Design and analysis of amino acid supplementation in hepatocyte culture using in vitro experiment and mathematical modeling:

Extracorporeal bioartificial liver (BAL) devices, involving primary hepatocytes, represent a promising option to provide temporary support for patients with liver failure. Current use of BAL is primary challenged by development of techniques for long-term culture of hepatocytes during plasma exposure, as occurs during clinical application. Previous in vitro studies and mathematical modeling analysis have shown that supplementation of amino acids to the plasma enhances liver-specific functions and reduces lipid accumulation. However, further improvement would be enhanced greatly by development of a rational strategy to design the profile of amino acid supplementation and by better understanding of the metabolic objectives of hepatocytes, and how they vary as a function of amino acid supplementation. In order to address these issues, a rational design approach was first developed using flux balance analysis (FBA) to determine a profile of amino acid supplementation to achieve a specific cellular objective (urea production) in cultured hepatocytes exposed to plasma. Experiments based on the designed supplementation showed that both urea and albumin production were increased compared with previously reported (empirical) amino acid supplementation. However, the experimental values did not match our theoretical prediction mainly due to the insufficient constraints imposed to the modeling. In an attempt to improve the model accuracy, we incorporated pathway energy balance (PEB) constraints, and amino acids transport constraints. It is found that both PEB and transport constraints significantly reduce the feasible region of the flux space. Moreover, metabolic objective prediction (MOP) model reveals that hepatocytes respond to variations in available amino acid supplementation by changing their metabolic objectives and pathway utilization. In particular, the analysis shows that fatty acid oxidation is vital to reduce the rate of lipid accumulation and to increase liver-specific functions with amino acid supplementation. This study leads to a better understanding of amino acid supplementation effects on hepatocytes during plasma exposure based on the integration of in vitro experiments and mathematical modeling. The approach enables the metabolic manipulation of hepatocytes with rationally designed amino acid supplementation to improve the targeted liver cell functionality and improve the long-term technique of hepatocytes applied for BAL devices.Ph.D.Includes bibliographical references (p. 154-164)by Hong Yan