Engineering High Content and Multicellular Human Liver Platforms for Drug Development

The diverse functions of the liver can be severely compromised by several diseases (e.g., non-alcoholic fatty liver disease and hepatitis C/B virus infections); furthermore, drug-induced liver injury is a leading cause of preclinical/clinical drug attrition and acute liver failure. While animal models are utilized to investigate disease mechanisms and screen drugs, there are significant differences between animals and humans in the liver’s molecular pathways. Therefore, there have been increased efforts over the last decade to develop in vitro models of the human liver using primary human liver cells and, more recently, induced pluripotent stem cell-derived hepatocyte-like cells (iHeps). However, there is a need to fully decipher the biophysical and biochemical microenvironmental cues that modulate hepatic functions and regeneration in physiologic and diseased contexts. Unfortunately, using traditional cell culture techniques is too slow and costly to explore such microenvironments in a combinatorial manner. In contrast, high-throughput cellular microarrays that enable precise and independent control over the extracellular matrix (ECM) protein composition and stiffness can be used to elucidate how these parameters affect cell function. Similarly, micropatterning primary human hepatocytes (PHHs) in coculture with supporting non-parenchymal cell (NPC) types enables precise manipulation of homotypic and heterotypic cell-cell interactions toward determining effects on cell’s phenotypic longevity. This thesis aims to adapt these tools to address fundamental gaps in the field of in vitro human liver models. First, high-throughput cell microarrays are being used to determine how ECM protein composition and stiffness act independently and in synergistic combinations to regulate the functions of PHHs and iHeps over at least 2 weeks in culture. Second, micropatterned cocultures are used to elucidate the role of liver NPCs in the modulation of long-term hepatic functions and reciprocal effects on NPCs. Third, soluble factors (e.g., growth factors and small molecules that modulate key liver pathways) are used to devise strategies to regenerate PHHs in vitro towards mimicking the impressive proliferative potential of these cells in vivo. In conclusion, this thesis will explore for the first time how precisely controlled biophysical, biochemical, and cellular factors affect human hepatic functions or mimic regeneration towards improving platforms for drug development and, ultimately, regenerative medicine.