78 Cell Biol Toxicol (2011) 27:69–81 Fig. 6 Stratification of HaCaT cells. HaCaT cells were treated at low (0.5 mM) and high (5 mM) of NaF for 3 weeks on a nitrocellulose membrane to achieve stratification. C, non- treated cells. In overviews, control (a) and low dose (b) membrane showed partial stratification with cubic basal cells and polygonal suprabasal cells. At high dose (c), HaCaT cells that cell contacts are compromised by misexpression of other sensible components. The altered keratins were expressed in cell clusters, whereas the surround- ing cells remained unlabeled. Expression of uniformly distributed keratins K13 and K19 remained un- changed. We hypothesize that this cell grouping represents early stages in the stratification process. Fluoride would interrupt the process, before K1/10 and K15 start to accumulate. Concerning K13, its expression in parceled small groups of cells was not influenced by fluoride, which would locate the fluoride- sensitive state after the initial clustering itself. Fluoride concentrations in our experiments have been chosen according to previous work that resulted in measurable effects on culture cells (Dogan et al. 2002). They are well below those used for topic application in the clinic (Walsh et al. 2010); however, the time of treatment was longer than applied by the dentist. We used serum-free conditions for fluoride treat- ment with the rationale to minimize cell proliferation activity during the assay period. In this way, any effects of superconfluence on cell vitality, with possible interference with gene expression and protein or RNA intactness, were avoided. During the experi- ments described in Figs. 1, 2, and 3, cells remained appear as monolayer. Detailed views (d–f) showed normal cells with intact nucleus (N), nuclear membrane (NM), and desmo- somes (D). The cytoplasm of treated cells is normal with normal organelles (Golgi, ER, etc.; not shown), although there are granules (f, black arrows) uniformly in a subconfluent state. Clear distinction of individual cells was essential for Fig. 3 in order to reveal the heterogeneity of the cultures in a sense that particular assemblies expressed stratification-specific keratins. The stratification assay (Fig. 6) required however superconfluent cultures that proliferated during the assay period. In this experiment, it is possible that cell density was influenced by the presence of fluoride at high dose (Dogan et al. 2002). According to ultrastructural criteria, apoptosis or necrosis of cells did not occur (compare Wurtz et al. (2008)). Concerning cell differentiation, we found a clear difference between basal cell morphology and that of the stratification layer in the control and low dose samples, whereas in the high dose sample, cell morphology was uniform and resembled that of undifferentiated, growing cells (Fig. 6). HaCaT cells were earlier employed in tissue engineering systems (Boelsma et al. 1999) for the development of artificial skin equivalents, because of their stratification capacity in monoculture (Raynov et al. 2008). We confirmed their capability to stratify; however, fluo- ride at high dose abolished this property. Apparently, the correct culture conditions are essential for the maintenance of the cellular phenotype. Low doses did