Die Entwicklung eines perfundierten 3D Leber-Gefäß Modells und dessen Einsatz zum Verständnis immunologischer Prozesse in Lebererkrankungen

• The development of a perfused 3D liver sinusoid model and its use to understand immunologic processes durig liver diseases

Frank, Anna Katharina; Tacke, Frank (Thesis advisor); Jahnen-Dechent, Wilhelm (Thesis advisor); Spehr, Marc (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020

Abstract

Acute drug-induced hepatoxicity and chronic non-alcoholic fatty liver disease are a major burden for patients, clinics and public health systems worldwide. Macrophage populations have been shown to be important for the progression and resolution of these diseases. The exact roles and functions of these cell populations during the onset of inflammatory cascades, during disease progression and during liver recovery however are controversial. Development of suitable in vitro models might aid the understanding of fundamental cell specific mechanisms of disease progression and result in the development of novel pharmaceutical therapies. Therefore, in this thesis a novel in vitro liver co-culture system, which mimics the three-dimensional architecture of the murine liver sinusoid, was established and tested for its use in studying immune mechanism processes during acute and chronic liver disease. Primary murine hepatocytes, liver sinusoidal endothelial cells, Kupfer cells and hepatic stellate cells were isolated from murine livers and seeded into the biochip. Fluorescence microscopy, immunofluorescence staining, and mass spectrometry analysis were performed to confirm survival and functionality of the biochip-cultured cells. Next, two in vitro liver disease models were developed using the liver sinusoid system. Acute drug-induced hepatoxicity was introduced through incubation with acetaminophen and hepatic steatosis was modelled by excess free fatty acids in cell culture medium. Fluorescence microscopy, qPCR analysis and measurements of specific cell damage markers were performed to investigate individual cellular responses to the hepatotoxic reagents. It could be shown that biochip-cultured hepatic cells showed similar reactions to hepatotoxic reagents and counteracting pharmaceutics than murine cells in vivo. In a third step, the liver biochip system was then tested for its value as a tool for studying macrophage-related immune-mechanisms during liver disease in vitro. Therefore, the influence of monocytes and Kupfer cells on the outcome of overall cell damage after administration of hepatotoxic reagents was compared. The in vitro behavior of Kupfer cells and monocytes during hepatic cell damage was then set in context with their unique polarization. It could thereby be shown that macrophage populations have a beneficial influence during early stages of hepatic diseases in vitro.