ABSTRACT Alcohol-associated liver disease (ALD) is the major cause of alcohol-related mortality and encompasses steatosis, steatohepatitis with or without progressive fibrosis, hepatocellular injury, and loss of liver function. Despite considerable research, the specific mechanisms underlying ALD development and progression have not been fully elucidated, partly due to the lack of in vitro and in vivo model systems that recapitulate human ALD. While live mice are used to model features of human ALD, current models are labor-intensive, induce significant mortality (unlike humans), include diets that induce confounding non-alcoholic steatohepatitis, and/or cannot reproduce all features of human ALD. In contrast, we developed a simple-to-administer 16-week western diet alcohol (WDA) model that recapitulates the inflammatory, fibrotic, and gene expression aspects of human alcohol-associated steatohepatitis (ASH). However, it is difficult to investigate the direct effects of alcohol on multiple liver cell types and elucidate the cell-cell interactions important for ALD pathogenesis in vivo. In contrast, while primary liver cells can be isolated with high purity from livers to build in vitro models, they rapidly lose phenotypic functions in 2D monocultures. To mitigate this limitation, we utilized high-throughput droplet microfluidics to fabricate highly monodisperse extracellular matrix (ECM)-based engineered 3D liver microtissues containing hepatocytes and liver non-parenchymal cells (NPCs) that functionally outperform conventional self- assembled cell spheroids and cells embedded within bulk gels. Here, we will leverage the above advances to test the novel hypothesis that microtissues containing multiple primary mouse liver cells can recapitulate the critical features of ASH as in the WDA mouse. In aim 1, we will fabricate and optimize long-term (4+ weeks) 3D liver microtissues containing primary mouse hepatocytes, liver endothelial cells, hepatic stellate cells, and Kupffer cells; the role of ECM and tissue size will be investigated towards inducing high (physiologic) and stable cell functions. We will further assess the effects of in vitro ethanol exposure on each cell type within microtissues. In aim 2, we will investigate cell-cell interactions in liver microtissues derived from cells isolated from the WDA mice. Microtissues will be cultured with or without ethanol and cellular functions as well as single cell RNA sequencing data will be compared to control microtissues and existing RNA sequencing data from freshly isolated cells from WDA mice. We will further examine the role of each of the alcohol-specific NPC type in maintaining hepatic function and regulating inflammatory responses and fibrogenesis in microtissues. Our efforts will yield a first-of-its-kind in vitro organotypic mouse liver model with long-term functions, which will be utilized to elucidate the direct effects of alcohol on multiple liver cell types, the extent to which the AL...