As described previously with modification [7 (link)], mucosal tissue was digested overnight with 0.04% trypsin solution (Life Technologies, Carlsbad, CA, USA) with 19.25 μg/mL of gentamicin (Life Technologies) and 0.765 μg/mL of fungizone (Life Technologies) at room temperature, and transferred into 0.0125% trypsin-inhibitor (Life Technologies). Dissociated oral keratinocytes were resuspended in a chemically defined culture system, complete Epilife (Life Technologies) and seeded into one T-25 flask. For serial cultures, cells were detached in 0.025% trypsin/EDTA (Life Technologies). For analysis, monolayer culture cells were fed adding 30mL of Epilife/150mm Petri culture dish every other day and collected after detachment in Enzyme Free Cell Dissociation Solution (Millipore, Billerica, MA, USA). ePUKs culture was previously described [2 (link)]. The cells were fed adding 60mL of medium/150 mm Petri culture dish, every 24 hour. At confluence, the monolayers continued to proliferate; pushing keratinocytes into the overlying medium and the cells in suspension were collected as ePUKs. The monolayer cells underlying ePUKs culture were collected after detachment in Enzyme Free Cell Dissociation Solution (Millipore).