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Preservation of hepatic phenotype and functions in vitro has always been a great challenge for the reconstruction of liver tissue engineering and in pharmaceutical research studies. Human induced hepatocytes (hiHeps) generated from fibroblasts can be reproducible with almost normal levels of liver specific functions, which are considered as a new source of hepatocytes for biomedical applications. Moreover, paper has served as an attractive biocompatible material for cell-based applications. In this study, we established a simple paper-based scaffold array for creating a 3D liver co-culture model that enabled the assessment of drug induced hepatotoxicity. The hiHeps co-cultured with HUVECs exhibited a 3D like morphology and maintained the liver specific functions of producing albumin and urea for up to 2 months. In addition, the hiHeps in this co-cultured model maintained a higher expression of cytochrome P450 genes as compared with a monolayer culture on a plate and a single culture on paper of hiHeps, revealing a marked enhancement of hepatic functions in the 3D liver co-culture model. Moreover, the 3D liver co-culture model was exposed to acetaminophen (APAP) and pioglitazone, exhibiting near physiological hepatotoxic responses compared to those of the monolayer cultures. Taken together, the low-cost and bioactive paper scaffold could offer great opportunities as 3D in vitro platforms for tissue engineering applications and high-throughput drug testing.

Paper supported long-term 3D liver co-culture model for the assessment of hepatotoxic drugs