Establishment of a Human Cell‐Based In Vitro Test System for Investigating and Evaluating Cytotoxic and Immunomodulatory Effects of Indoor Mold

Mold growth in damp indoor environments is a widespread problem associated with respiratory health effects, though causal mechanisms remain incompletely understood. We established a human cell-based in vitro test system using bronchial epithelial cells (NuLi-1) and macrophages (THP-1) to assess inhalation toxicity and immunomodulatory effects of indoor molds, focusing on cellular responses and mycotoxin patterns from contaminated building materials. The system integrates endpoints and impedance-based real-time cell viability analysis, quantification of released cytokines, and targeted mycotoxin profiling for comprehensive toxicological insights. Methanolic extracts from plasterboard, woodchip wallpaper, and malt extract agar (sterile PBS-treated or contaminated with Alternaria botrytis, Aspergillus versicolor, Penicillium chrysogenum, and Stachybotrys chartarum) were prepared via direct extraction of the material or surface swabs of the contaminated material. The system detected fungus- and material-specific effects, with pronounced cytotoxicity and immunomodulation, especially in Stachybotrys chartarum-contaminated samples. This material increased cytotoxicity in both tested cell types, decreased GM-CSF release from NuLi-1 cells, as well as increased interleukin-1β release from THP-1 macrophages. Therefore, mycotoxin composition and concentration were analyzed and confirmed elevated levels of the mycotoxins Roridin L2, Verrucarin J, and Stachybotrylactam. These findings reveal a substantial inhalation toxicity potential from Stachybotrys chartarum-contaminated indoor materials, highlighting health risks in damp environments and necessitating further in vivo and epidemiological studies.