EVALUATING SKIN SENSITISING POTENTIAL OF CHEMICALS WITH A SIMPLE AND RAPID IN VITRO METHOD
Buzzella A.1, Mori M.3, Vicini R.3, Angelinetta C.3, Mazzini G.2, Pastoris O1.
1 Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.
2 Institute of Molecular Genetics, CNR, Pavia, Italy.
3 BioBasic Europe s.r.l., Milan, Italy.
The human cell line activation test (h-CLAT) measures in vitro dendritic cell activation using THP-1 cell line. This method evaluates the variation in the expression of two specific membrane antigens (CD54 and CD86) induced by sensitizing substances by means of flow cytometric analysis. Antigens are detected via specific monoclonal antibodies FITC-labelled. Unfortunately, the emitted fluorescence is overlapped by a natural level of cell fluorescence and this generate the need of troubleshooting in data analysis and interpretation. The aim of this study was to find an alternative cytofluorimetric parameter more sensitive than fluorescence to evaluate skin sensitisation potency of chemicals. For this purpose, cells were seeded at density of 0.2x106 cells/ml in culture flask and cultured for 48 hours. Then cells were transferred in a 24 well plate (1x106 cells/well) and treated with different allergens and non-allergens. For control analysis, a set of cells was not exposed to any chemical. After 24 hours, cells were washed twice in PBS and flow cytometric analysis was performed. We observed a significant change in the forward scatter (FSC) of cells treated with sensitizers. No changes were observed in control cells and in cells treated with non-sensitizers. Our data suggest that well-known sensitizing chemicals are able to induce morphological changes in THP-1 cells, as demonstrated by variations in the FSC. For these considerations, the measure of FSC can be used as a sensitive, fast and low-cost method for discriminating between sensitizers and non-sensitizers.
Keywords: skin sensitisation, THP-1; morphology; flow cytometry; in vitro method