Dear all,

I have been trying for a while to generate 3D oral tissues from primary human keratinocytes cultures at the air-liquid interface with various calcium concentration (0.5 - 1.5 mM) in the basal medium feeding the cells. While I observe differentiation of the cells into a keratin layer, the tissues are very thin, composed of almost a single layer and covered with several layers of keratinocytes.

For some reasons I can't end up with tissues composed of several layers of differentiating cells (stratum spinosum). As anyone a suggestion? We use primary human gingival keratinocytes, and Transwell inserts, with a standard keratinocyte medium.