Introduction: It is increasingly evident that the currently available in vivo and in vitro methodologies for disease modelling are sub-optimal in recapitulating the complexity of human pathophysiology, as confirmed by the high failure rate of drug candidates due to lack of efficacy and safety. Moreover, hepatocyte transplantation has been tested as an alternative to liver transplantation for the treatment of liver diseases, but its applicability is hampered by the limited source of hepatocytes and poor hepatocyte engraftment. Aims: to develop human liver ECM hydrogels as novel in vitro platform for target identification/drug screening and for cell transplantation. Methods: Human livers unsuitable for transplantation were decellularized. The resulting ECM scaffold was then lyophilized and the resultant liver ECM powder was solubilised and mixed with three different biomaterials such as agarose, inert bio-ink or a synthetic thermo-responsive copolymer for hydrogel development. Samples were bioengineered with human hepatic cell lines (HepG2, LX2 or SNU-449), stem cells (IPSCs) or human primary hepatocytes. Validation of the hepatocellular carcinoma (HCC) model was investigated through treatment of SNU-449 samples with Sorafenib and TGF-β1. Furthermore, HepG2 bioengineered hydrogels were implanted for 3 weeks in immune-deficient mice. Samples were analysed by histology, immunofluorescence, immunohistochemistry, viability assays, gene expression and metabolic activity. Results: Bioengineered human liver ECM-based hydrogels with human liver cells showed an increase in cell survival, engraftment, proliferation and functionality compared to agarose, inert bio-ink or synthetic thermo-responsive copolymer. Viability assays of SNU-499 cells, upon Sorafenib treatment, revealed differences between 2D and 3D modelling in HCC. Implanted HepG2 ECM-hydrogels, retrieved from mice, showed that cells were still alive and engrafted. In vitro, ECM hydrogels combined with synthetic thermo-responsive copolymer showed the highest cell viability, better reproducibility, required less ECM volume and a smaller number of cells compared to ECM hydrogels combined with agarose or inert bio-ink. Conclusion: This study describes the development and the technical validation of human liver ECM hydrogels for in vitro and in vivo applications
