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Updated: Feb 16, 2026
08:25
Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure
Published on: June 5, 2020
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A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies
Ume-Kulsoom Shah1, Jefferson de Oliveira Mallia1, Neenu Singh2
1In vitro Toxicology Group, Institute of Life Science, College of Medicine, Swansea University,Singleton Park, Swansea, Wales, SA2 8PP, UK.
Summary
This study developed a 3D liver model using HepG2 cells in hanging-drop spheroids for improved genotoxicity testing. The 3D model demonstrated enhanced sensitivity to genotoxic agents compared to 2D cultures, offering a more accurate prediction of human toxicology.
Area of Science:
- Toxicology and Pharmacology
- Biotechnology and Bioengineering
- Cell Biology
Background:
- The liver is crucial for chemical metabolism, making it a key tissue for toxicity testing.
- Current toxicity testing methods (animal and 2D cell models) have limitations in mimicking in vivo liver cell behavior, affecting predictive accuracy for human toxicology.
- Three-dimensional (3D) in vitro liver models present a promising alternative for more relevant toxicity assessments.
Purpose of the Study:
- To develop and characterize a 3D liver model using HepG2 cells cultured in hanging-drop spheroids.
- To evaluate the suitability of this 3D model for genotoxicity testing, specifically using the cytokinesis-blocked micronucleus assay.
- To compare the performance of the 3D hanging-drop spheroid model with traditional 2D cultures for genotoxicity assessment.
Main Methods:
- Development of HepG2 spheroids using the hanging-drop technique with an initial seeding density of 5000 cells/20 µL drop.
- Adaptation of the cytokinesis-blocked micronucleus assay for micronucleus detection in 3D spheroids.
- Evaluation of dosing in hanging vs. non-hanging drops and comparison of automated (Metafer) vs. manual scoring for micronucleus detection.
Main Results:
- HepG2 hanging-drop spheroids maintained >75% cell viability at day 4, with optimal albumin secretion and CYP1A1/CYP1A2 gene expression in the 3D environment.
- Exposure to genotoxicants (B[a]P and PhIP) induced significant increases in CYP1A1 and CYP1A2 enzyme activity in 3D hanging spheroids.
- Micronucleus (MN) induction in response to B[a]P and PhIP was greater in 3D hanging spheroids than in 2D cultures, indicating higher sensitivity to genotoxic agents.
Conclusions:
- HepG2 hanging-drop spheroids represent a novel and improved in vitro system for genotoxicity studies.
- The 3D model exhibits enhanced structural and physiological properties, leading to more sensitive detection of genotoxic effects compared to 2D cultures.
- This 3D liver model offers a more accurate platform for predicting human toxicology and chemical safety assessment.
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