DEVELOPMENT OF IN VITRO LIVER CULTURE TECHNIQUES TO INTEGRATE A PHYSIOLOGICALLY RELEVANT MICROENVIROMENT RECAPITULATING LIVER LOBULE ZONATION

During drug-discovery, cell monolayers fail to accurately predict drug-induced liver injury– a common reason for discontinuation during clinical trials. One reason is that monolayer cultures lack many aspects of the tissue microenvironment. The liver is composed of subunits known as lobules. In the lobule, a sinusoid connects a portal triad and central vein and is surrounded by hepatocytes to maximize nutrient exchange. A unidirectional flow leads to extracellular gradients of oxygen, nutrients, hormones, and morphogens. These gradients in the lobule impact hepatocyte function through the differential expression of hepatocytic pathways in the periportal (PP) and perivenous (PV) regions. To improve in vitro hepatocyte cultures, a platform that incorporates structural and microenvironmental factors is needed to recapitulate the liver. Factors include: coculturing liver-specific cells; inclusion of liver-specific extracellular gradients, specifically nutrients and morphogens; incorporating dynamic nutrient flow and distribution; and inclusion of appropriate three-dimensional hepatocyte organization, and a relevant extracellular matrix (ECM). In this work, I integrated physiological aspects– oxygen, morphogens, extracellular matrix stiffness, and a 3D paper scaffold culture– into in vitro cultures of different cell types with increasing hepatocyte-like functions: HepG2, HepaRG, and primary human hepatocytes (PHHs). I evaluated cell health, liver-specific function, metabolic enzyme activity, and transcriptional regulation as each physiological condition was introduced individually, and in concert. This work displayed that oxygen improves in vitro zonal patterning of SULT and UGT transcript, but oxygen alone could not achieve the drug metabolizing enzyme’s zonal distribution. The inclusion of Wnt alone increases CYP, UGT and SULT activity, as compared to a control, while the magnitude of enzymatic activity increase is modulated by oxygen. Overall, the HepaRG cell’s basal and inducible drug metabolizing enzyme activity is similar across a wide range of ECM stiffness suggesting that the HepaRG cells are adaptable to various ECMs, unlike PHHs. Lastly, direct comparisons of monolayers and paper-based cultured in PP and PV microenvironments, indicated zonation-like trends between the PP and PV conditions. The paper-based PV conditioned cells exhibited the highest drug metabolizing enzyme activity. This work developed a more representative in vitro liver models by incorporating tissue-specific features.Doctor of Philosoph