A preliminary model of an oral dysplastic lesion on a chip

In vitro models of oral dysplasia fail to recapitulate physiologically relevant tissue-tissue interfaces and other microenvironmental cues. This study aimed to present a preliminary organ-on-a-chip (OoC) model of a precancerous oral cavity lesion (OD-OoC). The objective was to reproduce in a two-channel microfluidic device an in vitro tridimensional (3D) model characterized by an organized interaction between endothelial cells, fibroblasts, and dysplastic oral keratinocytes on a collagen I-coated membrane. On day 1, human umbilical vein endothelial cells (ECs) were introduced in the bottom channel, and the chip was inverted to allow cell adhesion to the lower side of the membrane. The chip was then inverted back to the original position, and human gingival fibroblasts (hGFs) were introduced into the top channel. On day 2, the laminar flow was activated, while uniform layers of hGFs and ECs were forming in the respective channels. On day 3, dysplastic oral keratinocytes (DOKs) were inoculated in the top channel above the hGFs layer. On day 5, the chip was fixed with 4 % paraformaldehyde and stained with antibodies targeting podoplanin, Trop2, and VE-cadherin for staining of hGFS, ECs, and DOKs, respectively. Confocal microscopy confirmed the presence of all cell types, showing fibroblast migration from the top channel to the bottom channel of the chip, where they localized between the membrane and the ECs. DOKs confined to the top channel showed slight and uneven E-cadherin and EpCAM (Epithelial Cell Adhesion Molecule) positivity, but evident positivity for Trop-2, confirming that their phenotype differed from that of healthy epithelial cells. The presented OD-OoC could enable in vitro monitoring of epithelial cell phenotype changes and cell migration across the membrane, suggesting its potential applicability in future oral cancer research.