Endothelial Cell Isolation and Manipulation

Abstract

In vitro characterization of primary vascular cells requires their isolation and purification from tissue. Considering the diversity in composition and function between the distinct types of aortic, arterial, capillary, venous, and lymphatic vessels, it is essential to retrieve vascular cells from the vessel type of investigation and conduct experiments at early passages to largely retain their differentiation. Hence, a variety of protocols for endothelial cell isolation from distinct species, tissues, and vessel types have been developed which will be listed in the following chapter, including two detailed standard protocols for the isolation of microvascular and lymphatic endothelial cells from human skin and for the isolation of human umbilical vein endothelial cells. Furthermore, mechanistic studies frequently require the overexpression or silencing of a single gene which is achieved by cell transfection or transduction methods. Adaptation of protocols for primary endothelial cells has been required as they are not readily amenable to gene transfer. In the following, several chemical, physical transfection, and viral transduction techniques will be introduced which were successfully applied to vascular cells in vitro and in vivo. The latter paved the route to vascular gene therapy, which is an area of particular interest that will be discussed in more detail. Again, a standard protocol for in vitro endothelial manipulation by electroporation is provided.