Morphological changes in rat tracheal cells during the adaptive and early growth phase in primary cell culture

The purpose of our studies was to determine the fate of different cell types present in early primary cultures of tracheal epithelial cells and, if possible, to elucidate the role they play in the establishment of the cultures. Epithelial cells were isolated from rat tracheas with 0.5% Pronase and were cultured on collagen-coated dishes as described previously. Light and transmission electron microscopic studies showed that the cell population harvested from rat trachea was composed of approximately 30% ciliated cells, 50% granule-containing cells and 20% undifferentiated cells (presumably basal cells). Upon seeding the tracheal cell suspensions into culture, approximately 40% of the cells attached. Cell attachment was virtually complete after 16 h. Roughly 60% of the cells attaching during the first 12 h were neither ciliated nor granulated, suggesting that undifferentiated cells played a major role in establishment of the early cultures. Between 20 and 35% of the cells attaching during this time were identified as granulated cells (mucous cells). Ciliated cells did not start to attach in significant numbers until 8 h after seeding. They never amounted to more than 8-12% of the attached cell population. After 12 h of culture, the cell population underwent a progressive loss of differentiation. The number of poorly differentiated cells (i.e. those showing neither cilia nor mucous granules) increased correspondingly. This loss of differentiation preceded the onset of DNA synthesis and cell growth which began at about 24 and 40 h, respectively. Continuous [3H]thymidine-labelling studies showed that at 48 h after the start of culture about 90% of all attached cells had entered DNA synthesis at least once. This finding is consistent with the interpretation that the ciliated cells are terminally differentiated cells and are probably the only part of the tracheal cell inoculum not participating in the growth of the cultures. At 72 h, the cultures (now in mid-log growth phase) were composed of uniformly undifferentiated cells lacking cilia and mucous granules. The cells nevertheless showed unequivocal epithelial characteristics such as tight junctions and desmosomes. The studies suggest that both basal and mucous cells are responsible for the establishment and growth of the rat tracheal epithelial cell cultures.