Preparation of large numbers of uniform tracheal organ cultures for long term studies

Summary

Rat tracheas were each sectioned into fourteen rings of equal size with a slicing device which holds evenly spaced razor blades in register. The razor blades were positioned to minimize shearing of tissues during sectioning so that there was no gross tissue disruption or cell death distant from cut edges. Hundreds of these fragments can be conveniently prepared for studies requiring replicate samples. The cultures can be established in McCoy’s 5a (modified) medium with or without calf serum. Cultures grown in the presence of calf serum were compared with those grown in serum-free medium, using vital phase microscopy, transmission and scanning electron microscopy, and light microscopic autoradiography of thymidine incorporation. When there is calf serum in the medium, epithelization of the entire surface of the ring occurs rapidly with the cells flattening and migrating as a sheet of closely apposed cells. Until migration is complete, mitoses are limited to the original mucosa near the cut edge. Without calf serum, migration is slow. The cells do not flatten or become closely apposed. Mitoses appear later but are present on all areas of the surface before migration is complete. In both serum-containing and serum-free media, ciliated cells are included in the migrating population and differentiation into pseudostratified epithelium occurs on newly epithelialized surfaces. The differing pattern of mitotic activity makes culture in serum-containing media more suitable for studies of wound healing and culture in serum-free medium more useful for some cytotoxicity and carcinogenicity studies.