HepG2 Cells: Are 3D Spheroids the Future of Liver Toxicity Testing?
"Exploring how 3D cell cultures could revolutionize drug safety assessment."
For decades, the pharmaceutical industry has grappled with the challenge of accurately predicting drug-induced liver injury (DILI) during preclinical development. Traditional methods often fall short, leading to costly clinical failures and potential risks to patients. This has spurred a search for more reliable in vitro models that better mimic the complexities of the human liver.
One promising avenue of research involves the use of three-dimensional (3D) cell cultures, specifically spheroids, which are aggregates of cells that more closely resemble the structure and function of real tissues than traditional two-dimensional (2D) monolayers. Among the cell types used in these 3D cultures, HepG2 cells, a well-established human liver cell line, are frequently employed.
A recent study published in Toxicology Letters investigates the proteomic responses of HepG2 cells grown in both 2D and 3D cultures when exposed to various hepatotoxins. By comparing the protein profiles of these cells, researchers aimed to determine whether 3D spheroids offer a more physiologically relevant model for assessing liver toxicity and predicting drug safety.
HepG2 at the Center of Hepatotoxicity Screening
Drug-induced hepatotoxicity is a leading cause of drug withdrawal from the market, making high-throughput screening with in vitro liver models critical for early-stage liver toxicity testing. HepG2 is an extensively used cell line in assessing human health risks and in exploring the underlying molecular mechanisms of hepatotoxicants. The scale of this work is evident in a study that combined mass-spectroscopy metabolomics with an in vitro HepG2 liver model, treating cells with 35 test substances and generating 1114 cell supernatants and 3556 intracellular samples for metabolomic analysis. Such assays are also applied to environmental contaminants, including three typical benzotriazoles (UV-328, UV-329, UV-P), underscoring HepG2's reach beyond pharmaceuticals. Ongoing comparisons continue to examine the expression of key liver-specific genes and proteins in widely used hepatic cancer cell lines such as HepG2, C3A, SNU449, and SNU475 against primary human hepatocytes.
Monolayer Models and Their Known Shortcomings
The standard approach to liver toxicity testing has long relied on monolayer cultures of primary human hepatocytes or immortalized liver cell lines such as HepG2 and HepaRG. Immortalized cell lines and primary isolated liver cells are currently the most widely used in vitro models, but they share common shortcomings, including limited throughput, loss of viability, and decreases in liver-specific functionality and gene expression. HepaRG, for example, has emerged as a successful model because these cells can be differentiated in vitro into mature and functional hepatocyte-like cells, although a head-to-head study comparing the Sison 2017 and Gripon et al. 2002 (Biopredic) protocols found that even this system requires careful culture optimization. As Yang et al. and others note, monolayer-cultured liver cells sometimes lack physiological relevance, which has driven growing interest in three-dimensional liver models as a more physiologically accurate and higher-throughput alternative for chemical testing.
From a 1975 Tumor Biopsy to 40 Years of Research
HepG2 is an immortal human liver cancer cell line derived in 1975 from the liver tissue of a 15-year-old Caucasian male from Argentina with a well-differentiated hepatoblastoma carcinoma. Over four decades, it has become a popular hepatic cell line used in a wide range of studies, from oncogenesis to assessing the cytotoxicity of substances on the liver. A significant milestone in its toxicology use came with the development of CYP-overexpressing HepG2 cells: according to XL Biotec, these engineered lines flag compounds that only turn harmful once the liver's own enzymes metabolize them, catching risks a plain HepG2 assay would likely miss. That history explains why the line, despite its age, remains a touchstone across liver research and drug discovery.
2D vs. 3D: Unraveling the Proteomic Puzzle
The study meticulously compared the proteomes of HepG2 cells cultured as conventional monolayers and as 3D spheroids. Proteomics, the large-scale study of proteins, provides a comprehensive snapshot of cellular activity and function. The researchers used sophisticated techniques to identify and quantify thousands of proteins in both culture types, revealing significant differences in their protein expression patterns.
- Increased abundance of liver-specific proteins: 3D spheroids exhibited higher levels of alpha-fetoprotein, alpha-2-HS-glycoprotein, and serum albumin, all of which are indicative of a more mature hepatic phenotype.
- Altered cytoskeletal arrangement: Proteins involved in cell structure and organization, such as keratins and tubulins, were also more abundant in 3D spheroids, reflecting the distinct morphology and cell-cell interactions within these cultures.
- Inconsistent proteomic transitions: Despite the overall differences, the researchers noted variability in the proteomic changes across different replicates of the 3D spheroid cultures. This suggests that the process of spheroid formation and maturation can be somewhat inconsistent, leading to diverse cellular phenotypes.
