In May 2025, Tim Ravnjak, PhD student at NIB, visited In vitro toxicology group of the Swansea University Medical School in United Kingdom. The research visit programme was focused on the hands-on training on establishing and maintaining hepatic 3D mono and cocultures in in vitro conditions. 3D hepatic cocultures are emerging cell models in the field of genetic toxicology and have several advantages over traditionally used monolayer (2D) cell models and monocultures in 3D, especially in their resemblance to the human hepatic tissue. Application of advanced in vitro cell models, such as hepatic cocultures, is also in accordance with EU REACH legislation 3R strategy (Reduction, Replacement, Refinement), which aims to minimize number of in vivo experiments through development of advanced in vitro systems and methodologies, that also have higher relevance for human health.

Established 3D hepatic cocultures of HepG2 and Kupffer cell line were exposed to selected chemicals for 24h. Then, several methods were used to assess various endpoints, including ELISA (Enzyme-Linked Immunosorbent Assay) for quantitative determination of inflammatory markers, Albumin and Urea assays to determine liver functionality of coculture cell model, LDH (lactate dehydrogenase) assays for cytotoxicity assessment and comet assay for evaluation of genotoxicity. Additionally, 3D coculture cell models were also fluorescently stained and analysed with confocal microscopy. Overall, the research visit was very successful with Tim gaining valuable experience and new knowledge, which resulted in a plan to continue with experiments, initiated at Swansea University, at his home institution.