In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-associated Steatotic Liver Disease
Abstract
The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading to significant health challenges. Previous reports have studied the establishment of animal and cellular models for MASLD, highlighting differences between them. In this study, a cellular model was created by inducing fat accumulation in MASLD. HepG2 cells were stimulated with the unsaturated fatty acid oleic acid at various concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) to emulate MASLD. The model's efficacy was assessed using cell counting kit-8 assays, Oil Red O staining, and lipid content analysis. This study aimed to create a simple-to-operate cellular model for MASLD cells. Results from the cell counting kit-8 assays showed that the survival of HepG2 cells was dependent on the concentration of oleic acid, with a GI50 of 1.875 mM. Cell viability in the 0.5 mM and 1 mM groups were significantly lower than those in the control group (P < 0.05). Furthermore, Oil Red O staining and lipid content analysis examined fat deposition at varying oleic acid concentrations (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) on HepG2 cells. The lipid content of the 0.25 mM, 0.5 mM, and 1 mM groups was significantly higher than that of the control group (P < 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P < 0.05).
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To induce steatosis in HepG2, supraphysiological concentrations of FFAs (0.5-2.0 mM) are often used.
“Second, to induce steatosis, supraphysiological concentrations of FFAs (concentrations of 0.5–2.0 mM) are often used in HepG2 experiments [28,91,125,126,127,128,129,130].”
From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [128]
The range of 0.1 to 2.0 mM for the combination of FAs is the standard range for modeling MASLD in 2D HepG2 monoculture.
“The range of 0.1 to 2.0 mM for the combination of FAs is the standard range for modeling MASLD in 2D HepG2 monoculture [93,100,125,126,127,128].”
From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [128]
In HepG2 cells, treatment with OA at concentrations of 0.125-0.25 mM induces significant triglyceride accumulation without a decrease in viability, whereas concentrations ≥ 0.5 mM already exhibit signs of toxicity.
“Thus, in HepG2 cells, treatment with OA at concentrations of 0.125–0.25 mM induces significant triglyceride accumulation without a decrease in viability, whereas concentrations ≥ 0.5 mM already exhibit signs of toxicity [126,128,156].”
From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [128]
Fatty acids are conjugated with bovine serum albumin (BSA, 1-4.5%, or fat-free BSA) to ensure solubility and mimic physiological transport; alternatively, DMSO is used.
“Fatty acids are conjugated with bovine serum albumin (BSA, 1–4.5%, or fat-free BSA) to ensure solubility and mimic physiological transport. Alternatively, DMSO is used [128].”
From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [128]
In 2D monocultures, FFA concentrations of 0.5-2.0 mM are more commonly used, which are higher than physiological values; this is a widely accepted compromise that allows for steatosis to be induced in a short amount of time (24-48 h).
“In 2D monocultures, concentrations of 0.5–2.0 mM are more commonly used, which are higher than physiological values [28,125,126,127,128]. This is a widely accepted compromise that allows for steatosis to be induced in a short amount of time (24–48 h).”
From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [128]
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The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease
Kotlyarova, A., Iskrina, A., & Kotlyarov, S. (2026) · International journal of molecular sciences · doi:10.3390/ijms27083399 · Cited by 5 · see citing works
- Authors
- Bao, Q., Zhang, X., Chen, Y., Wang, T., & Siqin, B.
- Affiliations
- Inner Mongolia Medical University (CN)
- Venue
- Journal of Visualized Experiments
- Publisher
- MyJove Corporation
- Published
- 2024-07-19
- Issue
- 209
- ISSN
- 1940-087X (ISSN-L)
- OpenAlex
- W4400861252
- Cited by
- 1
- References
- 37
- Citations by year
- 2026: 1
- FWCI
- 0.31
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Visualized Experiments (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Epidemiology › Liver Disease Diagnosis and Treatment
- Health Sciences › Medicine › Hepatology › Liver physiology and pathology
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Diet, Metabolism, and Disease
- Keywords
- non-alcoholic fatty liver disease, cell model, fat deposition, oleic acid, HepG2 cells, hepatocyte lipid accumulation, triglyceride levels, cell viability, in vitro disease model
- MeSH terms
- Azo Compounds, Fatty Liver, Humans, Models, Biological, Oleic Acid, Lipid Metabolism, Hep G2 Cells
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