Organ culture of arteries for experimental studies of vascular endothelium in situ

The objective of this study was to determine whether organ culture of arteries could be used as a more physiological model than endothelial cell culture for the study of vascular endothelium in vitro. Small pieces of artery from rat, pig, piglet and man were cultured in 24-well plates for up to seven or eight days to study the characteristics of the vascular endothelial cell layer during the first week of culture, in particular its integrity, viability and propensity for cell division. Using conventional and confocal microscopy, silver-stained endothelial cell boundaries were shown to be intact at all time points, up to and including day 7. However, occasional very small gaps between endothelial cells were seen with the scanning electron microscope under high power at day 7. Using the bromodeoxyuridine technique, no endothelial cell division was seen at day 4 in any species, except for the occasional endothelial cell in rat aorta. At day 7, pig, piglet and human arteries showed only very occasional dividing endothelial cells, but many endothelial cells had divided by day 7 in rat aorta. Viability of the endothelium was assessed using fluorochromes and examination of the endothelial layer en face using confocal microscopy. Viability was always excellent (> 95%) up to day 4. By day 7, occasional patches of dead cells could be seen, which were most obvious in rat aorta. This study demonstrates that endothelial cells can be studied in situ in organ culture with intact morphology, lack of cell division and excellent viability for a minimum of four days. For many research questions involving vascular endothelium-for example the pathophysiology of hyperacute rejection-short-term organ culture of vessels is likely to represent a more physiological model than endothelial cell culture.