Name: a model of experimental steatosis in vitro: hepatocyte cell culture in lipid overload-conditioned medium
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This work was funded by Consejo Nacional de Ciencia y Tecnolog&#237;a (Conacyt, CB-221137). Adriana Campos is a doctoral student at Programa de Doctorado en Ciencias Biom&#233;dicas, Universidad Nacional Aut&#243;noma de M&#233;xico, and was supported by Conacyt (CVU: 1002502).

1. Standard and conditioned medium preparation
<ol>
	<li>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 &#176;C.Sterilize by using 0.22 &#181;m filters.</li>
	<li>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...

<ol>
	<li>Younossi, Z. 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J., George, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFLD:+A+consensus-driven+proposed+nomenclature+for+metabolic+associated+fatty+liver+disease.">MAFLD: A consensus-driven proposed nomenclature for metabolic associated fatty liver disease.</a> Gastroenterology. 158 (7), 1999-2014 (2020).</li><li>Chalasani, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+and+management+of+nonalcoholic+fatty+liver+disease:+Practice+guidance+from+the+American+association+for+the+study+of+liver+diseases.">The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American association for the study of liver diseases.</a> Hepatology. 67 (1), 328-357 (2018).</li><li>Calzadilla Bertot, L., Adams, L. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+palmitic+acid+on+TNF-alpha-induced+cytotoxicity+in+SK-Hep-1+cells.">Effects of palmitic acid on TNF-alpha-induced cytotoxicity in SK-Hep-1 cells.</a> Toxicology In Vitro: An International Journal Published in Association with BIBRA. 26 (6), 783-790 (2012).</li><li>Stellavato, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assessment+of+nutraceutical+compounds+and+novel+nutraceutical+formulations+in+a+liver-steatosis-based+model.">In vitro assessment of nutraceutical compounds and novel nutraceutical formulations in a liver-steatosis-based model.</a> Lipids in Health and Disease. 17 (1), 24 (2018).</li><li>Pachikian, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+trans-11,+trans-13+conjugated+linoleic+acid+in+the+development+of+hepatic+steatosis.">Implication of trans-11, trans-13 conjugated linoleic acid in the development of hepatic steatosis.</a> PLoS One. 13 (2), 0192447 (2018).</li><li>Ferreira, T., Rasband, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyze.">Analyze.</a> ImageJ User Guide. , 132-135 (2012).</li><li>Geng, Y., Wu, Z., Buist-Homan, M., Blokzijl, H., Moshage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hesperetin+protects+against+palmitate-induced+cellular+toxicity+via+induction+of+GRP78+in+hepatocytes.">Hesperetin protects against palmitate-induced cellular toxicity via induction of GRP78 in hepatocytes.</a> Toxicology and Applied Pharmacology. , 404 (2020).</li><li>Geng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+metformin+against+palmitate-induced+hepatic+cell+death.">Protective effect of metformin against palmitate-induced hepatic cell death.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1866 (3), 165621 (2020).</li><li>Sarnyai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cis-+and+trans-monounsaturated+fatty+acids+on+palmitate+toxicity+and+on+palmitate-induced+accumulation+of+ceramides+and+diglycerides.">Effect of cis- and trans-monounsaturated fatty acids on palmitate toxicity and on palmitate-induced accumulation of ceramides and diglycerides.</a> International Journal of Molecular Science. 21 (7), 2626 (2020).</li><li>Chen, J. 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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...

