Development and characterisation of in vitro human oral mucosal equivalents derived from immortalised oral keratinocytes.

Tissue engineered oral mucosal equivalents (OME) are being increasingly used to measure toxicity, drug delivery, and to model oral diseases. Current OME are mainly comprised of normal oral keratinocytes (NOK) cultured on top of a normal oral fibroblasts (NOF)-containing matrix. However, the commercial supply of NOK is limited, restricting widespread use of these mucosal models. In addition, NOK suffer from poor longevity and donor-to-donor variability. Therefore, we constructed, characterised and tested the functionality of oral mucosal equivalents based on commercial TERT2-immortalised oral keratinocytes (FNB6) in order to produce a more readily available alternative to NOK-based OME. FNB6 OME cultured at an air-to-liquid interface for 14 days exhibited expression of differentiation markers cytokeratin 13 in the suprabasal layers and cytokeratin 14 in basal layer of the epithelium. Proliferating cells were restricted to the basal epithelium and there was immuno-positive expression of E-cadherin confirming the presence of established cell-to-cell contacts. The histology and expression of these structural markers paralleled those observed in the normal oral mucosa and NOK-based models. Upon stimulation with TNFα & IL-1, FNB6 OME displayed a similar global gene expression profile to NOK-based OME with increased expression of many common pro-inflammatory molecules such as chemokines (CXCL8), cytokines (IL-6) and adhesion molecules (ICAM-1) when analysed by gene array and qPCR. Similarly, pathway analysis showed that both FNB6 and NOK models initiated similar intracellular signalling upon stimulation. Gene expression in FNB6 OME was more consistent than NOK-based OME that suffered from donor variation in response to stimuli. Mucosal equivalents based on immortalised FNB6 cells are accessible, reproducible and will provide an alternative animal experimental system for studying mucosal drug delivery systems, host-pathogen interactions and drug-induced toxicity.