JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

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

Published: May 18th, 2021

DOI:

10.3791/62543

1Laboratorio de Hígado, Páncreas y Motilidad, Unidad de Medicina Experimental, Facultad de Medicina-UNAM, Hospital General de México “Dr. Eduardo Liceaga”

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.

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.

Fatty liver associated with metabolic dysfunction is highly prevalent worldwide1,2; 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 disease3,4.

Regardless of the n....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Hepatocytes cultured in the steatogenic medium display growth all over the surface of the well; however, fatty hepatocytes show lower growth rate compared with cells cultured in control medium. The proposed ratio and concentration of OA and PA, guarantee cell survival during culture. Seeding 1 x 105 cells per well in 24-well plates provides optimum confluence as shown in Figure 1.

Viability in cultured cells was lower in the steatogenic groups, Mild and.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was funded by Consejo Nacional de Ciencia y Tecnología (Conacyt, CB-221137). Adriana Campos is a doctoral student at Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, and was supported by Conacyt (CVU: 1002502).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

  1. Younossi, Z. M. Non-alcoholic fatty liver disease - a global public health perspective. Journal of Hepatology. 70 (3), 531-544 (2019).
  2. Younossi, Z., et al. Global perspectives on nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology. 69 (6), 2672-2682 (2019).
  3. Eslam, M., et al. A new definition for metabolic dysfunction-associated fatty liver disease: An international expert consensus statement. Journal of Hepatology. 73 (1), 202-209 (2020).
  4. Eslam, M., Sanyal, A. J., George, J. MAFLD: A consensus-driven proposed nomenclature for metabolic associated fatty liver disease. Gastroenterology. 158 (7), 1999-2014 (2020).
  5. Chalasani, N., et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American association for the study of liver diseases. Hepatology. 67 (1), 328-357 (2018).
  6. Calzadilla Bertot, L., Adams, L. A. The natural course of non-alcoholic fatty liver disease. International Journal of Molecular Science. 17 (5), 774 (2016).
  7. Reccia, I., et al. Non-alcoholic fatty liver disease: A sign of systemic disease. Metabolism. 72, 94-108 (2017).
  8. Tomita, K., et al. Free cholesterol accumulation in hepatic stellate cells: Mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice. Hepatology. 59 (1), 154-169 (2014).
  9. Byrne, C. D., Targher, G. Nafld: A multisystem disease. Journal of Hepatology. 62, 47-64 (2015).
  10. Ipsen, D. H., Lykkesfeldt, J., Tveden-Nyborg, P. Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease. Cellular and Molecular Life Sciences. 75 (18), 3313-3327 (2018).
  11. Diehl, A. M., Day, C. Cause, pathogenesis, and treatment of nonalcoholic steatohepatitis. New England Journal of Medicine. 377 (21), 2063-2072 (2017).
  12. Zeng, X., et al. Oleic acid ameliorates palmitic acid induced hepatocellular lipotoxicity by inhibition of ER stress and pyroptosis. Nutrition and Metabolism. 17, 11 (2020).
  13. Ore, A., Akinloye, O. A. Oxidative stress and antioxidant biomarkers in clinical and experimental models of non-alcoholic fatty liver disease. Medicina (Kaunas). 55 (2), 26 (2019).
  14. Paradies, G., Paradies, V., Ruggiero, F. M., Petrosillo, G. Oxidative stress, cardiolipin and mitochondrial dysfunction in nonalcoholic fatty liver disease. World Journal of Gastroenterology. 20 (39), 14205-14218 (2014).
  15. Aravinthan, A., et al. Hepatocyte senescence predicts progression in non-alcohol-related fatty liver disease. Journal of Hepatology. 58 (3), 549-556 (2013).
  16. Gomez, M. J., et al. A human hepatocellular in vitro model to investigate steatosis. Chemico Biological Interactions. 165 (2), 106-116 (2007).
  17. Wu, W. K. K., Zhang, L., Chan, M. T. V. Autophagy, NAFLD and NAFLD-related HCC. Advances in Experimental Medicine and Biology. 1061, 127-138 (2018).
  18. Levy, G., Cohen, M., Nahmias, Y. In vitro cell culture models of hepatic steatosis. Methods in Molecular Biology. 1250, 377-390 (2015).
