Isolation and characterization of large numbers of endothelial cells for studies of cell signaling.

Studies of endothelial cell signaling involving cholesterol-rich domains require large numbers of cells of bona fide origin. The growth of any cell in culture, particularly for extended periods, results in an altered phenotype that could include changes in the properties of caveolae and lipid raft structures. While continuously propagated cells are used to study specific questions because their origin is known and because proteins of interest are still expressed, such reasoning is an oversimplification and can lead to findings that are descriptive of the cell's adaptation to culture rather than its original phenotype. We are particularly cognizant of this concern as we examine caveolar signaling domains in endothelial cells. Here we present a reproducible method for the isolation and characterization of large numbers of bona fide endothelial cells suitable for studies of the regulation of receptor signal transduction. Digestion of guinea pig hearts with collagenase results in the liberation of cells that adhere to collagen-coated plastic and express platelet-endothelial cell adhesion molecule 1 (PECAM-1) and binding sites for Ulex europaeus agglutinin 1 (UEA-1) that permit segregation of cells using fluorescence-activated cell sorting. Growth of cells over 7 doublings results in enrichment in the expression of both PECAM-1 and UEA-1 and retention of functional low-density lipoprotein receptor. The ability of cells to differentiate into endothelial tubes at any stage during their characterization up to 20 doublings in culture suggests that this method can be employed to generate endothelial cells that are minimally altered from their site of origin.