A Rapidly Moving Literature
Ongoing research continues to generate new studies and reviews on HepG2 cells and their use in liver toxicity testing, so this field evolves quickly. Much of the recent work centers on improving the physiological relevance and predictive accuracy of cell-based assays, often through engineered modifications or advanced culture formats. Because the literature is moving fast, the findings summarized here should be read alongside the latest peer-reviewed publications.
Strengths, Drawbacks, and Critical Limitations
HepG2 cells are widely used in toxicology testing to evaluate the harmfulness of chemicals and environmental agents: by exposing the cells to various substances, scientists can assess their cytotoxicity and genotoxicity, providing insights into liver injury mechanisms. Yet sources that celebrate the line also catalog its drawbacks, with the review titled "HepG2 Cells: Properties, Applications, and Drawbacks" and AcademicPath's overview of the line's origins both highlighting critical limitations alongside its benefits. These constraints can limit how directly results translate to human liver responses, so findings from HepG2-based screens are often interpreted with caution. In practice, researchers commonly weigh these known weaknesses when designing toxicity studies around the cell line.
2D, Primary Cells, and Spheroids Compared
Direct, quantitative comparisons between HepG2 monolayers, primary human hepatocytes, and 3D spheroid systems are an active area of investigation, though no specific comparative figures fall within the source set used for this section. In general terms, the broader literature suggests that moving from flat 2D culture toward spheroid formats better preserves liver-cell phenotype and function. Readers should treat such comparisons as emerging rather than settled, given the variability in cell sources, culture protocols, and measured endpoints across studies.
The Future of Liver Toxicity Testing: A 3D Perspective
The study underscores the potential of 3D HepG2 cell cultures as a valuable tool for liver toxicity testing. By more closely mimicking the in vivo environment, these spheroids offer a more nuanced and physiologically relevant model for studying drug-induced liver injury.
Upgrading HepG2 Rather Than Abandoning It
Expert commentary increasingly points toward upgrading HepG2 rather than replacing it outright. One 2024 proposal describes a 3D culture system using HepG2 cells in spheroid form encapsulated in GelMA hydrogels, which reportedly preserves hepatocyte functionality and improves the accuracy of drug-induced liver toxicity assessment compared with 2D culture methods. A complementary body of work repositions the line through engineering, developing HepG2 cells that simultaneously express five P450 enzymes for screening hepatotoxicity and identifying bioactivable drugs and the potential mechanisms of toxicity involved. Upgraded HepG2 cells that recreate known inter-individual variations in hepatic CYP and conjugating activities due to genetic factors (such as polymorphisms) or environmental factors (such as induction and inhibition) have been described as a suitable model for bioactivable drug identification and hepatotoxicity risk assessment, a strategy that could eventually generate customized cells for specific subgroups.
Toward Hybrid and Validated Systems
The trajectory of HepG2-based testing points toward hybrid systems that combine 3D architecture with engineered enzyme activity. Researchers are likely to keep pushing spheroid and scaffold-based cultures toward higher throughput while refining cell lines to better mimic human drug metabolism. Regulatory acceptance of such models will depend on continued validation against real-world toxicity outcomes, a goal that remains firmly in the future as of this writing.
Scaling Up Metabolomics-Driven Toxicity Testing
At a systemic level, the push toward more predictive toxicity testing depends on generating large, standardized datasets across many compounds. One effort in this direction, reported in "Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells," applied metabolomics to an in vitro HepG2 liver model, analyzing metabolite profiles of cells treated with 35 test substances that yielded 1114 cell supernatants and 3556 intracellular samples. Datasets of this size highlight both the ambition of in vitro toxicity programs and the practical challenges of sample handling, analysis, and interpretation at scale.
Why Predictive Testing Matters for People
Behind every cell line are patients and clinicians whose care depends on safer medicines, and improved liver toxicity testing ultimately aims to prevent drug-induced liver injury in people. For patients, better in vitro models could mean fewer late-stage drug failures and more therapies reaching the market with a clearer safety profile. Regulators, sponsors, and the public all stand to benefit if laboratory findings translate more faithfully to human livers. As of this writing, realizing those benefits remains an ongoing effort.
However, the research also highlights the challenges associated with 3D cultures, particularly the variability in proteomic transitions during spheroid formation. To overcome this limitation, the authors suggest that optimizing the duration of spheroid culture may be essential to achieve reproducible proteomic transitions and establish a stable 3D spheroid phenotype.
As technology advances and our understanding of cellular behavior deepens, 3D cell cultures are poised to play an increasingly important role in preclinical drug development, ultimately leading to safer and more effective therapies.