Improved Metabolic Pathways of Glycolysis, Glycogen Synthesis, the Urea Cycle, and Cytochrome Peroxidase Oxidative...

Bioreactor-based bioartificial liver support systems have had limited success in a translational setting and at preclinical stages. None of the existing systems monitor the metabolic pathways of glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption. Herein, we designed a bioreactor that mimics the human liver microenvironment in vivo and monitors different hepatic metabolic pathways in order to help establish in vitro culture conditions for improved glycolysis, glycogen synthesis, the urea cycle, cytochrome peroxidase oxidative reabsorption and improved hepatic functions in a miniature bioartificial liver. An abnormality in such pathways negatively influences survivability and hepatic functions, including spontaneous liver regeneration.

We investigated the metabolic functions of primary mouse adult hepatocytes cultured in a three-dimensional configuration under direct oxygenation conditions (5%, 10%, 20%, and 40% O₂) for 14 days in the bioreactor. We analyzed the expression of the genes of hepatic metabolic pathways, such as glycolysis (glucokinase, phosphofructokinase, and pyruvate kinase), glycogen synthesis (glycogen synthetase, UTP glucose-1-phosphate uridylylisomerase, phosphoglucomutase, and glycogen phosphorylase), the urea cycle (arginase, ornithine carbomoyltransferase, fumarate hydratase), oxidative reabsorption (peroxidase), and cytochrome peroxides (catalase and superoxide dismutase), and compared it with the level in vivo. The metabolic mini-map was used to represent the above-mentioned metabolic genes.

Increased urea secretion under normoxia and hyperoxia conditions (20% and 40% O₂, respectively) was observed, while albumin secretion was decreased in hyperoxic cultures. Lactate formation was up to 15 mg/L^-g/h^-h/10⁶ cells, 2 mg/L^-g/h^-h/10⁶ cells, and 0.2 mg/L^-c/h^-h/10⁶ cells in 5%, 20%, and 40% O₂ conditions, respectively while glucose consumption was enhanced under hypoxic conditions (5% and 10% O₂). Cellular membrane integrity was estimated by lactate dehydrogenase assay and was found to be negligible in only 20% and 40% O₂ conditions. The expression of the phase II enzyme UDP-glucuronosyltransferase was only upregulated in 20% oxygenation.

Taken together, 20% O₂ was found to be an optimal condition for the long-term culture (up to 14 days) of hepatocytes that promoted the expression of genes in metabolic pathways such as glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption, and improved hepatic functions in a miniature bioreactor for bioartificial liver construction.