DEVELOPMENT OF AN EPIORAL IN VITRO HUMAN TISSUE MODEL FOR ORAL IRRITANCY TESTING.
This study by researchers at British American Tobacco and Advanced Technologies Ltd. demonstrated that MatTek’s EpiOral in vitro human buccal (inner cheek) tissue equivalent is an excellent in vitro model for analyzing the human oral irritation potential of tobacco products. The EpiOral™ tissue model (ORL-200), developed by the MatTek Corporation (Ashland, MA, USA), consists of normal human epidermal keratinocytes cultured to form a multilayered, highly differentiated model of human oral epithelium, analogous to that found in vivo. The tissues have a buccal phenotype and provide the opportunity for in vitro irritancy testing on human tissues. Methods and Materials: Initial conditions used for the experiments were those provided by the MatTek Corporation with the following modifications. A range of concentrations and two time points were used in our studies compared to a single concentration over a range of time points in Kubilus et al. The cells received nutrients from media fed through the porous membrane below. Substances were applied topically at 0.3-5000ìg/ml for both 1 and 20 hours in duplicate. Trton X-100 (1%) and sodium dodecyl sulphate (SDS, 1%) were included as positive controls at both exposure periods. Dimethyl sulphoxide (DMSO, 0.5%) was used a negative control at both exposure periods. Dimethyl Thiazoyl Tetrazolium (MTT) conversion to formazan, as a measure of tissue viability, was assessed by extraction of formazan from the tissues and measuring OD(570). Mitochondria in viable cells convert MTT (yellow) to formazan (purple). Irritancy was determined by measuring tissue viability. Under our experimental conditions, a reduction in tissue viability of 25%, relative to negative controls, was considered to be significant. This contrasts with the benchmark of 50% reduction recommended by MatTek. Reproducibility: The MTT tissue viability assay, when applied to the EpiOral tissue model, proved to be a highly reliable and reproducible method for assessing tissue viability and irritancy testing with consistent positive and negative control data. Future Studies: Future work will accumulate historical data for the current procedure and will test a range of compounds to collate a database of relative cytotoxicities. In addition, tissue supernatants collected following compound treatments will be analysed for pro-inflammatory cytokines, e.g. IL-8, IL-1á, IL-1â, TNF-á.
EpiOral, MTT, MTT tissue viability, ORL-200, Oral irritancy, Reproducibility
1% SDS, 1% Triton-X, Bergamot oil, Cinnamaldehyde, DMSO, Eugenol, Geranium oil, Phenyl acetic acid
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