<em>In Vitro</em> Modeling of Fat Deposition in Metabolic Dysfunction-associated Steatotic Liver Disease

Qinglan Bao; Xiaoxiao Zhang; Yingsong Chen; Tegexibaiyin Wang; Bateer Siqin

doi:10.3791/66810

JoVE Journal > Biology

Biology

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-associated Steatotic Liver Disease

Published: July 19, 2024

doi:

10.3791/66810

Qinglan Bao, Xiaoxiao Zhang, Yingsong Chen*^1,3,4, Tegexibaiyin Wang*⁵, Bateer Siqin*⁶

¹Inner Mongolia Medical University, ²Xilin Gol League Mongolian Medical Hospital, ³National and local Joint Mongolian Medicine R & D Engineering Center, ⁴Inner Mongolia Minzu University, ⁵Affiliated Hospital of Inner Mongolia Minzu University, ⁶Traditional Chinese & Mongolian Medical Research Institute of Inner Mongolia

Summary

This article describes the use of oleic acid-induced HepG2 cells as a model for 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).

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a range of conditions, including simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma¹^,²^,³^,⁴^,⁵^,⁶, all attributed to factors other than alcohol consuption⁷. MASLD is the most prevalent liver disease caused by metabolic liver injury, affecting nearly one-quarter of the global population⁸^,⁹^,¹⁰^,¹¹^,¹². While the precise pathogenesis of MASLD has not yet been elucidated, various theories attempt to explain its development. One prevailing notion suggests a departure from the classic "two-hit" theory towards a "multiple-hit" model¹. Central to these hypotheses is the role of insulin resistance, which is believed to be pivotal in MASLD pathogenesis¹³. Research indicates that insulin resistance in hepatocytes leads to increased levels of free fatty acids, subsequently forming triglycerides stored within the liver¹⁴^,¹⁵.

Researchers have used both in vivo and in vitro models to simulate fat deposition in MASLD; yet fully replicating its pathomechanism remains challenging. Despite this limitation, these models have been instrumental in studying potential therapeutic targets for MASLD. However, the development of a stable model of MASLD is crucial. While animal models are effective, they are time-consuming and expensive, thus highlighting the growing interest in in vitro cellular models. These models often use single or multiple free fatty acids such as oleic acid (OA) and palmitic acid to recreate diet-induced MASLD. Among these, the human hepatoblastoma cell line HepG2 is often used to establish in vitro cellular models of MASLD.

OA induction stimulates HepG2 cells to replicate fatty deposition akin to MASLD, a method with a well-established history. The aim of this study was to demonstrate the viability, Oil Red O (ORO) staining, lipid content, and triglyceride (TG) level of HepG2 cells treated with 0.25 mM OA. The objective of this experiment was to provide further evidence for the development of MAFLD modeling studies.

Protocol

NOTE: See the Table of Materials for details related to all materials, instruments, and reagents used in this protocol.

1. Cell culture

Culture HepG2 cells in culture flasks containing Dulbecco's Modified Eagle Medium (DMEM) (containing 10% fetal bovine serum [FBS], 100 units/mL penicillin, and 100 µg/mL streptomycin). Maintain the culture flasks at 37 °C in a 5% CO₂ incubator.

2. Effect of oleic acid on cell viability as measured by cell counting kit-8

Dissolve a specific volume of OA in dimethyl sulfoxide (DMSO) to achieve a concentration of 200 mM. Store the solution at -20 °C for future use.
Seed HepG2 cells in a 96-well plate at a cell density of 6 × 10³ cells per well. Add 100 µL of DMEM to each well. Incubate and culture the cells at 37 °C in a 5% CO₂ incubator for 24 h. Divide the HepG2 cells into two groups:
1. Control group: add cell culture medium.
2. OA group: add OA to the cell culture medium to achieve final concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM.
After 24 h of initial incubation, discard the supernatant from each well and add OA according to the specified grouping, adding 100 µL per well. For the control group, add 100 µL of cell culture medium to each well. Continue to incubate the cells for another 24 h.
NOTE: Ensure each group has six replicate wells. To prevent evaporation, add 100 µL of phosphate-buffered saline (PBS) to the outer ring of wells in the 96-well plate.
After 24 h of incubation, add 10 µL of cell counting kit-8 (CCK-8) to each well, mix gently, and incubate for 2 h in the dark. Remove the 96-well plate from the incubator, place it in the microplate reader, and measure the absorbance value at 450 nm (A₄₅₀). The GI₅₀values were counted according to the OD.

