A rational design approach for amino acid supplementation in hepatocyte culture

Abstract

Improvement of culture media for mammalian cells is conducted via empirical adjustments, sometimes aided by statistical design methodologies. Here, we demonstrate a proof of principle for the use of constraints-based modeling to achieve enhanced performance of liver-specific functions of cultured hepatocytes during plasma exposure by adjusting amino acid supplementation and hormone levels in the medium. Flux balance analysis (FBA) is used to determine an amino acid flux profile consistent with a desired output; this is used to design an amino acid supplementation. Under conditions of no supplementation, empirical supplementation, and designed supplementation, hepatocytes were exposed to plasma and their morphology, specific cell functions (urea, albumin production) and lipid metabolism were measured. Urea production under the designed amino acid supplementation was found to be increased compared with previously reported (empirical) amino acid supplementation. Not surprisingly, the urea production attained was less than the theoretical value, indicating the existence of pathways or constraints not present in the current model. Although not an explicit design objective, albumin production was also increased by designed amino acid supplementation, suggesting a functional linkage between these outputs. In conjunction with traditional approaches to improving culture conditions, the rational design approach described herein provides a novel means to tune the metabolic outputs of cultured hepatocytes.

Nitrogen anabolism underlies the importance of glutaminolysis in proliferating cells

“…22,32,34 Gln is a major transport form of nitrogen in vivo, being the most abundant amino acid in circulation. 35 Indeed DM-α-KG rescues Hep3B proliferation, and α-KG from glutaminolysis can be utilized by the Krebs cycle. However, it is also proposed that reduced, not enhanced, Krebs cycle activity and oxidative phosphorylation provides proliferative advantage for tumor growth in vivo.…”

CHO gene expression profiling in biopharmaceutical process analysis and design

“…In the pursuit to develop highyield cell culture media, typically, statistical approaches such as principal component analysis (De Alwis et al, 2007) or design of experiments (Lin et al, 2007) are used for media and process optimization. Increasingly, in silico technologies using metabolic models and metabolic flux analysis (MFA) or flux balance analysis (FBA) are applied to investigate cellular metabolism (Selvarasu et al, 2009) and to derive rational optimization strategies (Yang et al, 2009). From an experimental point of view, genomics and proteomics approaches (Gupta and Lee, 2007) provide novel opportunities for data-driven media and process improvements.…”

Stoichiometry Based Steady-State Hepatic Flux Analysis: Computational and Experimental Aspects

“…Similarly, Yang et al . used FBA approaches to design an optimum amino acid supplementation [66,68,69]. They experimentally verified that urea and albumin production under the designed amino acid supplementation was found to be increased compared with previously reported (empirical) amino acid supplementation [59,89].…”

“…Yang et al . [68] used a FBA model where the urea output had been maximized in order to optimize amino acid supplementation and hormone levels in the medium. The aim is to increase the performance of the liver-specific function, i.e.…”