Application of triple co-cultured cell spheroid model for exploring hepatotoxicity and metabolic pathway of AFB1

The toxicity evaluation suffers from the absence of suitable models capable of replicating in the co-cultured cell microenvironment and the function of specific tissues in vitro. Motivated by this urgent need, this study aimed to describe a novel three-dimensional (3D) liver spheroid model. The model consisted of a triple co-culture of HepG2, EA.hy 926, and LX-2. Subsequently, it was used for the toxicity evaluation of aflatoxin B1 (AFB1), and its advantages over the two-dimensional (2D) model and the mono-type cell spheroid model were assessed. This study examined the effects of AFB1 on cell viability, proliferation, mitochondria, oxidative stress, and cell membranes. The results revealed that AFB1 greatly affected 2D cell membranes and oxidative stress levels (0.01 μg/mL; 24 h), and could also significantly affect 2D cell viability, proliferation, and mitochondria levels (1 μg/mL; 24 h). On the contrary, 3D cells were less susceptible to AFB1. Combined with the analysis of gene expression, both metabolic activation (cytochrome P450; CYP450) and detoxification efficiency (drug-metabolizing enzymes) were found to be higher in 3D cells than in 2D cells. Moreover, 3D cells in triple co-culture outperformed mono-type cell spheroids. Therefore, the advanced 3D co-cultured spheroid model constructed in this study allowed us to more realistically simulate the microenvironment in vitro, and was a valuable and precise model to study mycotoxins.