DIFFERENTIATION OF HUMAN SURFACE UPPER AIRWAY EPITHELIAL-CELLS IN PRIMARY CULTURE ON A FLOATING COLLAGEN GEL

The differentiation of human nasal surface epithelial cells in primary three-dimensional (3D) culture was studied. The dissociated cells were seeded on type I and IV collagen gel and grown in a serum-free medium supplemented with hormones and growth factors. During the first days of culture, epithelial cells were infrequently differentiated. Detachment and retraction of collagen by the cells generally occurred after 8-10 days of culture, allowing the formation of a floating collagen gel. This induced the differentiation of epithelial cells on 3D cord-like structures consisting of a collagen core surrounded by well-differentiated cells. Under scanning and transmission electron microscopy, we observed the formation of a pseudostratified respiratory-type epithelium consisting of columnar mature ciliated cells and secretory cells, epithelial cells in the process of ciliogenesis, and small pyramidal basal cells. The videomicroscopic analysis of the ciliated cells showed that the mean ciliary beating frequency (12.2 +/- 1 Hz) was close to the values obtained on polyp explants (11.7 +/- 0.8 Hz). Immunocytochemical localization of secretion with mucin-specific antibodies showed the expression of mucous cell function. In addition, the epithelial cells within the cord-like structures maintained a differentiated morphology and active beating of ciliated cells for more than 35 days in primary culture. Conversely, when the cells were grown on a collagen gel attached to plastic, they remained more flattened and the number of differentiated cells was lower. These results suggest that human upper airway epithelial cell differentiation in culture, as assessed by mucociliary function, is enhanced by the 3D organization of the cells around the floating collagen gel substrate.