Adaptation of a skin sensitization assay to a chemically defined culture

Abstract

There are currently three in vitro methods adopted by the Organization for the Economic Co-operation and Development for testing chemicals based on the third key event of the skin sensitization adverse outcome pathway, the activation of dendritic cells. All of them use culture medium supplemented with fetal bovine serum (FBS), which brings technical disadvantages and animal welfare concerns. The objective of this study was to analyze the possibility of eliminating the use of FBS in the human Cell Line Activation Test (h-CLAT). After successful implementation of the h-CLAT using THP-1 cells cultured in FBS-containing medium, several attempts to adapt THP-1 cells to four different serum-free media were made. The best results were obtained with gradual adaptation to RPMI-1640 medium with HL-1™ Supplement and to X-VIVO™ 10. Adapted cells were cryopreserved and submitted to the reactivity check. After being approved, they were used in dose finding and proficiency assays. Despite minor adjustments in the original protocol, it was possible to correctly predict the sensitizing potential of the ten proficiency substances using THP-1 cells adapted to X-VIVO™ 10, which indicates that it is possible to eliminate the use of FBS in the h-CLAT, using a chemically defined medium.