The metabolic capabilities and constraints of cultured hepatocytes are of great interest for the development of bioartificial liver (BAL) devices. Metabolic flux analysis (MFA) has been to provide a comprehensive view of intracellular metabolism in hepatocyte cultures under various conditions to quantify the intracellular fluxes and to investigate the effects of insulin and amino acids supplementation of primary rat hepatocytes in culture medium following plasma exposure [1]. Our work presents an optimal framework for optimal supplementation by a comprehensive analysis of culture hepatocytes' metabolism in order to achieve the best performance of liver-specific functions of hepatocytes during plasma exposure. And results calculated by mathematic models are further validated using experimental data.

First, a flux balance analysis (FBA) is developed to determine the optimal flux distribution of hepatic metabolism and to design an amino acid supplementation protocol given the flux bounds encompassing a range of physiological states [1]. The effects of several factors governing the specific liver functions, such as insulin level in the precondition of culture, amino acid supplementation and hormone level during plasma exposure, are then investigated for cryopreserved hepatocytes. Hepatocyte morphology, specific cell functions (urea and albumin production), glucose production/consumption and lipid accumulation are studied as markers to validate our hypothesis that liver specific functions are improved and hepatic pathway may change by modulation of hepatic network using amino acids supplementation. Based on the analysis of variance and a factorial design of experiments, amino acid supplementation and hormone level are the most significant factors under the experimental ranges examined during plasma exposure. Both the computationally predicted values and experimental measurements of optimal level of urea production were found to be increased by two fold compared to those that have been observed in the literature [1-3]. Metabolic Flux Analysis is used to calculate internal fluxes given the measured external fluxes in order to validate that higher liver-specific function is achieved simultaneously with an increase of fluxes of gluconeogenesis/glycolysis, TCA cycle, urea cycle fluxes, pentose phosphate pathway and uptake of some amino acids.

The obtained result helps our understanding of hepatocyte metabolism that can be used to develop liver models to test hepatotoxicity of various substances and to improve an extracorporeal bioartificial liver device during different patient treatments.

Reference:

[1] Chan, C., Hwang, D., Stephanopoulos, G. N., Yarmush, M. L. and Stephanopoulos, G., 2003c. Application of multivariate analysis to optimize function of cultured hepatocytes. Biotechnol Prog 19(2), 580-598.

[2] Chan, C., Berthiaume, F., Lee, K. and Yarmush, M. L., 2003. Metabolic flux analysis of hepatocyte function in hormone- and amino acid-supplemented plasma. Metab Eng 5(1), 1-15.

[3] Washizu, J., Berthiaume, F., Chan, C., Tompkins, R. G., Toner, M. and Yarmush, M. L., 2000. Optimization of rat hepatocyte culture in citrated human plasma. Journal of Surgical Research 93(2), 237-246.