The three‐dimensional (3D) cell culture models serve as the interface between conventional two‐dimensional (2D) monolayer culture and animal models. 3D culture offers the best possible model system to understand the pathophysiology of human pathogens such as hepatitis C virus (HCV), which lacks a small animal model, due to narrow host tropism and non‐permissiveness of murine hepatocytes. In this study, functionally robust spheroids of HCV permissive Huh7.5 cells were generated, assisted by the temperature or pH‐responsive polymers PNIPAAm and Eudragit respectively, followed by the long‐term growth of the multilayered 3D aggregates in poly(ethylene glycol) (PEG)–alginate–gelatin (PAG) cryogel. The human serum albumin (HSA), marker of hepatic viability was detected up to 600 ng/ml on 24th day of culture. The 3D spheroid culture exhibited a distinct morphology and transcript levels with the upregulation of hepato‐specific transcripts, nuclear factor 4α (HNF4α), transthyretin (TTr), albumin (Alb), phase I and phase II drug‐metabolizing genes. The two most important phase I enzymes CYP3A4 and CYP2D6, together responsible for 90% metabolism of drugs exhibited up to 9‐ and 12‐fold increment, respectively in transcripts. The 3D culture was highly permissive to HCV infection and supported higher multiplicity of infection compared to monolayer Huh7.5 culture. Quantitation of high levels of HSA (500–200 ng/ml) in circulation in mice for 32 days asserted integration with host vasculature and in vivo establishment of 3D culture implants as an ectopic human hepatic tissue in mice. The study demonstrates the 3D spheroid Huh7.5 culture as a model for HCV studies and screening potential for anti‐HCV drug candidates.

Sorafenib (SFB), a multi-kinase inhibitor, is the only approved drug for treating hepatocellular carcinoma (HCC). However, SFB shows low efficacy in many cases. HCC related mortality therefore remains to be high worldwide. SFB, a multi-kinase inhibitor is also known to modulate the redox homeostasis in cancer cells. To understand the effect of SFB on the redox status, a quantitative understanding of the system is necessary. Kinetic modeling of the relevant pathways is a useful approach for obtaining a quantitative understanding of the pathway dynamics and to rank the individual factors based on the extent of influence they wield on the pathway. Here, we report a comprehensive model of the glutathione reaction network (GSH ), consisting of four modules and includes SFB-induced redox stress. We compared GSH simulations for HCC of six different etiologies with healthy liver, and correctly identified the expected variations in cancer. Next, we studied alterations induced in the system upon SFB treatment and observed differential H O dynamics in all the conditions. Using metabolic control analysis, we identified glutathione S-transferase (GST) as the enzyme with the highest selective control coefficient, making it an attractive co-target for potentiating the action of SFB across all six etiologies. As a proof-of-concept, we selected ethacrynic acid (EA), a known inhibitor of GST, and verified ex vivo that EA synergistically potentiates the cytotoxic effect of SFB. Being an FDA approved drug, EA is a promising candidate for repurposing as a combination therapy with SFB for HCC treatment.