Ultrastructural Observation of Rabbit Tracheal Epithelial Cells Cultured on Floating Collagen Gel

Abstract

Morphology of the rabbit tracheal epithelial cells cultured on attached collagen gel (AG) and floating collagen gel (FG) was studied in comparison. Dispersed epithelial cells with 0.5%protease (26.5 units/ml) were seeded on the both collagen gels (0.4%) at a density of 2×105cells/ml/dish. They were cultured in Waymouth MB 752/1 medium supplemented with insulin (5μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (10 ng/ml) and fetal bovine serum (0.3%). After 6 days in culture, the cells on FG became cuboidal in shape, while the cells on AG showed flattened profiles. The intracellular features of the cells, such as Golgi apparatus, mitochondria, rough endoplasmic reticulum, lysosomes, etc., cultured with both methods were similar. By the 13th day, the cells cultured on AG remained flattened and Golgi apparatus was poorly developed. In contrast, the cells on FG became columnar and showed morphological features reminiscent of tracheal epithelium in vivo. These columnar cells had numerous microvilli and junctional complexes (tight junction, intermediate junction and desmosome) at their medium-facing surface and contained well developed Golgi apparatus. These findings are characteristic of a secretory cell. In the histocytochemical observation for thiamine pyrophosphatase (TPPase) which is one of the marker enzymes for Golgi apparatus, a reaction product was demonstrated in one or two trans Golgi cisternae of the cells cultured on FG during cultivating period. On the contrary, the cells cultured on AG have lost TPPase activity by the 13 day in culture.