Abstract: Objective To investigate whether the novel hepatic maturation medium (HMM) can improve the hepatocyte function of the liver tumor cell line of HepG2, and to develop a more suitable culture system for HepG2 in combination with 3-dimensional (3D) culture technique.Methods Human hepatoma cell line HepG2 was obtained from the cell bank. Subsequently, the cells were cultured both in 2-dimensional (2D) and 3D, using Dulbecco's modified Eagle medium (DMEM) or HMM. Functional evaluation was performed by quantitative polymerase chain reaction (qPCR), periodic acid-schiff staining, urea synthesis assay. Finally, the sensitivity of cultured HepG2 to chlorpromazine was detected in the 3D condition combined with DMEM and HMM, respectively. Results HMM could increase the glycogen synthesis ability of HepG2, and upregulate the expression levels of Cytochrome P450 3A4(CYP3A4), Na⁺-taurocholate cotransporting polypeptide (NTCP), albumin (ALB), Carbamoyl-phosphase synthetase 1(CPS1) and Glucose-6-phosphatase (G6PC), by 4.7±0.18, 1.3±0.10, 1.4±0.10, 4.9±0.20 and 5.6±0.10 times, respectively. More importantly, in HepG2 cultured using HMM in 3D compared with those in 2D , the expression levels of CYP3A4, NTCP, CPS1, G6PC, and the ability of urea synthesis were further increased by 6.5±0.6, 2.0±0.03, 2.3±0.2, 77±1.2 and 479±196 times, respectively. In addition, HMM significantly improved the sensitivity of HepG2 to chlorpromazine toxicity in 3D culture. Conclusion HMM can improve the hepatocyte function of HepG2 in both 2D and 3D conditions. HMM combined with 3D culture will be helpful in HepG2-based researches.

Key words: HepG2, 3-dimensional, Hepatocyte maturation medium, Hepatocytes, Hepatotoxicity