Development of a Tracheal Implant Xenograft Model to Expose Human Bronchial Epithelial Cells to Toxic Gases

Abstract

A tracheal implant model was developed which enabled exposure of differentiated normal human bronchial epithelial cells (NHBE) to a single exposure of a model toxic gas (formaldehyde). NHBE cells were grown in vitro in explant culture under defined serum-free conditions from previously frozen bronchial segments, harvested, and used to repopulate de-epithelialized rabbit tracheas. Rabbit tracheal segments repopulated with NHBE cells were implanted into the subcutis of congenitally athymic nude mice. Following graft vascularization, the xenografted NHBE cells differentiated and formed a mucociliary epithelial surface which lined approximately 50% of the surface of the implant lumen. Eight weeks post-implantation both ends of the implanted grafts were cannulated, and formaldehyde (HCHO) vapor (0, 6, or 15 ppm) in humidified air was passed through the tracheal lumens. Representative epithelial surfaces were examined by light and scanning electronmicroscopy, and autoradiography prior to, immediately after, and 48 hr following a 1-hr exposure to the test vapors. Light microscopic examination of implant sections immediately following exposure to 6 and 15 ppm HCHO detected cessation of ciliary activity, which recovered by 48 hr postexposure. Scanning electron microscopic examination of the epithelial surface demonstrated mild morphologic changes, restricted to those implants exposed to 15 ppm. Findings immediately following HCHO exposure included swelling and exfoliation of individual cells, deciliation, and mucus release. Changes present after 48 hr included presence of flattened cells with few short microvilli and focal increase in the number of S-phase nuclei in the basal epithelium. These results demonstrate the utility of tracheal implants for single short-term exposure of differentiated human bronchial epithelial cells to gaseous agents. This system should prove useful for mechanistic studies of human upper airway toxicity.