Isolation and Characterization of Retinal Endothelial Cells

Primary vascular endothelial cell cultures provide powerful systems to investigate the molecular architecture and regulation of the blood-brain and blood-retinal barriers. Most investigators agree that in vitro models of endothelial cells alone do not completely recapitulate the strong resistance barrier achieved in vivo by the blood vessels in these neural tissues. However, in vitro models provide a number of advantages that make this a highly useful system to study the transport of molecules across an endothelial monolayer. First, the system is highly defined; the investigator has control over the cell types that are present as well as the timing and degree of the perturbation applied to the system. Thus, the direct effect of a hormone or physical stress on endothelial cell transport properties can be determined and highly precise measures for time course and dose response can be made. Second, precise rate measures can be made and compared between different molecules. The effect of size and charge on solute transport rate may be determined and the rate of water, ion, and solute flux can be directly compared. Also, with the appropriate system, real-time measures of changes in transport rate after a specific perturbation may be characterized. Third, in vitro systems allow a means to rapidly dissect the molecular mechanisms employed to regulate endothelial cell barrier properties. Through the use of specific cell-signaling inhibitors, neutralizing antibodies, and transfection experiments an investigator can readily move to an understanding of the molecular mechanisms employed in endothelial cells to develop, maintain, and regulate the blood-brain and blood-retinal barrier. In combination with in vivo studies, cell culture models continue to provide an important research tool in the arsenal of the investigator.