Introduction: Mortality caused by liver disease and its complications is on the rise, representing a significant global health issue. Transplantation is the only efficient treatment for end-stage liver disease but is limited by the shortage of organ donors. Bioengineering represents a promising option, with researchers aiming at developing suitable organ replacements for transplantation. Tissue engineered organs rely (i) on a proper source of cells, able to support organ’s functionality long term, and (ii) on bioreactors, for the culture of whole-organ constructs. Amnion epithelial cells (AECs) can be isolated from full-term membrane with no ethical concerns. AECs can mature into hepatocyte-like cells (HLC), representing a promising source of hepatocytes for liver regenerative medicine and toxicological evaluations. Extracellular matrix (ECM) proteins promote cell maturation and long-term function; organ-specific ECMs can be obtained using decellularization, which allows eliminating cells from a tissue while maintaining ECM composition and 3D-architecture.
Here, we induced maturation of human AEC into functional HLC by culturing the cells into a 3D decellularised liver construct in a custom-made bioreactor, and evaluated differentiation and functionality of the HLC obtained.
Methodology: 40-50 million human AEC (isolated from full-term amnion membrane and characterized via FACS and qPCR) were seeded into decellularised rat liver scaffolds obtained via established detergent-enzymatic treatment. Constructs were cultured in custom-made bioreactors for up to 40 days (10 in expansion and 30 in hepato-specific culture conditions), with static cultured scaffolds used as control. Metabolites (e.g. lactate and glucose) and hepatic activities were monitored at different time points via NMR, ELISA and EROD assays, and compared to primary human hepatocytes. Transcriptome and proteomic analysis at intermediate and final time points were used to confirm the functional analysis.
Results: Freshly isolated AECs were positive for epithelial markers but negative for mature hepatocytes markers. AEC seeded in ECM-scaffolds adhered and proliferated to some extent when exposed to proliferative conditions. After 2 weeks in hepatic maturation media, AEC expressed immature hepatocyte marker alpha-feto protein, while 2 additional weeks generated CK18+-HLC characterized by secretive (Albumin) and functional (CYP3A4) protein expression. Albumin and urea concentration in the culture media increased in bioreactor-cultured constructs in respect to static culture, and NMR showed a shift in metabolite production over the course of the maturation. Finally, phase-1 hepatic metabolism was quantified via EROD assay at different time points.
Conclusion: Here we show that liver ECM-scaffolds efficiently supported the maturation of AEC into functional HLC in a 3D liver model. Remarkably, both cell distribution and hepato-specific activities and functionality were enhanced when human AEC were cultured in bioreactor than in static ECM scaffold. The bioreactor technology may provide an advantage for cell differentiation thanks to a more even distribution of oxygen and nutrients in comparison to static conditions. The technology here presented can serve as a paradigm for hepatic maturation in a 3D model of the liver composed by natural ECM and can help to investigate the role of ECM-specific protein in cell maturation and functionality.