Differentiation of endothelial cells in vivo
Endothelial cells (EC) cover the inside of all blood vessels as a monolayer. In the adult EC contact smooth muscle cells (SMC) or pericytes (PC) through breaks in the undelying basal membrane (BM). Under normal conditions these EC do not proliferate and are referred to as "quiescent". 



Stimulatory events such as ovarian corpus luteum angiogenesis (physiological condition) or wound healing (pathological condition) activate EC to degrade their underlying basal membrane, to migrate into the surrounding matrix, to proliferate and to establish new anastomosing networks which were again covered by SMC or PC. Activated EC are referred to as "angiogenic".



Differentiation of endothelial cells in vitro
In vitro primary EC were usually cultured as two dimensional flat monolayers which reflects very much some properties of the endothelial in vivo phenotype. Never- theless EC cultured in that way are not completely quiescent. Even in a confluent monolayer up to 10% of the EC proliferate and most of the EC tend to loose their differentiation over time (i.e. less tight junctions, downregulation of CD34, upregulation of Ang-2/PDGF/VEGF-R2). 


In order to keep the EC quiet and to preserve their differentiation in vitro we developed a novel method to culture EC as three dimensional spheroids. Single suspended EC are seeded under non adhesive conditions in round bottom 96-well plates. After 18-24h all EC seeded in one well contribute to the formation of a single spheroid. The spheroids organize over time (1d) to establish a quiescent, non proliferating surface monolayer of endothelial cells enclosing a core of also quiescent, unorganized cells which die by apoptosis and disappear after prolonged incubation (7d). In our further studies we used these spheroids to analyze endothelial differentiation, angiogenesis and apoptosis.