A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Summary

This protocol is intended to be a tool to study steatosis and the molecular, biochemical, cellular changes produced by the over exposure of hepatocytes to lipids in vitro.

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

Introduction

Fatty liver associated with metabolic dysfunction is highly prevalent worldwide^1,2; it is estimated that up to 25% of the population is affected³. This disease previously known as non-alcoholic fatty liver disease (NAFLD), has updated its nomenclature to metabolic dysfunction associated fatty liver disease (MAFLD) to accurately reflect the pathogenesis related with obesity, insulin resistance, diabetes type 2, and dyslipidemia, as well as the possible managements of the disease^3,4.

Regardless of the n....

Protocol

1. Standard and conditioned medium preparation

1. To prepare standard RPMI 1640, supplement RPMI 1640 culture medium with 10% (v/v) of fetal bovine serum (FBS, previously heat inactivated) and 1% (v/v) of Penicillin-Streptomycin solution. Store the medium at 4 °C.Sterilize by using 0.22 µm filters.
2. To prepare palmitate stock solution, prepare a 50 mM solution of palmitate in the standard RPMI 1640 previously supplemented with 1% of bovine serum albumin (lipid free). A volume of 5-10 mL of thi.......

Discussion

This protocol is intended to provide a strategy to study steatosis in vitro. Cell culture is a powerful tool to study cellular, molecular, biochemical, and toxicological aspects of the cells exposed to different conditions. With this approach, steatosis can be visualized not only as a stage of the complex disease that is MAFLD, but also as the hepatocyte overexposure to lipids and the possible outcomes resulting from such exposure. Therefore, its application is not restricted to the physiopathology of MAFLD, but.......

Materials

Name	Company	Catalog Number	Comments
Biosafety cabinet	ESCO Airstream	AC2-452+C2:C26	Class II Type A2 Biological Safety Cabinet
Bottle top filter	Corning, US	430513	Non-pyrogenic, polystyrene, sterile. 1 filter/Bag. 0.22 μm, 500 mL.
Bovine serum albimun (BSA)	Gold Biotechnology, US	A-421-10	BSA Fatty Acid Free for cell culture
Culture media RPMI 1640	ThermoFisher-Gibco, US	31800-022	-
Fetal Bovine Serum (FBS)	ThermoFisher-Gibco, US	A4766801	-
Hemocytometer	Marienfeld, DE	640010	-
HepG2 cell line	ATCC, US	HB-8065	Hepatocellular carcinoma human cells.
Humidified incubator	Thermo Electronic Corporation,US	Model: 3110	Temperature (37 °C ± 1 °C), humidity (90% ± 5%) , CO2 (5% ± 1%)
Inverted microscope Eclipse	NIKON, JPN	Model: TE2000-S	-
Isopropanol	Sigma-Aldrich, US	I9030-4L	-
Oil Red O Kit	Abcam, US	ab150678	Kit for histological visualization of neutral fat.
Paraformaldehyde	Sigma-Aldrich, US	P6148-500G	-
Penicillin/streptomycin	ThermoFisher-Gibco, US	15140-122	Antibiotics 10,000 U/mL Penicillin, 10,000 μg/mL Streptomycin
pH meter	Beckman, US	Model: 360 PH/Temp/MV Meter	-
Phosphate buffered saline	ThermoFisher-Gibco, US	10010-023	-
Serological Pipettes	Sarstedt, AUS	86.1253.001	Non-pyrogenic, sterile, 5 mL
Serological Pipettes	Sarstedt, AUS	86.1254.001	Non-pyrogenic, sterile, 10 mL
Sodium bicarbonate	Sigma-Aldrich, US	S5761-1KG	Preparation of culture media
Sodium oleate	Santa Cruz Biotechnology, US	sc-215879A	-
Sodium palmitate	Santa Cruz Biotechnology, US	sc-215881	-
Syring filter	Corning, US	431219	Non-pyrogenic, sterile, 28 mm, 0.2 μm.
Trypan Blue	Sigma-Aldrich, US	T6146-25G	-
Trypsin 0.05% /EDTA 0.53 mM	Corning, US	25-052-Cl	-
24 well cell culture cluster	Corning, US	3524	Flat bottom with lid. Tissue culture treated. Nonpyrogenic, polystyrene, sterile. 1/Pack.
96 well cell culture cluster	Corning, US	3599	Flat bottom with lid. Tissue culture treated. Nonpyrogenic, polystyrene, sterile. 1/Pack.