3. Oil Red O staining to observe intracellular lipid droplet formation

Seed HepG2 cells in 6-well cell culture plates at a density of 5 × 10⁵ cells per well and culture the plates in a constant-temperature incubator for 24 h. Refer to Figure 1 for a visual representation of the described steps.
After 24 h of cell culture, add 2 mL of cell culture medium containing OA to each well, achieving final concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM. After an additional 24 h, remove the cell culture medium from each well and wash twice with PBS. Add 1 mL of ORO fixative to each well and incubate for 30 min.
Prepare the ORO staining solution by mixing staining solution A with staining solution B at a ratio of 3:2. Allow the mixture to stand at room temperature for 10 min, then filter it once through a 0.45 µm filter. Store the filtered solution in a centrifuge tube protected from light until use.
Discard the fixative and wash twice with distilled water. Add 1 mL of 60% isopropanol to each well and incubate for 30 s. Discard 60% of the isopropanol solution and add 1 mL of freshly prepared ORO stain solution to each well before incubating for 20 min. Discard the ORO staining solution, add 1 mL of 60% isopropanol to each well, and incubate for 30 s. Wash 5x with water to remove excess dye.
Cover the cells with distilled water and observe under a microscope. Once the images are collected, discard the liquid in the plate and allow it to dry. Then, add 2 mL of isopropanol to each well and shake the plate on an orbital shaker for 10 min. Transfer the liquid to a new 96-well plate, with 16 wells in each group, adding 100 µL per well. Calculate the lipid content by measuring the optical density (OD) of each well using a microplate reader at 510 nm (A₅₁₀).

4. Effects of different concentrations of oleic acid on total triglyceride in HepG2 cell supernatant

Equilibrate the kit at room temperature for 20 min and prepare the required plates for the experiment.
Collect the cell supernatant and centrifuge at 1,570 × g for 10 min. Set standard wells and testing sample wells. Add 50 µL of standard ([S0 → S5] concentration followed by: 0, 0.5, 1, 2, 4, 8 mmol/L) to standard wells. In addition to the blank and standard wells, add 10 µL of different samples to the sample wells, followed by adding 40 µL of sample diluent to each well. Add 100 µL of detection antibody-horseradish peroxidase to each well, seal with a plate membrane, and incubate at 37 °C for 1 h in a constant temperature oven.
Discard the supernatant, blot dry on dust-free paper, and wash each well with 1x washing solution. Leave to stand at room temperature for 1 min. Repeat the washing process 5x.
Add 50 µL of substrate A and 50 µL of substrate B to each well. Mix gently and incubate for 15 min at 37 °C. Add 50 µL of termination solution to each well and measure the OD value of each well at 450 nm (A₄₅₀) within 15 min.
Plot the concentration of the standard along the x-axis and the corresponding absorbance (OD) value along the y-axis to perform linear regression and derive the curve equation to calculate the concentration value of each sample.

5. Statistical analysis

Determine significant differences in quantitative data.
Calculate the mean ± standard deviation (SD) and graphically represent the data. Consider P < 0.05 to be statistically significant.

Representative Results

Effect of oleic acid on cell viability
HepG2 cells were exposed to varying concentrations of OA (0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM), resulting in a decrease in cell survival rates at 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM compared to 0 mM. Statistical significance was observed at 0.5 mM (P < 0.05) and 1 mM (P < 0.05) when compared to 0 mM. The results of OA's impact on cell viability, as assessed by the CCK-8 kit, are shown in Figure 2. The GI₅₀ value for OA-treated HepG2 cells was calculated as 1.875 mM.

