A System for Culture of Endothelial Cells in 20–50‐μm Branching Tubes

Abstract

To construct an in vitro endothelial cell culture system which would mimic the geometry and hemodynamic conditions of the arteriolar microcirculation. Using a photolithography technique, semicircular channels (20-50 microns in diameter) were etched in mirror-image patterns on pairs of borosilicate microscope slide glass. One-half of each plate pair was predrilled with perfusion port holes at funnel-shaped fluid entrance regions. The perfusion system was constructed of micropipette glass and Teflon tubing, and imbedded in Sylgard. Two types of endothelial cells were grown to confluence within the half-channels: rabbit lung microvascular endothelial cells (a gift of Dr. M.E. Gerritsen, Miles Inc.) and human umbilical vein endothelial cells. After the cells were confluent, the two mirror images were aligned and clamped together to form a complete branching system of tubes lined with endothelial cells. This cell culture system can be perfused at physiological flow rates corresponding to wall shear stress values in the range 0.03-48 dyn/cm2. The fluid velocity profiles can be measured in this system by tracking the velocity and flow paths of 0.5-micron fluorescently labeled microspheres. Endothelial cells which grow within the channel exhibit F-actin alignment along the long axis of the channel by 3 days after seeding. Scanning electron micrographs indicate that 4 hr after seeding, endothelial cells commonly form cellular projections extending across the half-channel; by 5 days after seeding, the projections appear to have flattened out along the bottom of the channel. An in vitro endothelial cell culture system was constructed which mimics the geometry and hemodynamics conditions of resistance arterioles. This system can be used to examine endothelial cell responses to flow and flow gradients under defined and controllable conditions which mimic the arteriolar microcirculation.