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 name, the disease includes a wide spectrum of histopathological changes characterized by abnormally high accumulation of lipids in the liver (&#62;5% of fat in the hepatocytes5) and might progress through the lipid accumulation typically found in simple steatosis to steatohepatitis, which in turn might lead to the development of fibrosis, cirrhosis, hepatocellular carcinoma, and liver failure5,6,7,8. Due to its increasing prevalence, MAFLD is expected to become the first indication of liver transplantation and the leading cause of hepatocellular carcinoma9.
Although it has been considered as a benign or mild form of fatty liver disease, hepatic steatosis is in fact the metabolic key in MAFLD10. Different metabolic pathways are affected by lipid accumulation in the liver, including but not limited to lipid synthesis, exportation, and metabolism10. Insulin resistance, oxidative stress, reticular stress, and cellular dysfunction are strongly associated to hepatic lipotoxicity11,12. On the other hand, fatty hepatocytes are the target of reactive oxygen species, rendering metabolites as lipid peroxides, protein carbonyls, and adducts of nucleic acids13. At the cellular level, fatty hepatocytes might undergo mitochondrial damage14, cellular senescence15, apoptosis16, pyroptosis12, and autophagia17, among other events.
Hepatocytes are highly responsible for metabolism, detoxification, and synthesis of a wide range of molecules. Many of these functions might be compromised by the lipid accumulation observed in steatosis. Therefore, it is of great importance to have reproducible tools that allow an accurate evaluation of steatosis. In this sense, in vitro models are readily applicable and highly reproducible. Steatosis in vitro has been used with different goals16,18,19. The HepG2 cells are widely used as hepatocyte cell line. It has advantages such as being easy to culture and well characterized. Perhaps, the only disadvantage of HepG2 cells is the fact that it is a carcinogenic cell line, so this must be considered when analyzing the outcomes. Here, the application of a mixture of fatty acids widely used in cell culture: palmitic acid (PA) and oleic acid (OA) is shown. Both PA and OA offer different outcomes in culture20. PA (C 16:0) is the most common saturated fatty acid obtained from the diet16. PA is considered as a biomarker of de-novo lipogenesis, a crucial step in the development of NAFLD21. PA is shown to be highly toxic22; therefore, it might not be recommended to induce steatosis in vitro. OA (C 18:1) is a monounsaturated fatty acid. In contrast to PA, OA has been suggested to possess anti-inflammatory and anti-oxidant properties, being able to counteract PA12. Both PA and OA are the main fatty acids present in the triglycerides, regardless of the condition of health or disease16. Table 1 provides examples of the hepatocyte culture with PA, OA, and their mixture, as well as the outcomes reported12,23,24,25,26,27. Other fatty acids have also been used in hepatocyte culture, including stearic acid (C 18:0)28,29,30, linoleic acid (C 18:1)28,30,31 and its conjugates (CLA)28,32, palmitoleic acid (C 16:1)29. However, their use is least frequently reported in the literature, perhaps because their hepatic abundance is lower than PA and OA16.
In conjunction, both fatty acids resemble steatosis in vitro, providing proliferating cells, with increased cell death and lower viability compared with control conditions. It is worth mentioning that the respective salts of these fatty acids are available and can be used as well. One of the main problems when assessing lipid overload in hepatocyte cell culture is given in the differentiation between toxicological models and a model that best represent steatosis. Many models can be accounted in the first case. In fact, the use of PA alone might be considered among them, and the high mortality is the most evident outcome12,16,23,24,25,26,27. The use of high doses even in the case of OA can also be considered as a toxicologic model. The protocol shown here is in higher accordance with steatosis development since it shows low mortality compared with that observed in other models and allows it to be followed during several days with progressive lipid accumulation as it occurs in NAFLD. The possibility to assess mild and severe steatosis through experimental conditions is considered another advantage.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fatty&#160;acids</td>
