Functional Comparison of HepaRG Cells and Primary Human Hepatocytes in Sandwich and Spheroid Culture as Repeated-Exposure Models for Hepatotoxicity

Abstract

Introduction:

Reliable and effective predictions of hepatotoxicity resulting from repeated exposure to chemicals are of critical importance for the safety assessment of chemicals; however, in vitro long-term repeated-exposure studies are difficult to conduct using conventional hepatocyte monolayer cultures due to de-differentiation and subsequent loss of hepatocyte function. Alternative in vitro culture techniques have emerged to address this challenge and have improved the longevity of hepatocyte cultures.

Materials and Methods:

Two promising cell models, HepaRG and primary human hepatocytes (PHHs), were compared in both spheroid and sandwich cultures to evaluate their potential as predictive models for repeated-exposure hepatotoxicity.

Results:

When compared to sandwich cultures, untreated cells in spheroid culture maintained higher levels of viability for a longer duration. Likewise, functional characterization revealed higher levels of xenobiotic metabolic activity (CYP1A2, CYP2B6, and CYP3A4) and urea production in spheroid than in sandwich cultures. In repeated-exposure studies (7 days), both PHH and HepaRG spheroid cultures showed increased sensitivity to a set of hepatotoxic compounds compared to their respective sandwich cultures, and the sensitivity was further elevated in an extended exposure duration (14 days).

Discussion and Conclusion:

Spheroid cultures can maintain robust hepatic functions during prolonged culture periods and predicted hepatotoxicity at toxicologically relevant levels after repeated exposure. In addition, HepaRG spheroids exhibited similar sensitivity as PHH spheroids, especially in extended exposure, suggesting that HepaRG spheroids could be a reliable surrogate for PHH spheroids and serve as a repeated-exposure model to predict hepatotoxicity.