  19. Kanuri, G., Bergheim, I. In vitro and in vivo models of non-alcoholic fatty liver disease (NAFLD). International Journal of Molecular Science. 14 (6), 11963-11980 (2013).
  20. Ricchi, M., et al. Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. Journal of Gastroenterology and Hepatology. 24 (5), 830-840 (2009).
  21. Lee, Y., et al. Serial biomarkers of de novo lipogenesis fatty acids and incident heart failure in older adults: The cardiovascular health study. Journal of the American Heart Association. 9 (4), 014119 (2020).
  22. Alnahdi, A., John, A., Raza, H. Augmentation of glucotoxicity, oxidative stress, apoptosis and mitochondrial dysfunction in Hepg2 cells by palmitic acid. Nutrients. 11 (9), 1979 (2019).
  23. Chen, X., et al. Oleic acid protects saturated fatty acid mediated lipotoxicity in hepatocytes and rat of non-alcoholic steatohepatitis. Life Sciences. 203, 291-304 (2018).
  24. Xing, J. H., et al. NLRP3 inflammasome mediate palmitate-induced endothelial dysfunction. Life Sciences. 239, 116882 (2019).
  25. Yan, H., et al. Insulin-like Growth Factor Binding Protein 7 accelerates hepatic steatosis and insulin resistance in non-alcoholic fatty liver disease. Clinical and Experimental Pharmacology and Physiology. 46 (12), 1101-1110 (2019).
  26. Wang, J., Hu, R., Yin, C., Xiao, Y. Tanshinone IIA reduces palmitate-induced apoptosis via inhibition of endoplasmic reticulum stress in Hepg2 liver cells. Fundamental and Clinical Pharmacology. 34 (2), 249-262 (2020).
  27. Xiao, Z., Chu, Y., Qin, W. IGFBP5 modulates lipid metabolism and insulin sensitivity through activating ampk pathway in non-alcoholic fatty liver disease. Life Sciences. 256, 117997 (2020).
  28. Avila, G., et al. In vitro effects of conjugated linoleic acid (CLA) on inflammatory functions of bovine monocytes. Journal of Dairy Science. 103 (9), 8554-8563 (2020).
  29. Malhi, H., Bronk, S. F., Werneburg, N. W., Gores, G. J. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. The Journal of Biological Chemistry. 281 (17), 12093-12101 (2006).
  30. Oh, J. M., et al. Effects of palmitic acid on TNF-alpha-induced cytotoxicity in SK-Hep-1 cells. Toxicology In Vitro: An International Journal Published in Association with BIBRA. 26 (6), 783-790 (2012).
  31. Stellavato, A., et al. In vitro assessment of nutraceutical compounds and novel nutraceutical formulations in a liver-steatosis-based model. Lipids in Health and Disease. 17 (1), 24 (2018).
  32. Pachikian, B. D., et al. Implication of trans-11, trans-13 conjugated linoleic acid in the development of hepatic steatosis. PLoS One. 13 (2), 0192447 (2018).
  33. Ferreira, T., Rasband, W. Analyze. ImageJ User Guide. , 132-135 (2012).
  34. Geng, Y., Wu, Z., Buist-Homan, M., Blokzijl, H., Moshage, H. Hesperetin protects against palmitate-induced cellular toxicity via induction of GRP78 in hepatocytes. Toxicology and Applied Pharmacology. , 404 (2020).
  35. Geng, Y., et al. Protective effect of metformin against palmitate-induced hepatic cell death. Biochimica et Biophysica Acta - Molecular Basis of Disease. 1866 (3), 165621 (2020).
  36. Sarnyai, F., et al. Effect of cis- and trans-monounsaturated fatty acids on palmitate toxicity and on palmitate-induced accumulation of ceramides and diglycerides. International Journal of Molecular Science. 21 (7), 2626 (2020).
  37. Chen, J. W., et al. Tetrahydrocurcumin ameliorates free fatty acid-induced hepatic steatosis and improves insulin resistance in HepG2 cells. Journal of Food and Drug Analysis. 26 (3), 1075-1085 (2018).
  38. Burhans, M. S., et al. Hepatic oleate regulates adipose tissue lipogenesis and fatty acid oxidation. Journal of Lipid Research. 56 (2), 304-318 (2015).
  39. Abenavoli, L., Milanovic, M., Milic, N., Luzza, F., Giuffre, A. M. Olive oil antioxidants and non-alcoholic fatty liver disease. Expert Reviews in Gastroenterology and Hepatology. 13 (8), 739-749 (2019).
  40. Nagarajan, S. R., et al. Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: A comprehensive resource for in vitro studies of hepatic metabolism. American Journal of Physiology - Endocrinology and Metabolism. 316 (4), 578-589 (2019).
  41. Hodson, L., Skeaff, C. M., Fielding, B. A. Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake. Progress in Lipid Research. 47 (5), 348-380 (2008).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved