Use of microcarriers to isolate and culture pulmonary microvascular endothelium

Microcarriers of known diameter can be used to collect endothelial cells from microvessels of the same or slightly smaller internal diameter. The procedure is illustrated by collection of endothelial cells from rabbit pulmonary pre-capillary vessels. The lungs are perfused free of blood with physiological saline and then cold vessels. The lungs are perfused free of blood with physiological saline and then cold (4 degrees C) saline (containing EDTA, 0.02%, and microcarriers 600/ml; 40-60 micrometers diameter) is perfused via the pulmonary artery. The direction of flow is reversed periodically to collect the bead-cell harvest from the arterial side. Cold shock and EDTA cause the endothelial cells to detach from the vessels under conditions such that the cells remain attached to the microcarriers. The selective attachment to microcarriers is apparently aided by the tight fit of the beads within vessels of the same diameter. Beads do not emerge on the venous side, all being trapped at the pre-capillary level. Electron microscopic examination of lungs fixed during the perfusion shows that the beads lodge in terminal arterioles and pre-capillary vessels (approximately 40-60 micrometers in diameter, with one, sometimes incomplete, muscle layer). Endothelial cells recovered on microcarriers can be allowed to migrate on to flasks and back on to beads. The resultant cultures have an endothelial morphology and possess high levels of angiotensin coverting enzyme and carboxypeptidase N activity.