3D liver cell spheroids in a futuristic lab.

HepG2 Cells: Are 3D Spheroids the Future of Liver Toxicity Testing?

Jordan Keane in Health & Wellness December 2025 • 4 min read.

"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.

2D vs. 3D: Unraveling the Proteomic Puzzle

3D liver cell spheroids in a futuristic lab.

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.

Specifically, the research highlighted that approximately 34% of the proteins present differed between monolayer and 3D spheroid cultures. This substantial variation suggests that the cellular environment profoundly influences the behavior and characteristics of HepG2 cells. The transition from an exponential growth phase in monolayers to an equilibrium phase in spheroids appears to drive these proteomic shifts.

Key differences observed include:

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.

Interestingly, when the HepG2 cells were exposed to known hepatotoxins like ketoconazole and troglitazone, the researchers did not observe specific subsets of proteins consistently linked to the drugs' mechanisms of toxicity. This finding suggests that while 3D spheroids may better mimic certain aspects of liver cell physiology, further optimization is needed to fully capture the complex responses to toxic compounds.

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.

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.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.1016/j.toxlet.2018.10.030, Alternate LINK

Title: Proteomic Responses Of Hepg2 Cell Monolayers And 3D Spheroids To Selected Hepatotoxins

Subject: Toxicology

Journal: Toxicology Letters

Publisher: Elsevier BV

Authors: Tracey Hurrell, Kathryn S. Lilley, Allan Duncan Cromarty

Published: 2019-01-01

Everything You Need To Know

What exactly are HepG2 cells and why are they used in liver toxicity testing?

HepG2 cells are a well-established human liver cell line that scientists use in both two-dimensional (2D) and three-dimensional (3D) cell cultures to study liver function and toxicity. They are particularly valuable because they retain many characteristics of normal liver cells, making them useful for in vitro studies. Traditional 2D cultures grow as a single layer, while 3D cultures form spheroids, which more closely mimic the structure and function of real liver tissue. This makes HepG2 cells a cornerstone of research aimed at improving drug safety assessment and understanding liver diseases. Further investigation could explore genetic modification of HepG2 cells to better mimic specific liver conditions.

Could you explain what 3D spheroids are and how they improve liver toxicity testing with HepG2 cells?

3D spheroids are aggregates of cells that grow in three dimensions, forming a more tissue-like structure compared to traditional two-dimensional (2D) cell cultures. In the context of liver toxicity testing, HepG2 cell spheroids are used to mimic the in vivo environment of the liver more accurately. Compared to 2D monolayers, 3D spheroids exhibit enhanced cell-cell interactions, altered protein expression, and a more mature hepatic phenotype, leading to a more physiologically relevant model for studying drug-induced liver injury (DILI). Additional research into scaffold materials and co-culture systems could further enhance the utility of 3D spheroids.

What is proteomics, and how does it help us understand the differences between 2D and 3D HepG2 cell cultures?

Proteomics is the large-scale study of proteins, providing a comprehensive snapshot of cellular activity and function. In the study of HepG2 cells, proteomics is used to compare the protein profiles of cells grown in 2D and 3D cultures. Researchers can identify and quantify thousands of proteins, revealing significant differences in protein expression patterns. These differences help determine whether 3D spheroids offer a more physiologically relevant model for assessing liver toxicity and predicting drug safety. Future studies might integrate proteomics with genomics and metabolomics for a more holistic understanding.

What key differences were found in the protein composition of HepG2 cells grown in 2D versus 3D cultures, according to the Toxicology Letters study?

The Toxicology Letters study revealed that approximately 34% of the proteins present differed between monolayer (2D) and 3D spheroid cultures of HepG2 cells. Key differences included increased abundance of liver-specific proteins such as alpha-fetoprotein, alpha-2-HS-glycoprotein, and serum albumin in 3D spheroids, indicative of a more mature hepatic phenotype. Additionally, proteins involved in cell structure and organization, like keratins and tubulins, were more abundant in 3D spheroids, reflecting the distinct morphology and cell-cell interactions within these cultures. However, the study also noted variability in proteomic changes across different replicates of the 3D spheroid cultures, suggesting some inconsistency in spheroid formation and maturation. These observations highlight the importance of considering the cellular environment when assessing liver toxicity and suggest that 3D spheroids more closely mimic in vivo conditions compared to 2D monolayers.

What are the limitations of using 3D HepG2 cell spheroids for liver toxicity testing, as indicated by the Toxicology Letters study?

While 3D HepG2 cell spheroids show promise for improving liver toxicity testing, the Toxicology Letters study found that when exposed to known hepatotoxins like ketoconazole and troglitazone, specific subsets of proteins consistently linked to the drugs' mechanisms of toxicity were not observed. This suggests that while 3D spheroids may better mimic certain aspects of liver cell physiology, further optimization is needed to fully capture the complex responses to toxic compounds. This could involve refining culture conditions, incorporating other cell types, or using more advanced analytical techniques to better understand the molecular mechanisms of drug-induced liver injury (DILI). The study implies that while 3D models offer advantages, they are not a complete solution and require further development to accurately predict drug safety.