			<td colspan="2">Conditions</td>
			<td colspan="2">Outcomes</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td rowspan="3">PA</td>
			<td colspan="2">Concentration:&#160;200&#160;&#956;M</td>
			<td colspan="2">Lipid accumulation</td>
			<td rowspan="3">Yan et al, 201925.</td>
		</tr>
		<tr>
			<td colspan="2">Time exposure:&#160;24 h</td>
			<td colspan="2">Hepatocyte damage</td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td colspan="2">Transaminases elevation</td>
		</tr>
		<tr>
			<td rowspan="2">PA</td>
			<td colspan="2">Concentration:&#160;50, 100&#160;and&#160;200&#160;&#956;M</td>
			<td colspan="2" rowspan="2">Lipid accumulation</td>
			<td rowspan="2">Xing et al, 201924.</td>
		</tr>
		<tr>
			<td colspan="2">Time exposure:&#160;24 h</td>
		</tr>
		<tr>
			<td rowspan="2">PA</td>
			<td colspan="2">Concentration:&#160;250 &#956;M&#160;, 500 &#956;M&#160;, 750 &#956;M&#160; and 1,000&#160;&#956;M</td>
			<td colspan="2">Lipid accumulation</td>
			<td rowspan="2">Wang et al, 202026.</td>
		</tr>
		<tr>
			<td colspan="2">Time exposure:&#160;24 h</td>
			<td colspan="2">Progressive reduction of cell viability</td>
		</tr>
		<tr>
			<td rowspan="3">Mix of&#160;OA/PA</td>
			<td colspan="2">Concentration:&#160;1 mM</td>
			<td colspan="2">Lipid&#160;accumulation</td>
			<td rowspan="3">Xiao et al, 202027.</td>
		</tr>
		<tr>
			<td colspan="2">Time exposure:&#160;24 h</td>
			<td colspan="2">Does not report&#160;lipotoxicity</td>
		</tr>
		<tr>
			<td colspan="2">Rate:&#160;2OA:1PA</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td rowspan="4">Mix of&#160;OA/PA</td>
			<td colspan="2">First stimulation with&#160;200 &#956;M&#160;&#160;and&#160;400&#160;&#956;M of PA&#160;and then second stimulation with&#160;200&#160;&#956;M of OA</td>
			<td colspan="2">Lipid&#160;accumulation.&#160;</td>
			<td rowspan="4">Zeng et al, 202012.</td>
		</tr>
		<tr>
			<td colspan="2">Concentration:400&#160;&#956;M PA: 200&#160;&#956;M OA</td>
			<td colspan="2">Evidence of&#160;lipotoxicity&#160;induced by PA was reduced by stimulation of OA.</td>
		</tr>
		<tr>
			<td colspan="2">Rate:&#160;2PA:1OA</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td colspan="2">Time exposure:&#160;24 h</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td rowspan="3">Mix of&#160;OA/PA</td>
			<td colspan="2">Concentration:&#160;400&#160;&#956;M PA: 200&#160;&#956;M OA</td>
			<td colspan="2" rowspan="3">Lipid accumulation</td>
			<td rowspan="3">Chen et al, 201823.</td>
		</tr>
		<tr>
			<td colspan="2">Rate:&#160;2PA:1OA</td>
		</tr>
		<tr>
			<td colspan="2">Time exposure:&#160;24 h</td>
		</tr>
		<tr>
			<td rowspan="3">Mix of&#160;OA/PA</td>
			<td colspan="2">Concentration&#8239;:50&#160;and 500&#160;&#956;M</td>
			<td colspan="2">Generation&#160;of&#160;two&#160;types of&#160;steatosis:&#160;mild&#160;steatosis&#160;and 
			&#160;severe&#160;steatosis.</td>
			<td rowspan="3">Campos and Guzm&#225;n 2021</td>
		</tr>
		<tr>
			<td colspan="2">Rate: 2PA:1OA</td>
			<td colspan="2">Simulates&#160;chronic&#160;exposition of&#160;lipid overload</td>
		</tr>
		<tr>
			<td colspan="2">Time exposure: 24 h, 2 days,3 days and 4 days.</td>
			<td colspan="2"></td>
		</tr>
	</tbody>
</table>
Table 1. Hepatocyte culture in steatogenic conditions. The table presents the type of fatty acid used, the conditions maintained, and the observed outcomes in hepatocyte culture. PA: Palmitic acid. OA: Oleic acid.
Finally, this model is applicable not only to the study of steatosis and fatty liver, but also to the hepatic metabolic, synthetic, and detoxification pathways in the context of steatosis. Also, in vitro induced steatosis might provide evidence for the identification of potential markers of the disease as well as therapeutic targets.

