single-cell suspensio ns that were analyzed by flow cytometry. Approximately 25% of the cell s were positive for the endothelial cell (EC) markers MECA-32 and Sca- 1 and for uptake of diIAcLDL. Similar results were obtained when spong es were implanted in several different mouse strains, although there w as 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 s upplemented with cis-hydroxyproline and FGF-1. These cells have been u tilized in preliminary studies of T cell-EC binding. Thus we have deve loped a generalized method for the recovery and culture of organ-deriv ed murine endothelial cells. This technique should greatly improve the feasibility of studies of the interactions between murine endothelial and immune effector cells.