Cell density affects spreading and clustering, but not attachment, of human keratinocytes in serum-free medium

Attachment, spreading and clustering of second-passage human human keratinocytes in serum-free medium have been evaluated within 24 h after plating, as a function of the density of the inoculum and of time, in two different strains. The results show that attachment is unaffected by cell density and differs significantly from strain to strain. Cell density affects the distribution of attached keratinocytes among three morphologically distinct classes: unspread, spread and clustered cells. The percentage of unspread keratinocytes shows a linear decrease at increasing cell density, and that of spread keratinocytes an increase, resulting from statistically significant increases in the percentages of both single and clustered cells. Spreading on uncoated surfaces appears therefore as an inducible phenomenon. The use of media conditioned by keratinocytes, fibroblasts and HeLa cells shows that keratinocytes specifically secrete a diffusible ‘spreading factor’. We term this phenomenon ‘autocrine induced spreading’. Preliminary physicochemical characterization suggests that a protein could be responsible for the spreading activity of conditioned media. The ‘spreading factor’ seems to act directly on the cells, and not through a modification of the plastic surface of the dishes, since most (greater than 70%) of the spreading activity can be recovered in the conditioned media used in pre-coating experiments. The percentages of clusters follow ‘saturation’ kinetics at increasing cell density, while the percentage of clustered cells increases linearly with the density of inoculum. Time-course experiments show that the rate of spreading is cell density- and strain-independent. The percentages of clusters and of total clustered cells are time-independent, suggesting that cluster formation takes place in suspension.(ABSTRACT TRUNCATED AT 250 WORDS)