University of Veterinary Medicine Vienna The Intricacies of Inflammatory Bowel Disease: A Preliminary Study of Redox Biology in Intestinal Organoids We evaluated the redox status, precisely glutathione levels, which have a major impact in cellular detoxification and antioxidant defence in IBD-derived and healthy intestinal organoids. Therefore, we wanted to explore the differences in terms of their redox balance and mitochondrial fitness. To this end, we introduced a Grx1-roGFP2 construct into the organoids by lentiviral transduction before performing a stress assay by treating the organoids with hydrogen peroxide and examined the GSH/GSSG ratio using confocal imaging. Using ratio imaging, we could detect statistically significant differences between healthy and IBD-derived samples. To gain more insight, we also performed a GSH/GSSG assay, which directly measured glutathione levels. This analysis revealed that both organoid lines had higher levels of oxidized glutathione due to the stress treatment demonstrated by a lower GSH/GSSG ratio compared to the untreated control. Nevertheless, the results showed no significant difference between healthy and IBD-derived organoids. We further challenged organoids with hydrogen peroxide after incubation with MitoTracker® to see if mitochondrial fitness might be different in IBD-derived organoids. However, these results were also very comparable. In summary, our preliminary findings indicate that both organoid lines demonstrate a well-functioning system in terms of analysis but show no clear difference between healthy and IBD-derived samples. en TEXT article intestinal organoid; redox biology; IBD; glutathione; oxidative stress; ROS; redox imaging 2023 2023 Csukovich, Georg (University of Veterinary Medicine Vienna) Burgener, Iwan Anton (University of Veterinary Medicine Vienna) Pratscher, Barbara (University of Veterinary Medicine Vienna) Huainig, Janina (University of Veterinary Medicine Vienna) Troester, Selina (University of Veterinary Medicine Vienna) MDPI