Mechanical–enzymatic isolation and characterization of primary human gingival epithelial cells for reproducible in vitro oral mucosa models

The establishment of reliable in vitro oral mucosa models is essential for advancing studies in epithelial barrier function, wound healing, and host-microbe interactions. However, the widespread use of immortalized cell lines such as HaCaT or TR146 limits physiological relevance owing to altered differentiation profiles and genetic drift. In this study, we developed a robust mechanical-enzymatic protocol for isolating and expanding primary human gingival keratinocytes from healthy gingival explants without feeder layers. The resulting cells demonstrated high viability, maintained consistent proliferative capacity across passages, and exhibited characteristic cobblestone morphology. Comprehensive phenotypic validation included immunofluorescence and immunohistochemistry confirming strong expression of epithelial markers CK18, AE1/AE3, and MUC1, with absence of the mesenchymal marker vimentin. Transcriptomic analysis using RT-qPCR corroborated epithelial lineage fidelity, revealing stable MUC1 expression and lack of MUC5AC transcripts, indicative of a nonglandular phenotype. Metabolic competence was supported by WST-1 assays that correlated strongly with manual cell counts, underscoring functional viability. Importantly, AGS and 293T/17 cell lines were processed in parallel as orthogonal controls to confirm assay specificity and lineage discrimination. Under rigorously standardized, within-laboratory conditions, our workflow yielded high interdonor concordance in epithelial identity and growth kinetics across a young-adult cohort (n = 3), supporting its use as a practical primary-cell platform for downstream applications. Generalizable reproducibility-across age strata, operators, and sites-will require formal, preregistered multicenter validation. By mitigating limitations inherent to immortalized lines, this approach enables more accurate investigations of epithelial biology and strengthens the reliability of in vitro experimental systems relevant to oral regenerative medicine and mucosal immunology.