Clearing intrahepatic lipids to aid in toxicity testing

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DESCRIPTION (provided by applicant): Vesta Therapeutics is one of the leading procurers of non-transplantable human livers in the United States and has developed proprietary technologies for the isolation, cryopreservation, and regenerative application of isolated primary hepatocytes. As Vesta processes cells for our research programs, a significant number of surplus mature hepatocytes are collected, [which are made available to academic, clinical and pharmaceutical researchers. Primary human hepatocytes are valuable and crucial models used by pharmaceutical companies to test the absorption, distribution, metabolism, excretion (ADME) and toxicity (T) properties of novel drug candidates. However,] with the increasing rates of obesity in the United States, a high percentage of the non-transplantable organs offered by Organ Procurement Organizations [for research purposes] are steatotic (fatty), which adds an uncontrollable layer of variability to the toxicity of potential test compounds. [As a consequence, pharmaceutical companies are reluctant to utilize steatotic hepatocytes, and are forced to use non-physiological animal and tumor cell models for drug development.] Research over the last few years has shown that a number of factors that target lipid biosynthesis, oxidation, or intrahepatic export can reverse steatosis in animals and in human patients. [However, the feasibility of applying these methodologies to primary human hepatocytes for the purpose of generating a standardized supply of non-steatotic hepatocytes has not been explored.] Vesta is uniquely positioned to assess these technologies given our consistent supply of steatotic livers and our extensive cryopreserved stocks of steatotic hepatocytes. We propose to decrease steatosis levels in human hepatocytes using bile acids, PPAR agonists, and choline singly or in combination, which have been shown to reverse steatosis in animal and human studies. We will document the optimal conditions to clear lipids, the time course of lipid clearing, the stability of clearance, and the functional characteristics of "lipid-free" hepatocytes. In the latter stages of the proposed studies, we will apply these protocols to isolate hepatocytes from livers containing levels of steatosis that typically preclude their use in drug discovery applications. Our overall goal is to determine the least "invasive" protocol to clear hepatocytes of excess lipids to address the acute shortage of primary human hepatocytes for academic, clinical, and pharmaceutical applications. PUBLIC HEALTH RELEVANCE: In the application "Clearing intrahepatic lipids to aid in toxicity testing," studies are aimed at examining the feasibility of targeting three pathways known to regulate lipid (fat) metabolism with the aim of reducing the fat levels in human hepatocytes isolated from donated livers. Vesta Therapeutics is one of the leading procurers of non-transplantable livers in the United States and has developed proprietary technologies for the isolation and preservation of primary hepatocytes from these livers. Currently, pharmaceutical companies are reluctant to utilize hepatocytes with high levels of fat, which add an uncontrollable layer of variability to the toxicity of test compounds. Devising strategies to eliminate fat from freshly isolated or cryopreserved hepatocytes would provide extremely valuable technologies for the full utilization of limited number of livers donated each year. Thus, if successful, this source of "fat-free" hepatocytes could benefit many goals of public health.