The key stimulus for differentiation is set by reducing the medium to a level below the surface of the model. So, the keratinocytes are supplied with nutrients through the membrane meanwhile the air contact effects stratification. The progress in differentiation can be monitored by immunodetection of the specific proteins expressed.

The major component of the basal lamina is Collagen IV. The distinctiveness of its expression is an important criterion for functionality and physiological comparability.

Mitotic cells of the basal layer express Cytokeratin 14. Due to ongoing differentiation its expression is ceased in cells of the suprabasal layers.

The crosslinking activity of Transglutaminase effects growing insolubility and as a consequence leads to formation of a functional barrier.

Transglutaminase links the most abundant protein of the upper layers - Involucrin - to membrane proteins of the cells. This sealing is the final step of keratinocyte differentiation.