Rapid detection of immunotoxicity and adverse reactions to new compounds using 3D skin equivalent models

Abstract

Safety testing procedures and risk assessment analysis of newly developed chemicals are mandatory to attest them as non-toxic for human handling. The European Union implemented several regulations enforcing safety testing and has recently banned the use of animals for toxicology studies for human safety. From in vitro cell-based assays to reconstructed human epidermis (RHE) models, there are several non-animal alternatives for toxicology studies to determine human safety. Cell-based assays are not accurate enough to predict the toxic potential of chemicals as they are a two-dimensional (2D) representation of skin cells. Three-dimensional (3D) RHE models are better, as they allow for differentiation of skin epidermal layers (i.e. stratum corneum). Commercially available RHE models are now the gold standard for toxicity studies for human safety and some of these have been validated by the Organization for Economic Cooperation and Development (OECD) for skin irritation and corrosion testing (two major categories of skin toxicity). The aim of this study was to develop an open source 3D epidermal skin culture based on Poumay’s work. Using the Alvetex Strata scaffold, cell seeding was optimised using 1x106 keratinocytes for a 30 day culture period for best differentiation of the stratum corneum layer (confirmed by immunofluorescence staining of involucrin). Further optimization of vitamin C and calcium supplements did not seem to improve distribution of keratinocytes throughout the scaffold. Ultimately, the best results were achieved by the addition of a human-based collagen coat to the Alvetex Strata Scaffold, where development of stratum corneum was confirmed by collagen and involucrin staining. Moreover, development of the stratum basale layer was confirmed by cytokeratin 14 immunofluorescence. Evaluation of the 3D epidermal skin culture was tested following OECD Testing Guideline (TG) 439 for skin irritation in 12 reference chemicals from the 20 reference chemicals list in TG 439. Results showed the 3D epidermal skin culture was able to distinguish non-irritant from irritant chemicals using 10 different keratinocyte donors. Comparative performance analysis with a monolayer (2D) keratinocyte culture, a commercial RHE model and a collagen-based RHE models were performed. Results showed similar outcomes between the different models, while the monolayer culture performed poorly in discriminating irritant from non-irritant chemicals. Overall, these results were indicative of the predicative capacity of the 3D epidermal skin culture for in vitro testing of skin irritation. The second aim of this project was to use a skin explant assay, developed by Alcyomics, as a novel in vitro test for assessment of immunotoxicity caused by aggregation of monoclonal antibodies (mAbs). mAbs are important therapeutics but their potential for aggregation has become a critical quality parameter that can turn into a potential health risk during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show that it can lead to immune responses via cell activation and cytokine release. Our results showed that aggregated mAbs caused adverse immune events by evidence of tissue damage, expression of cell death markers and overall increase of IFN-γ (pro-inflammatory cytokine). These results showed that the skin explant assay could be a promising tool for predicating immunotoxicity caused by mAb aggregation. In conclusion, this study has provided further insight on the assessment of skin toxicity and immunotoxicity using in vitro skin assays.