TDK conference 2020

Three-dimensional cell culture-based methods are gaining more importance in scientific research. The greatest advantage of these technique is that the cells are structured in a way that is more resemble to the living tissues than we could obtain with two-dimensional cell cultures. Their applications are especially significant in the area of tumour research, medicine development and toxicology.

In the present study, the hepatocellular inflammatory response to different bacterial cell wall components was compared using two- and three-dimensional cultures. Hepatocytes and non-parenchymal cells were isolated from chicken liver, followed by the establishment of two- and three-dimensional hepatocyte monocultures and co-cultures with cell ratio of 6:1 (hepatocytes to non-parenchymal cells). Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were used to induce inflammation on the cell culture models, applied in 10 and 50 μg/ml concentration for 24 hours. The metabolic activity of the cells was measured by CCK-8 test, and among the inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8) concentration in culture media were quantified by chicken specific sandwich ELISA tests.

According to the results of the CCK-8 assay, metabolic activity of the two-dimensional cell cultures was not changed significantly by treatment, however, significant increase was induced by each treatment in three-dimensional culture. The concentration of IL-6 and IL-8 in culture media was higher in two-dimensional models compared to three-dimensional cell cultures independently from the treatments. There was significant increase in IL-6 concentration in the two-dimensional hepatocyte monoculture after the application of 10 μg/ml LTA and in the co-culture following 50 μg/ml LTA treatment. In contrast, the same parameter was decreased in the three-dimensional hepatocyte monoculture after 10 μg/ml and 50 μg/ml LTA treatment, as well as in the co-culture as a result of 50 μg/ml LTA treatment. Production of IL-8 was not changed significantly in the two-dimensional hepatocyte monoculture, while it was significantly increased in the co-culture after 50 μg/ml LTA treatment. In contrast, there was a significant decrease in the IL-8 concentration in the three-dimensional hepatocyte monoculture following the application of 10 μg/ml and 50 μg/ml LTA treatment. Furthermore, every treatment resulted in a decreased IL-8 concentration in the co-culture.

According to our results, there could be remarkable differences between two- and three-dimensional cell cultures in the response to pro-inflammatory effects, which observation – considering the results of other studies, carried out in three-dimensional models - could be presumably related to differences in the gene expression patterns. The more detailed knowledge of these differences is especially important for the development of novel cell models and future research involving three-dimensional cell cultures.