AbstractOrganotypic epithelial structures can be cultured using primary or immortalized keratinocytes. However, there has been little detailed quantitative histological characterization of such cultures in comparison with normal mucosal architecture. The aim of this study is to identify morphological markers of tissue architecture that can be used to monitor tissue structure, maturation, and differentiation and to enable quantitative comparison of organotypic cultures (OCs) with normal oral mucosa. OCs of oral keratinocytes [immortalized H400 or primary rat keratinocytes (PRKs)] were generated using the three scaffolds of de‐epidermalized dermis (DED), polyethylene terephthalate (PET), and collagen gels for up to 14 days. Cultures and normal epithelium were analyzed immunohistochemically and by using the semi‐quantitative reverse transcriptase polymerase chain reaction (sq‐RT‐PCR) for E‐cadherin, desmoglein‐3, plakophilin, involucrin, cytokeratins‐1, ‐5, ‐6, ‐10, ‐13, and Ki67. The epithelial thickness of OCs was measured in stained sections using image processing. Histological analysis revealed that air–liquid interface (ALI) cultures generated stratified organotypic epithelial structures by 14‐days. The final thickness of these cultures as well as the degree of maturation/stratification (including stratum corneum formation) varied significantly depending on the scaffold used. For certain scaffolds, the immunohistochemical profiles obtained recapitulated those of normal oral epithelium indicating comparable in vitro differentiation and proliferation. In conclusion, quantitative microscopy approaches enabled unbiased architectural characterization of OCs. The scaffold materials used in the present study (DED, collagen type‐I and PET) differentially influenced cell behavior in OCs of oral epithelia. H400 and PRK OCs on DED at the ALI demonstrated similar characteristics in terms of gene expression and protein distribution to the normal tissue architecture. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:3227–3238, 2012.