Development of a highly reproducible three-dimensional organotypic model of the oral mucosa.

In this report we describe the development of a standardized three-dimensional (3D) system of the human oral mucosa based on an immortalized human oral keratinocyte cell line (OKF6/TERT-2). The procedure takes approximately 2-3 weeks to complete and includes three main stages: preparation of collagen-embedded fibroblasts, addition of the mucosal component and airlifting of cultures to ensure adequate differentiation/stratification. This procedure results in a multilayer epithelial structure in which layers are organized similarly to the cells in native oral mucosa. Specifically, this model system consists of a stratum basale, having one layer of columnar to round cells, a relatively flattened stratum spinosum and stratum granulosum, and a non-keratinizing stratum corneum. This 3D system resembles the commercially available system based on the cell line TR146 (SkinEthic), with the exception that our model system does not contain dyskeratotic changes and has a submucosal component, and thus better represents the normal human mucosa and submucosa.