P19-05 Primary cell-based 3D models for toxicity test and disease modeling

Studying organ structure and function is challenging using traditional in vitro models like monolayer cultures. Although current in vitro 3D models show promise, they often lack consistency between individual spheroids/organoids. To address this, we have developed and refined 3D models for toxicity and disease modeling, based on primary cells. In our approach, we modeled metabolic dysfunction-associated steatohepatitis (MASH) using rodent primary cells. After the primary hepatocytes formed normal spheroids, we induced liver spheroids by replacing the standard culture medium with a nutrient-rich medium and assessed the relevant factors. This led to the development of a 3D MASH spheroid model, which is suitable for MASH research and the development of related therapeutic drugs. In our C57 mouse primary cell-based 3D models, we first optimized the induction culture medium. After constructing the spheroids, we analyzed the relevant markers of 3D MASH spheroids using immunofluorescence and quantitative PCR. Finally, we assessed the potential application of the 3D MASH model in drug screening. Compared to healthy cell spheroids, 3D MASH spheroids show significantly higher levels of fibrosis. The expression of fibrosis markers, such as smooth muscle actin, and insulin resistance-related genes in the MASH group was notably higher than in the control group, with statistical significance. Additionally, triglyceride and albumin levels in 3D MASH spheroids were significantly elevated compared to healthy controls. Treatment with Selonsertib or Obeticholic acid reduced triglyceride and albumin levels in 3D MASH spheroids to the normal levels seen in healthy controls. Following treatment, MASH-related factors showed significant changes, indicating that the developed 3D MASH spheroid model is effective for screening NASH inhibitors. Improvements of in vitro 3D models for primary cell-based models increased their potential for toxicity and disease modeling. Conflict of interest: All authors are employees and stock shareholders of MILECELL BIOTECHNOLOGY INC. Related patents have been applied.