Characterisation of 2D and 3D oral keratinocyte cultures

Oral keratinocyte behaviour were analysed in two and three dimensional cultures of an immortalised human H400 cellline and primary rat keratinocytes (PRKs) using a novel method of quantitative microscopy, RT-PCR data and immunohistochemistry profiles. Monolayer cultures were established in high and low calcium media at different cell densities and analysed prior to generating 3D organotypic cultures (OCs) onde-epidermalised dermis (DED), polyethylene terephthalate porous membrane (PET) and collagen gels for up to 14 days.H400 and PRKs proliferation in monolayer cultures was greater in low calcium medium compared with high calcium medium.Gene expression analysis indicated that adhesion and structural molecules including E-cadherin, plakophilin, desmocollin-3, desmogleins-3 and cytokeratins-1, -5, -6, -10, -13 were up-regulated by days 6 and 8 compared with day 4in high calcium medium. Immunohistochemical profiles and gene expression data of OCs on DED recapitulated those of normal oral epithelium. The final thickness of OCs as well as the degree of maturation/stratification was significantly greater on DED compared with other scaffolds used. Quantitative microscopy approaches enabled unbiased architectural characterisation of OCs and the ability to relate stratified organotypic epithelial structures to the normal oral mucosa. H400 and PRK OCs on DED at the air liquid interface demonstrated similar characteristics in terms of gene expression and protein distribution to the normal tissue architecture.