Rat multicellular 3D liver microtissues to explore TGF-β1 induced effects.

Chronic liver damage can lead to fibrosis, encompassing hepatocellular injury, activation of Kupffer cells (KC), and activation of hepatic stellate cells (HSC). Inflammation and TGF-β1 are known mediators in the liver fibrosis adverse outcome pathway (AOP). The aim of this project was to develop a suitable rodent cell culture model for the investigation of key events involved in the development of liver fibrosis, specifically the responses to pathophysiological stimuli such as TGF-β1 and LPS-triggered inflammation. We optimized a single step protocol to purify rat primary hepatocytes (Hep), HSC and KC cells to generate 3D co-cultures based on the hanging drop method. This primary multicellular model responded to the profibrotic cytokine TGF-β1 (1 ng/mL) with signs of hepatocellular damage, inflammation and ultimately HSC activation (increase in αSMA expression). LPS elicited an inflammatory response characterized by increased expression of cytokines. 3D-monocultures comprising only Hep displayed different responses, underlying that parenchymal and non-parenchymal cells need to be present in the system to recapitulate fibrosis. The data also suggest that pre-activated HSC may reverse to a quiescent phenotype in 3D, probably due to the more physiological conditions.