High-pressure pulsation of central and microvessel pulmonary artery endothelial cells

We developed an in vitro method of pulsating central and microvessel pulmonary artery endothelial cells that would allow us to study the effects of increased distending pressures over a prolonged period of time. Preservation of the contact-inhibited monolayer was assessed on phase contrast microscopy and, in addition, scanning and transmission electron microscopy (SEM, TEM) were used to determine whether there were alterations in the surface characteristics or intracytoplasmic organelles that suggested cellular damage. The cells used were obtained from Rambouillet lambs, age 3-5 days, anesthetized with halothane and ventilated. The endothelium was harvested from the central pulmonary artery (CPA) by scraping the luminal surface and from the microvessels (MPA) by infusing microcarrier beads 40-140 microns external diameter. After the second passage in culture, the cells were seeded onto the translucent, flexible polyvinylchloride membrane of a transducer dome and grown to confluence. The cell dome was then connected to a blank dome with an attached quartz transducer, to a reservoir, and to stainless steel bellows tubing, all filled with culture medium and affixed to a pulsation generator. By varying the height of the reservoir, the amplitude of excursion of the bellows tubing, and the rate, the cells could be pulsated at a given distending pressure and frequency. Confluent CPA endothelial cells from three lambs and MPA cells from two others were studied after pulsation at both 100/60 and 20/10 mmHg, 60 times/min for 48 h and after nonpulsation. On phase contrast light microscopy and on SEM, the cells remained confluent.(ABSTRACT TRUNCATED AT 250 WORDS)