A simplified 3D oral epithelial model using an air-liquid interface culture system

Three-dimensional (3D) epithelial models are widely used to investigate epithelial biology, host-pathogen interactions, and topical therapeutics. However, many existing models rely on primary keratinocytes or specific cell lines, limiting accessibility, reproducibility, and scalability. Besides, reliable oral epithelial models based on stable keratinocyte cell lines remain limited, as most current systems depend on primary cells or organotypic cultures. In this study, we established simplified and reproducible 3D epithelial models using an optimized air-liquid interface (ALI) culture system with two human keratinocyte cell lines, HaCaT (epidermal) and HOK (oral). After culture condition optimization, both cell types maintained stable stratification and differentiation across multiple passages. Histological analysis showed well-organized epithelial architectures with distinct basal and suprabasal layers. Immunofluorescence staining confirmed spatially distinct expression of epithelial differentiation markers closely resembling native epithelia. Functionally, the reconstructed epithelia robust barrier integrity and responded to Candida albicans infection. Moreover, the models allowed evaluation of drug permeability across epithelial layers. Collectively, this simplified 3D culture platform provides a reproducible, accessible strategy for constructing epithelial models using standardized keratinocyte cell lines without primary cells. The system offers a practical tool for oral and cutaneous epithelial research, with potential in infection studies, disease modeling, and preclinical drug screening.