A Novel Serum-Free Medium Formulation for Human Liver Cultures with Applications in Drug Screening
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The liver is the largest internal organ of the human body and provides for a wide range of functions, which can be seriously compromised by toxic drugs and diseases. In order to develop novel and safer drugs, in vitro models of the human liver are increasingly adopted to complement drug screenings in animal models, known to have species-specific differences in liver pathways. Micropatterned co-cultures (MPCCs) of primary human hepatocytes (PHHs) and supportive 3T3-J2 murine embryonic fibroblasts maintain high levels of liver functions for several weeks in vitro and have been highly useful to screen for drug toxicity and mimic key mechanisms of liver diseases, such as non-alcoholic fatty liver disease (NAFLD), a condition that affects ~30% of patients in the US and is highly correlated to obesity. When culturing cells, bovine serum is commonly used; however, it is problematic given its variability and the mismatch of proteins across species. Nonetheless, avoiding the use of bovine serum in the cell culture medium results in reduced proliferation and number of fibroblasts, in turn causing a rapid decline in PHH functions. Therefore, in this thesis we firstly developed a novel animal serum-free culture medium by implementing low levels of both low density lipoproteins (LDL) and high density lipoproteins (HDL), able to induce and maintain MPCC functions for several weeks towards significantly higher levels than the current serum-free medium formulation. Secondly, we tested the newly developed serum-free medium formulation for drug screening against MPCCs induced towards a NAFLD phenotype (steatosis and insulin resistance) using either a mixture of fatty acids found in the human diet or human serum, evaluating the efficacy of clinical drugs (2,4- dinitrophenol and pioglitazone) at non-toxic concentrations. Although unable to reduce hepatic steatosis, insulin sensitivity improved transiently, whereas steatotic PHHs spontaneously purged intracellular lipids irrespective of drug therapy upon removal of fatty acids from the culture, suggesting that diet restriction may be critical for alleviating NAFLD in patients. Ultimately, our model system together with the newly developed serum-free medium can prove useful for developing novel and safe drugs against liver diseases towards alleviating the disease burden on the human population.