Liver sinusoidal endothelial cells (SECs) are generally refractory to extended in vitro culture. In an attempt to recreate some features of the complex set of cues arising from the liver parenchyma, we cocultured adult rat liver SECs, identified by the expression of the marker SE-1, with primary adult rat hepatocytes in a 3D culture system that provides controlled microscale perfusion through the tissue mass. The culture was established in a medium containing serum and VEGF, and these factors were then removed to assess whether cells with the SE-1 phenotype could be supported by the local microenvironment in vitro. Rats expressing enhanced green fluorescent protein (EGFP) in all liver cells were used for isolation of the SE-1-positive cells added to cocultures. By the 13th day of culture, EGFP-expressing cells had largely disappeared from 2D control cultures but exhibited moderate proliferation in 3D perfused cultures. SE-1-positive cells were present in 3D cocultures after 13 days, and these cultures also contained Kupffer cells, stellate cells, and CD31-expressing endothelial cells. Global transcriptional profiling did not reveal profound changes between 2D and 3D cultures in expression of most canonical angiogenic factors but suggested changes in several pathways related to endothelial cell function.--Hwa, A. J., Fry, R. C., Sivaraman, A., So, P. T., Samson, L. D., Stolz, D. B., Griffith, L. G. Rat liver sinusoidal endothelial cells survive without exogenous VEGF in 3D perfused co-cultures with hepatocytes.