Co-cultivation of rat pneumocytes and bovine endothelial cells on a liquid-air interface.

Abstract

The blood-air barrier is a most important functional element of the lung but little information is available about the cells constituting this barrier in vivo. The aim of the present study was to create an in vitro model of the blood-air barrier that would allow investigation of cellular interactions and alveolar metabolism, and would be suitable for in vitro drug screening. Rat pneumocytes and bovine microvascular endothelial cells were grown on opposite sides of microporous polycarbonate filters, as immersion, perfusion and liquid-air interface (LAI) cultures. The effects of culture conditions on cell morphology were examined by light and transmission electron microscopy. For immersion and perfusion co-cultures, both compartments were supplied with culture medium. In contrast, for liquid-air interface studies, only the endothelial cell compartment was continuously supplied with serum-free medium, whilst the type II pneumocytes were ventilated with air. The pneumocytes lost their morphological characteristics when using immersion or perfusion co-cultures. Under liquid-air interface conditions, they retained most of their characteristic morphological features when compared to the intact blood-air barrier. A subset of primary type II pneumocytes retained its differentiated phenotype, with cuboidal morphology, lamellar bodies and apical microvilli. These type II pneumocytes appeared to be connected by tight junctions to cells expressing morphological characteristics of type I pneumocytes. As shown herein, the liquid-air interface co-culture possesses many morphological characteristics of the intact blood-air barrier. In summary, this article describes the design of an artificial blood-air barrier, in which rat pneumocytes were cultivated with bovine microvascular endothelial lung cells on opposing sides of a microporous polycarbonate filter. We conclude that it might be a promising in vitro model for studies of molecular transport via the blood-air barrier, the investigation of repair mechanisms after alveolar injury, or as an in vitro screening system.