The introduction of new preclinical models for in vitro drug discovery and testing based on 3D tissue-specific extracellular matrix (ECM) is very much awaited. This study was aimed at developing and validating a co-culture model using decellularized human liver 3D ECM scaffolds as a platform for anti-fibrotic and anti-cancer drug testing. Decellularized 3D scaffolds obtained from healthy and cirrhotic human livers were bioengineered with LX2 and HEPG2 as single and co-cultures for up to 13 days and validated as a new drug-testing platform. Pro-fibrogenic markers and cancer phenotypic gene/protein expression and secretion were differently affected when single and co-cultures were exposed to TGF-β1 with specific ECM-dependent effects. The anti-fibrotic efficacy of Sorafenib significantly reduced TGF-β1-induced pro-fibrogenic effects, which coincided with a downregulation of STAT3 phosphorylation. The anti-cancer efficacy of Regorafenib was significantly reduced in 3D bioengineered cells when compared to 2D cultures and dose-dependently associated with cell apoptosis by cleaved PARP-1 activation and P-STAT3 inhibition. Regorafenib reversed TGF-β1-induced P-STAT3 and SHP-1 through induction of epithelial mesenchymal marker E-cadherin and downregulation of vimentin protein expression in both co-cultures engrafting healthy and cirrhotic 3D scaffolds. In their complex, the results of the study suggest that this newly proposed 3D co-culture platform is able to reproduce the natural physio-pathological microenvironment and could be employed for anti-fibrotic and anti-HCC drug screening.

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