Engineered culture platforms to uncover synergies between microenvironmental cues in modulating liver zonation - ABSTRACT Hepatocytes exhibit compartmental (zonated) functions along the sinusoid with as many as 50% of liver genes thought to be zonated. The initiation and progression of several diseases can show a zonated bias including drug-induced liver injury, non-alcoholic fatty liver disease, and hepatocellular carcinoma. The fetal liver is not zonated and thus zonation begins after birth due to gradients of O2, hormones, nutritional stimuli, and non- parenchymal cell (NPC) secretions acting on common pathways. As complementary tools to live animal studies, in vitro hepatocyte +/- NPC cultures subjected to specific factor gradients within fluidic devices can enable a more detailed understanding of the regulators and functional outcomes of zonation. However, previous in vitro platforms/studies have only been able to recapitulate limited features and an incomplete understanding of hepatic zonation. We have pioneered a droplet microfluidics platform for the high-throughput generation of reproducibly-sized 3D extracellular matrix (ECM) microgels (<300 µm) containing primary hepatocytes that display liver functions for 4+ weeks in vitro when the microgels are coated with primary liver sinusoidal endothelial cells (LSECs); microtissues can be further augmented with hepatic stellate cells (HSCs) and Kupffer cells (KCs). We have also developed microfluidic devices that enable precise microscale and spatial control over the O2 environment of cells with higher resolution than afforded for by conventional devices; such devices also uniquely allow decoupling of the effects of O2 from other soluble factor gradients that are typically induced via perfusion. Additionally, our data shows that primary rat and human hepatocytes display differential regulation of phenotypic functions when subjected to in vivo-like O2 tensions over prolonged culture. Here, we will test our novel hypothesis that microtissues can be used within custom microfluidic platforms to elucidate the roles of different soluble factor gradients, individually and in controlled combinations, on the long-term phenotypic responses of multiple liver cell types from rats and humans. In aims 1 and 2, we will elucidate the effects of physiological O2 tensions and the effects of hormonal and nutrient gradients on the long-term functions of rat and human liver microtissues, respectively. Lastly, in aim 3, we will utilize microfluidic devices that enable multiple overlapping soluble factor gradients to elucidate the effects of gradient crosstalk on multicellular zonation in rat and human liver microtissues. The detailed investigations here will be the first-of-their kind and will significantly increase our understanding of how key factor gradients and heterotypic cell-cell signaling affects liver zonation across human and rodents, which can be useful for building physiologically-relevant liver tissues for drug development across its various phases and ultimately regenerative medicine. Lastly, the microfluidic tools developed here can serve as a community resource to probe molecular mechanisms underlying liver zonation and how it affects the initiation and progression of several liver diseases and chemical-induced liver injury.
