Cell cycle kinetics of cultured human epidermal keratinocytes

When stratified epithelia maintained in culture are used for autoradiographic studies of labeling index, the emulsion is usually placed over the uppermost strata of the culture. In many cases the distance from the basal cell nucleus to the emulsion exceeds the average pathlength of beta-particle emissions from 14C or 3H. We describe a technique for inverting the cultures so that the emulsion can be brought into close association with the basal cells. Attempts to label cultured human epidermal keratinocytes using a pulse of [3H]- or [14C]-thymidine produced labeling only at the periphery of the colonies. This was noted when emulsion was laid on top of the colonies but also when the emulsion was in close contact with the "basal cells" adhering to the plastic culture vessel. Continuous labeling of the cultures produced nearly 100% labeling of all the basal layer, i.e., central and peripheral, indicating that the central cells were also in rapid cell cycle. The results are interpreted as indicating the presence of an efficient barrier to free diffusion over the center of the colonies, presumably due to the presence of several layers of corneocytes. Percent labeled mitoses (PLM) studies produced an unusual PLM curve with a well-defined third peak which showed a higher PLM than the second peak. These results may indicate that the cultures contain discrete cell populations with different cell kinetic phase durations.