Endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors does not prevent apoptosis of suspended EC. We have developed a three dimensional model of organoid endothelial spheroids for the study of differentiated endothelial functions, such as survival factor activities, organotypic differentiation, and sprouting angiogenesis. The spheroids differentiate over time to establish a surface monolayer of EC and a centre of unorganised EC that subsequently undergo apoptosis. Surface EC establish firm cell-cell contacts and can be induced to express differentiation antigens that are downregulated in monolayer EC. In contrast, the unorganised cells in the centre of the spheroids will rapidly undergo apoptosis if they are not rescued by exogenous survival factors, such as VEGF, Ang-1, and FGF-2. In addition to using EC spheroids as a cellular differentiation model, we developed a novel collagen gel based three dimensional in vitro angiogenesis assay based on the use of EC spheroids as a cellular delivery device. Gel embedded EC spheroids act as focal starting points for the sprouting of luminized capillary-like structures that can be induced to form complex anastomozing networks. Formation of capillary anastomoses is associated with directional sprouting of outgrowing capillaries towards each other. To analyse, if directional sprouting is dependent on cytokine gradients or tensegrity forces exerted through the extracellular matrix, we designed a “tension trimmer” that enables the application defined tensional forces on the extracellular matrix. Based on this assay, we present causal evidence that tensional forces on a fibrillar extracellular matrix, such as type I collagen but not fibrin, is sufficient to guide directional outgrowth of endothelial cells. Lastly, we have further developed the spheroidal cell culture system to establish organised coculture spheroids of endothelial cells and smooth muscles in order to study phenomena of vessel maturation as they are associated with the recruitment of mural cells. Coculture spheroids of EC and SMC differentiate spontaneously in a calcium-dependent manner to organise into a core of SMC and a surface layer of EC, thus, mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a downregulation of PDGF expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression upon VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a three dimensional in gel angiogenesis assay). Taken together, the data establish the three dimensional EC spheroid model as a powerful tool for the study of endothelial cell differentiation and angiogenesis.

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