High-Yield Method for Isolation and Culture of Endothelial Cells from Rat Coronary Blood Vessels Suitable for Analysis of Intracellular Calcium and Nitric Oxide Biosynthetic Pathways

We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed

Effects of Shear Forces and Pressure on Blood Vessel Function and Metabolism in a Perfusion Bioreactor.

Bovine saphenous veins (BSV) were incubated in a perfusion bioreactor to study vessel wall metabolism and wall structure under tissue engineering conditions. Group 1 vessels were perfused for 4 or 8 days. The viscosity of the medium was increased to that of blood in group 2. Group 3 vessels were additionally strained with luminal pressure. Groups 1-d through 3-d were similar except that BSV were endothelium-denuded before perfusion. Groups 1-a through 3-a used native vessels at elevated flow rates. Group 3 vessels responded significantly better to noradrenaline on day 4, whereas denuded vessels showed attenuated responses (p < 0.001). Tetrazolium dye reduction did not depend on perfusion conditions or time except for denuded vessels. pO(2) gradients across the vessels were independent of time and significantly higher in group 2 (p < 0.001). BSV converted glucose stoichiometrically to lactate except vessels of groups 3, 1-d, and 3-d which released more lactate than glucose could supply (p < 0.001). Group 1 vessels as well as all vessels perfused with elevated flow rates showed a loss of endothelial cells after 4 days, whereas group 2 and 3 vessels retained most of the endothelium. These data suggest that vessel metabolism was not limited by oxygen supply. Shear forces did not affect glucose metabolism but increased oxygen consumption and endothelial cell survival. Luminal pressure caused the utilization of energy sources other than glucose, as long as the endothelium was intact. Therefore, vessel metabolism needs to be monitored during tissue engineering procedures which challenge the constructs with mechanical stimuli