Organotypic co-cultures allow for immortalized human gingival keratinocytes to reconstitute a gingival epithelial phenotype in vitro.

We report here that the organotypic co-culture (OCC) system allows for significant preservation of the tissue-specific phenotype of human gingival keratinocytes (IHGK) immortalized with the E6/E7 gene of the human papillomavirus type 16 (HPV16). The approach adopted is based on the OCC system facilitating spatially separated cell growth and cell-to-cell interactions via diffusible growth factors. Generally, IHGK reveal transcription of the HPV16 E6/E7 gene at rising passages. Fluorescence in situ hybridization performed for chromosomes 1, 8, 10, and 18 demonstrates that disomic fractions differ between the tested chromosomes but otherwise remain fairly constant. Monosomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromosome 10 and 18 are detected in early passages 25 and 27. In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, proliferate, and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for growth factors interleukin 1beta, granulocyte-macrophage colony stimulating factor, fibroblast growth factor 7, and EGF in OCCs is different from that in MCs. When grafted onto nude mice, IHGK develop hyperplastic, differentiated surface epithelia devoid of malignant growth. We are not aware of any other OCC system comprising of IHGK, which allows for site-specific expression of gingival epithelial markers. This substantiates reconstitution of a gingival epithelial phenotype in vitro.