ORIGINAL ARTICLE Mariana Roesch-Ely . Thorsten Steinberg . F. Xavier Bosch . Eva Mu¨ssig . Noel Whitaker . Tina Wiest . Annette Kohl . Gerda Komposch . Pascal Tomakidi Organotypic co-cultures allow for immortalized human gingival keratinocytes to reconstitute a gingival epithelial phenotype in vitro Received February 15, 2006; accepted in revised form June 5, 2006 Abstract We report here that the organotypic co-cul- ture (OCC) system allows for significant preservation of the tissue-specific phenotype of human gingival kera- tinocytes (IHGK) immortalized with the E6/E7 gene of the human papillomavirus type 16 (HPV16). The ap- proach adopted is based on the OCC system facilitating spatially separated cell growth and cell-to-cell interac- tions 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 chromo- somes but otherwise remain fairly constant. Mon- osomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromo- some 10 and 18 are detected in early passages 25 and 27. In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, pro- liferate, and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for growth factors interleukin 1b, 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, differen- tiated 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 gin- gival epithelial markers. This substantiates reconstitu- tion of a gingival epithelial phenotype in vitro. Key words keratinocytes Á gingiva Á human papillomavirus type 16/HPV16 Á immortalization Á organotypic co-cultures/OCCs Introduction The development and maintenance of an epithelium re- sults from the complex interactions between the epithe- lium and its underlying mesenchyme. Specific epithelial–mesenchymal interactions are critical for à These authors have equally contributed to the manuscript. Mariana Roesch-Ely à Á Franz Xavier Bosch Ear-Nose-and Throat Hospital University of Heidelberg Im Neuenheimer Feld 400 D-69120 Heidelberg Germany Thorsten Steinberg à Á Eva Mu¨ssig Á Annette Kohl Á Gerda Komposch Á Pascal Tomakidi ( . *) Department of Orthodontics and Dentofacial Orthopedics Dental School University of Heidelberg Im Neuenheimer Feld 400 D-69120 Heidelberg Germany Tel: 149 (0) 6221 56 6574 Fax: 149 (0) 6221 56 5753 E-mail: pascal.tomakidi@med.uni-heidelberg.de Noel Whitaker School of Biotechnology and Biomolecular Sciences University of New South Wales NSW 2052 Sydney Australia Tina Wiest Department of Neurology University of Heidelberg Im Neuenheimer Feld 400 D-69120 Heidelberg Germany U.S. Copyright Clearance Center Code Statement: 0301–4681/2006/7409–622 $ 15.00/0 Differentiation (2006) 74:622–637 DOI: 10.1111/j.1432-0436.2006.00099.x r 2006, Copyright the Authors Journal compilation r 2006, International Society of Differentiation