Amplified response of drug-induced liver fibrosis via immune cell co-culture in a 3D in vitro hepatic fibrosis model

Abstract

Liver fibrosis, a critical consequence of chronic liver diseases, is characterized by excessive extracellular matrix (ECM) deposition driven by inflammation. This process involves complex interactions among hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells, the liver's resident macrophages. Kupffer cells are essential in initiating fibrosis through the release of pro-inflammatory cytokines that activate HSCs. Although various in vitro liver fibrosis models have been developed, there is a lack of models that include the immune environment of the liver to clarify the influence of immune cells on the progression of liver fibrosis. We developed an in vitro liver fibrosis model by co-culturing hepatocytes (HepaRG), a hepatic stellate cell line (LX-2), and macrophages (differentiated THP-1). The effects of liver fibrosis inducers, transforming growth factor-beta1 (TGF-β1) and methotrexate (MTX), on the inflammatory response and stellate cell activation were evaluated in this triple co-culture model. A triple co-culture condition was developed as a 3D in vitro model using gelatin methacrylate (GelMA), offering a more biomimetic environment and achieving liver fibrosis via immune cell activation associated ECM deposition. In this study, the developed triple co-culture model has the potential to elucidate cell progression roles in liver fibrosis and can be applied in drug screening and toxicity assessments targeting liver fibrosis.