Three-Dimensional Perfusion Bioreactor Culture Supports Differentiation of Human Fetal Liver Cells

The ability of human fetal liver cells to survive, expand, and form functional tissue in vitro is of high interest for the development of bioartificial extracorporeal liver support systems, liver cell transplantation therapies, and pharmacologic models. Conventional static two-dimensional culture models seem to be inadequate tools. We focus on dynamic three-dimensional perfusion technologies and developed a scaled-down bioreactor, providing decentralized mass exchange with integral oxygenation. Human fetal liver cells were embedded in a hyaluronan hydrogel within the capillary system to mimic an in vivo matrix and perfusion environment. Metabolic performance was monitored daily, including glucose consumption, lactate dehydrogenase activity, and secretion of alpha-fetoprotein and albumin. At culture termination cells were analyzed for proliferation and liver-specific lineage-dependent cytochrome P450 (CYP3A4/3A7) gene expression. Occurrence of hepatic differentiation in bioreactor cultures was demonstrated by a strong increase in CYP3A4/3A7 gene expression ratio, lower alpha-fetoprotein, and higher albumin secretion than in conventional Petri dish controls. Cells in bioreactors formed three-dimensional structures. Viability of cells was higher in bioreactors than in control cultures. In conclusion, the culture model implementing three-dimensionality, constant perfusion, and integral oxygenation in combination with a hyaluronan hydrogel provides superior conditions for liver cell survival and differentiation compared to conventional culture.