Oil Red O staining to observe intracellular lipid droplet formation
ORO staining is a commonly used method for lipid visualization¹⁶^,¹⁷. Consequently, the lipid droplet formation in HepG2 cells incubated with OA for 24 h was observed under a microscope following ORO staining. As shown in Figure 3 and Figure 4, the intensity of red staining in the OA-treated cells was higher than in the untreated cells. Red lipid droplets were present in the plasma of HepG2 cells after OA treatment, with the quantities of lipid droplets and lipids increasing with rising OA concentrations (Figure 5).

Effects of different concentrations of oleic acid on total triglyceride in HepG2 cell supernatant
The experimental findings, as illustrated in Figure 6, revealed that compared to the control group, treatment of HepG2 cells with varying concentrations of OA (0.125 mM, 0.25 mM, 0.5 mM, 1 mM) resulted in a significant increase in the TG content of the cell supernatant at OA group (P < 0.05).

Figure 1: Flow chart for Oil Red O staining. Abbreviations: OA = oleic acid; OD = optical density. Please click here to view a larger version of this figure.

Figure 2: Effect of oleic acid on cell viability, assessed using a cell counting kit-8 assay. Each bar represents the mean ± SD (n = 6). *P < 0.05. Abbreviation: OA = oleic acid. Please click here to view a larger version of this figure.

Figure 3: Oil Red O staining images of multiple time points (3, 6, 9, and 12 o'clock directions). Lipid droplet formation was detected using an inverted microscope for cell culture at 40x magnification. Scale bar = 500 µm. Please click here to view a larger version of this figure.

Figure 4: Effect of oleic acid on lipid droplets in the cells, assessed through Oil Red O staining. Lipid droplet formation was detected using an inverted microscope for cell culture at 40x magnification. Scale bar = 250 µm. Please click here to view a larger version of this figure.

Figure 5: Effect of oleic acid on lipid content in the cells, assessed by the value of optical density. Relative fold changes are presented in the values of optical density in different groups. Each column represents the mean ± SD (n = 16). *P < 0.05. Please click here to view a larger version of this figure.

Figure 6: Effects of various concentrations of oleic acid on total triglyceride levels in HepG2 cell supernatant. Relative fold changes are presented in the value of triglyceride levels in different groups. Each column represents the mean ± SD (n = 3). *P < 0.05. Please click here to view a larger version of this figure.

Discussion

MASLD is a clinicopathological syndrome characterized by excessive intracellular fat deposition in hepatocytes due to factors beyond alcohol and other established liver-damaging agents¹⁸. MASLD is intricately linked to acquired metabolic stress liver injury, notably associated with insulin resistance and genetic susceptibility. To effectively study and screen drugs for MASLD, it is crucial to select an appropriate experimental model. Establishing a cell model is particularly vital in MASLD research, facilitating a deeper understanding of pathological mechanisms and the evaluation of novel drug effects.

This protocol describes an in vitro model of MASLD established through OA-induced HepG2 cells. ORO staining and lipid content analysis serve as critical methods for creating this model. In addition, assessing cell viability and total TG content in cell supernatants is essential for evaluating the appropriate OA concentration. The diagnostic criterion for MASLD involves the presence of over 5% TG stored in liver cells¹⁹^,²⁰. MASLD cell models primarily rely on HepG2 cells²¹^,²²^,²³^,²⁴^,²⁵^,²⁶^,²⁷^,²⁸, known for their successful utilization in constructing fatty liver degeneration models in various domestic and international studies. Despite originating from hepatoma cells, HepG2 cells share many phenotypic characteristics with hepatocytes and are easily cultivated and propagated²⁹^,³⁰. Tests such as the CCK-8 kit, ORO staining, lipid content, and total TG measurements offer simple, objective, and reasonable approaches to assess cell viability, fat accumulation, and lipid content in the study subjects. In this context, OA aligns with the pathogenesis of MASLD³¹, where excess energy is stored in the form of lipids when energy intake exceeds consumption.

