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 repeatedexposure 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, Hepa-RG 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.

Current Perspective: 3D Spheroid Models Utilizing Human-Based Cells for Investigating Metabolism-Dependent Drug-Induced Liver Injury

“…Conventionally, this has involved culturing primary human hepatocytes or immortalized/cancer-derived cell lines as 2D monolayers. However, the cells used in these models will often be phenotypically different from in-vivo hepatocytes (i.e., Ammonia metabolism/urea secretion is higher in spheroids (73) Gene expression is higher and/or maintained for longer for a number of phase I proteins in spheroid cultures (68,141,144) Activity for CYP450 enzymes involved in xenobiotic metabolism is higher and /or maintained for longer in spheroids (71,73,89,141) Protein expression for a number of phase I, II, and III proteins is maintained for longer in spheroids (71) CYP1A2, 2C9, 2C19, and 3A4 more inducible to known pharmacological inducers (89) Gene expression for phase II and III proteins, nuclear receptors, and hepatocyte markers is higher and/or maintained for longer in spheroids (141,144) GSTP1 gene expression lower in spheroids (141) CYP4A11 and SLC27A5 expression lost more rapidly in spheroids (71) Lower protein expression for some basolateral transporters in spheroids (71) Proteomic and metabolite profile is more stable in 3D cultures over time (71,141) Spheroid cultures have greater sensitivity to detect toxicity for known hepatotoxic compounds at toxicologically relevant concentrations (71,73,81) Spheroid cultures show greater recovery from the dedifferentiation process associated with in-vitro culture, when considering gene expression (74) cancer-derived cell lines), or rapidly dedifferentiate, becoming metabolically incompetent, when cultured in vitro (i.e., PHH). This hinders their ability to investigate metabolism-dependent toxicities and limits their application for detecting DILI-positive compounds, particularly over chronic and repeat dose time courses that are more emulative of in-vivo manifestations of DILI.…”

“…Comparable activity was seen for CYP1A2 with 2D monolayers and 3D spheroids ( 70 , 73 ), although another study reported significantly higher CYP1A activity (i.e., using EROD assay) ( 119 ). CYP2B6 activity was shown to be higher for 3D spheroids ( 70 , 73 ). HepaRG spheroids display inducible CYP450 enzymes in response to known pharmacological inducers, indicating functional nuclear receptors (i.e., PXR, CAR, and AhR) ( 70 , 88 , 104 , 119 ).…”

“…Each cell type has its own advantages and disadvantages (discussed throughout), but when they are grown in vitro, they will often be phenotypically different from their in-vivo counterparts for which you are trying to make conclusions (62)(63)(64)(65)(66). To try to overcome these phenotypic differences, a number of techniques have been used to try to recapitulate the hepatocyte's native environment in vitro, and evidence is gathering to suggest that growing cells in 3D (67)(68)(69)(70)(71)(72)(73)(74) or exposing them to shear stresses and flow (75)(76)(77) can promote a more physiologically relevant model to study DILI, based on hepatocyte function and metabolic capabilities.…”

“…However, contradictory data is seen for CYP3A4 activity, with some studies showing higher activity in 3D spheroids (70,119) and another showing comparable activity (73). Comparable activity was seen for CYP1A2 with 2D monolayers and 3D spheroids (70,73), although another study reported significantly higher CYP1A activity (i.e., using EROD assay) (119). CYP2B6 activity was shown to be higher for 3D spheroids (70,73).…”

3D-bioprinted HepaRG cultures as a model for testing long term aflatoxin B1 toxicity in vitro

“…In the present study, we used human HepaRG hepatocytes due to their metabolic competence to evaluate the appropriateness of 3D constructs for toxicity testing. Our 3D constructs exhibited long-term stability and cell viability, which is suitable for chronic toxicity testing in vitro similar to the 3D organoids and spheroids [ 33 ]. It has been shown that AFB1 treatment induces cell toxicity and loss of metabolic activity of HepaRG spheroid model ([ 34 ], [ 35 ]).…”