Factors regulating the emergence of spontaneous and X-ray-induced variants in primary rat tracheal epithelial cell cultures

Abstract

A series of experiments have been carried out to identify those factors that affect the number of altered populations detected in control, nonexposed, and radiation-exposed primary cultures of rat tracheal epithelial cells. The number of colony forming cells per milliliter of culture medium and the frequency with which the culture medium is changed seemed to be the most critical factors regulating the emergence of induced and spontaneous variants. Increasing the number of cells plated so that of colony forming cells increase from 25 to 200 per ml, regardless of the dish size used, was associated with a 200-fold decline in the frequency of spontaneous variants and a 40-fold decline in X-ray-induced variants. Increasing the interval between medium changes from 3 to 7 days after the first week of culture was associated with a 10-fold decrease in the frequency of spontaneous variants. The frequency of spontaneous and induced variants is markedly less dependent on culture density at densities between 150 and 600 colony forming cells per ml. The type of medium used to establish primary cultures had little effect on the frequency of variants detected. Similarly, when assays were performed at densities in excess of 150 colony forming cells per ml the frequency of spontaneous and x-ray-induced variants was not affected by the absence of epidermal growth factor, increased levels of calcium (final concentration, 0.8 mM), or by removal of pyruvate from the selection medium.