Figure 3.

Manufacturing protocol of the 3D organotypic in vitro oral cancer model. The model was cultured in a submerged condition for the first six days (1)–(3), raised at the air–liquid interface on day 6, and cultured for another seven days (4). Depending on the manufacturing procedure, the model was fed with three different culture media: DMEM containing 10% FBS, serum-free EpiLife (0.06 mM Ca²⁺) containing Human Keratinocyte Growth Supplement (complete medium), and a 1:1 mixture of DMEM and complete medium containing 10% FBS. In the manufacturing protocol, three culture media of DMEM containing 10% FBS, complete medium, and a 1:1 mixture of DMEM and complete medium containing 10% FBS are shown in dark pink, light pink, and beige, respectively. Macroscopic appearances of the model are displayed (a–f). Oblique view at day 0 before seeding PD-CAFs and PD-NOFs (a). Top view at day 0 after seeding PD-CAFs and PD-NOFs (b). Oblique view on day 2 before seeding NOKs (c). Oblique view on day 4 before seeding HSC-3/HSC-4 cells (d). Top and side views on day 6 at the air–liquid interface, respectively (e,f). White dots indicate the partition’s original position (e).