Researchers have utilized OA²³^,²⁸^,³¹^,³²^,³³, palmitic acid³⁴^,³⁵, and combinations of oleic/palmitic acid²⁶^,³⁶ to replicate the MASLD model. Both palmitic acid and OA contribute to liver fatty degeneration and the accumulation of TG. Palmitic acid, the most abundant saturated fatty acid in the human body, exhibits significant lipotoxicity, leading to lipid accumulation in hepatocytes, increased intracellular reactive oxygen radicals, and cell death or necrosis³⁷. Conversely, OA leads to more pronounced intracellular TG deposition (Figure 6) and less cellular modulation³⁸. Thus, researchers often utilize OA-induced HepG2 cells to establish in vitro MASLD models. As described in protocol step 2.2.2, it is crucial to heat the OA solution when adding it. In this study, ORO staining and lipid content analysis were based on the lipid deposition of HepG2 cells, effectively supporting the exploration of in vitro models of MASLD with complex mechanisms and reducing the time and cost required for research.

ORO readily binds to TG in a droplet shape, producing orange to red staining indicative of fat, with color intensity depending on lipid concentration. However, the ORO staining solution is unstable and prone to precipitation, making it unsuitable for preparation in advance. Therefore, the ORO staining solution was prepared after protocol step 3.2, with precautions taken to avoid light exposure during preparation and incubation (protocol steps 3.2, 3.3). In protocol step 3.5, selecting images at multiple time points (3, 6, 9, and 12 o'clock directions) represents a key modification of the experiment that is essential for improving the accuracy of the results. Although elevated OA concentrations affect cell survival, thus representing a limitation of this method, it has been demonstrated that an appropriate OA concentration ensures cell viability.

This study employed a comprehensive approach, using a CCK-8 kit, ORO staining, lipid content analysis, and total TG measurement to establish and validate the model of MASLD. The CCK-8 kit was used to assess the effect of drugs on cell viability, serving as a crucial validation step. In the future, this model holds promise for drug screening and mechanism research aimed at understanding MASLD pathogenesis and identifying potential therapeutic targets. To further ensure the quality of reliability of validation, the expression levels of target proteins implicated in the model will be analyzed using western blotting and quantitative polymerase chain reaction analyses.

Disclosures

The authors have nothing to disclose.

Acknowledgements

The current study was granted by "Study on the key issues of curative effect of Koumiss on regional diseases of Mongolian medicine" in 2018 Supported Project of the science and technology program of the Department of Science and Technology of Inner Mongolia Autonomous Region.

Materials

0.22 µm filter	Millex
0.25% Trypsin-EDTA (1x) Trypsin-EDTA	Gibco	25200-056
0.45 µm filter	Millex
2 mL Crygenic Vials	CORNING	430659
25 cm² Cell Culture Flask	CORNING	430639
6-well cell culture plate	CORNING	3516
96-well cell culture plate	CORNING	3599
Blood Count Plate	Shanghai Jing Jing Biochemical Reagent & Instrument Co.	02270113
Cell Counting Kit-8 assays	Beijing Solarbio Science & Technology Co.,Ltd.	CA1210-1000T
CO₂ incubator	NUAIRE	NU-5710E
DMSO Dimethyl sulfoxide	Beijing Solarbio Science & Technology Co.,Ltd.	D8371
Dulbecco's Modified Eagle Medium	Gibco	8122691
Enzyme Labeling Equipment	Tecan	Spark
Fetal Bovine Serum, Qualified	Gibco	10099141
HepG2 cells line	Beijing North China Chuanglian Biotechnology Research Institute (BNCC)	221031
Human Triglyceride (TG) ELISA instruction	Nanjing Jiacheng Bioengineering Institute	20170301
Inverted Microscope for Cell Culture	Leica	DMi1
Isopropanol	Tianjin Zhiyuan Chemical Reagent Co.	2021030141
Oil Red Stain Kit, For Cultured Cells	Beijing Solarbio Science & Technology Co.,Ltd.	G1262
Oleic acid	Sangon Biotech (Shanghai) Co., Ltd.	A502071
Penicillin Streptomycin	Gibco	15140122
SPSS 24.0		Statistics software

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Bao, Q., Zhang, X., Chen, Y., Wang, T., Siqin, B. In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-associated Steatotic Liver Disease. J. Vis. Exp. (209), e66810, doi:10.3791/66810 (2024).