Human serum-supplemented cell culture conditions improve xenobiotic metabolism in hepatoma cell lines HepG2 and Huh7.

Hepatoma cell lines are established in vitro models for xenobiotic metabolism, yet their enzymatic capacity is typically lower than that of primary human hepatocytes. Although widely used as standard culture supplement, fetal bovine serum (FBS) poses ethical concerns and may not provide the optimal nutritional environment for human-specific metabolic functions. This study compared the metabolic activity of the hepatoma cell lines HepG2 and Huh7 cultured in medium supplemented with either human serum (HS) or FBS. Metabolic performance was tested using a dual cytochrome P450 (CYP) substrate cocktail in HepG2, with results compared to literature data for Huh7. For comprehensive evaluation of metabolic capacity, both cell lines were exposed to six synthetic cathinones: 4MeO-αP-VP (4'-methoxy-α-pyrrolidinovalerophenone), 4MeO-αP-BP (4'-methoxy-α-pyrrolidinobutyrophenone), 4MeO-NE-BP (4'-methoxy-N-ethylbutyrophenone), 4MeS-αMor-PrP (4'-methylthio-2-morpholinopropiophenone), 4MeS-αP-BP (4'-methylthio-α-pyrrolidinobutyrophenone), and 4MeS-NE-BP (4'-methylthio-N-ethylbutyrophenone) under both supplement conditions. HepG2 showed unchanged CYP2D6 activity and increased CYP3A4 activity following HS incubation. HS further led to a general increase in metabolite abundance across both cell lines. Particularly the N-oxide formation consistently increased for every compound tested and abundance of most hydroxy and oxo metabolites was higher, most likely due to elevated CYP3A4 activity. Compared to FBS, metabolite formation in the HS group increased significantly, up to fourfold in 12 of 13 reactions for HepG2, and up to fivefold in 8 of 11 reactions for Huh7. Although HepG2 is a more common hepatoma cell line in metabolic research, Huh7 demonstrated higher normalized metabolite formation for most reactions, suggesting it as preferred or highly complementary model. Supplementing culture media with HS significantly enhanced the metabolic competence of the tested hepatoma cell lines. HS can be recommended as a cost-efficient, ethically sound, and physiologically relevant alternative for improving in vitro metabolism assays.