Architectural Characterisation of H400 and Primary Rat Keratinocytes Organotypic Cultures

Introduction: Recent advances in quantitative microscopy now enable reproducible and quantitative morphological tissue characterisation. Organotypic epithelial structures can be engineered using primary or immortalised oral keratinocytes. However, their detailed quantitative histological characterisation in comparison with normal mucosal architecture has not been performed until now. Objective: To identify morphological markers of tissue arrangement that can be used to monitor tissue structure, maturation and differentiation and enable quantitative comparison of organotypic cultures with normal oral mucosa. Materials and methods: Organotypic cultures of oral keratinocytes (immortalised H400 or primary rat keratinocytes PRKs) were generated on a selection of scaffolds (de-epidermalised dermis DED, collagen gels and PET) for up to 14 days. Cultures were analysed using immunohistochemistry for structural and transmembrane proteins of oral epithelium including cytokeratins-1,-4,-5,-6,-10,-13, E-cadherin, desmoglein-3, involucrin, and Ki67. The epithelial thickness of organotypic cultures was measured in H&E sections using a semi-automated method of object extraction (SIOX). Results: Histological sections of organotypic cultures of H400 and PRKs on three different scaffolds DED, collagen and PET and their statistical analysis based on SIOX revealed that air-liquid interface cultures generated stratified organotypic epithelial structures after 14-days that were significantly different (P<0.001). The final thickness of the engineered epithelium as well as degrees of maturation/stratification (including stratum corneum formation) varied significantly depending on the scaffold used. For certain scaffolds, the immunohistochemical profiles obtained recapitulated those of normal oral epithelium indicating appropriate in vitro differentiation and proliferation. Conclusion: Quantitative microscopy approaches enable identification and extraction of cellular components, structural and spatial characteristics which help relating tissue structure to maturation marker expression. This model should facilitate a better understanding of the culture environment and the conditions that affect in the oral epithelium in vivo as well as in vitro.