SUMMARY/ABSTRACT This revised application is in response to the BX-23-042 BLRD Merit Review Pilot Project Awards to Validate Novel Animal or Cell-based Models of Diseases (121) and is centered on validating an in vitro microfluidic model of non-alcoholic steatohepatitis (NASH), Specifically, the proposed study is a 2-year pilot project that follows the RFA guidelines to validate cell/tissue culture models of diseases (e,g,, organs-on-chip) to replace currently used animal models of diseases that are important to the healthcare of Veterans, In accordance with the RFA, the proposal is a technology-driven project that is narrowly focused on assessing the fidelity of the in vitro model of human disease compared against two established-but limited-animal models, Non-alcoholic fatty liver disease (NAFLD), including the approximately 20% of fatty liver patients who develop its progressive form, NASH, are strongly associated with the metabolic syndrome, including obesity, insulin resistance and diabetes, dyslipidemia and hypertension that are all prevalent within the Veteran community, While there are currently no effective medications to treat NASH, no non-invasive biomarkers to effectively determine disease progression and no effective platforms for high-throughput or personalized drug screening, this validation study is necessary before an in vitro, personalized assay that replaces animal models or investigates mechanistic causes of disease can be employed to address progressive hepatic dysfunction, cirrhosis and liver failure due to NASH, The project utilizes de-identified iPSCs from patients with and without NASH to validate a system that incorporates a multi-lineage liver cell population in a multi-channeled microfluidic device that responds to gutassociated fatty acids and leads to fibrosis and increased lipid accumulation in the iPSC-derived liver organoid tissue, The degree of lipid accumulation correlates with the disease state of the donor, with increased lipid accumulation observed in iPSC-derived organoid tissue from patients with NASH compared to those with normal liver function, The in vitro microfluidic system is validated against a commonly used mouse model of NASH based upon high-fat, high calorie diets with supplemental fructose or fructose/sucrose and a novel mouse model of NASH that utilizes a humanized liver model via fumarylacetoacetate hydrolase (Fah)-deficient (Fah-1-/Rag2-1- /ll2rg-1-) mice, The study is innovative through its use of patient-specific iPSCs differentiated into multiple liver sub-types (hepatocyte, stellate, Kupffer, etc,) to develop an in vitro model of NASH-related read-outs (lipid accumulation, metabolomics/lipidomics, molecular analysis, fibrosis) that may be influenced by the disease state of the cellular source, The project brings together a complementary team of experts with experience in bioengineering, microfluidics, iPSC and organoid cell biology, lipid metabolism and hepatology to validate a novel NASH ...