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	<tbody>
		<tr>
			<td>Biosafety cabinet</td>
			<td>ESCO Airstream</td>
			<td>AC2-452+C2:C26</td>
			<td>Class II&#160;Type A2&#160;Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Bottle top filter</td>
			<td>Corning, US</td>
			<td>430513</td>
			<td>&#160;Non-pyrogenic, polystyrene, sterile. 1 filter/Bag. 0.22 &#956;m, 500 mL.</td>
		</tr>
		<tr>
			<td>Bovine serum albimun (BSA)</td>
			<td>Gold Biotechnology, US</td>
			<td>A-421-10</td>
			<td>BSA Fatty Acid Free for cell culture</td>
		</tr>
		<tr>
			<td>Culture media RPMI 1640</td>
			<td>ThermoFisher-Gibco, US</td>
			<td>31800-022</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher-Gibco, US</td>
			<td>A4766801</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Marienfeld, DE</td>
			<td>640010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HepG2 cell line</td>
			<td>ATCC, US</td>
			<td>HB-8065</td>
			<td>Hepatocellular carcinoma human cells.</td>
		</tr>
		<tr>
			<td>Humidified incubator</td>
			<td>Thermo Electronic Corporation,US</td>
			<td>Model: 3110</td>
			<td>Temperature (37 &#176;C &#177; 1 &#176;C), humidity (90% &#177; 5%) , CO2 (5% &#177; 1%)</td>
		</tr>
		<tr>
			<td>Inverted microscope Eclipse</td>
			<td>NIKON, JPN</td>
			<td>Model: TE2000-S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich, US</td>
			<td>I9030-4L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Oil Red O Kit</td>
			<td>Abcam, US</td>
			<td>ab150678</td>
			<td>Kit for histological visualization of neutral fat.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich, US</td>
			<td>P6148-500G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>ThermoFisher-Gibco, US</td>
			<td>15140-122</td>
			<td>Antibiotics 10,000 U/mL Penicillin, 10,000 &#956;g/mL Streptomycin</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Beckman, US</td>
			<td>Model: 360 PH/Temp/MV Meter</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>ThermoFisher-Gibco, US</td>
			<td>10010-023</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Sarstedt, AUS</td>
			<td>86.1253.001</td>
			<td>&#160;Non-pyrogenic, sterile, 5 mL</td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Sarstedt, AUS</td>
			<td>&#160;86.1254.001</td>
			<td>&#160;Non-pyrogenic, sterile, 10 mL</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich, US</td>
			<td>S5761-1KG</td>
			<td>Preparation of culture media</td>
		</tr>
		<tr>
			<td>Sodium oleate</td>
			<td>Santa Cruz Biotechnology, US</td>
			<td>sc-215879A</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium palmitate</td>
			<td>Santa Cruz Biotechnology, US</td>
			<td>sc-215881</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Syring filter</td>
			<td>Corning, US</td>
			<td>431219</td>
			<td>&#160;Non-pyrogenic, sterile, 28 mm, 0.2 &#956;m.</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma-Aldrich, US</td>
			<td>T6146-25G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin 0.05% /EDTA 0.53 mM</td>
			<td>Corning, US</td>
			<td>25-052-Cl</td>
			<td>-</td>
		</tr>
		<tr>
			<td>24 well cell culture cluster</td>
			<td>Corning, US</td>
			<td>3524</td>
			<td>Flat bottom with lid. Tissue culture treated. Nonpyrogenic, polystyrene, sterile. 1/Pack.</td>
		</tr>
		<tr>
			<td>96 well cell culture cluster</td>
			<td>Corning, US</td>
			<td>3599</td>
			<td>Flat bottom with lid. Tissue culture treated. Nonpyrogenic, polystyrene, sterile. 1/Pack.</td>
		</tr>
	</tbody>
</table>

a model of experimental steatosis in vitro: hepatocyte cell culture in lipid overload-conditioned medium

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.

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...

Watch this Scientific Journal Video about A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium at JoVE.com

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

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.

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

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver ...

This protocol is applicable to the study of liver steatosis as well as to the hepatic, metabolic, synthetic, and the toxification pathways in the context of steatosis. The main advantages of this method are its reproducibility and the short time needed to obtain results. Add in the fact that it offers two levels of steatosis, mild, and severe.In vitro induced steatosis might provide evidence for the identification of potential markers for the diagnosis of the disease, as well as therapeutic targets. This method is meant to be applied in the steatosis result. However, it can be adjusted to a wide range of different cells affected by lipid over-exposures, like during obesity and lipidemia.Prepare standard RPMI 1640. Supplement RPMI 1640 culture medium with 10%of heat activated FBS, and 1%of penicillin streptomycin solution. Sterilize it by using 0.22 micrometer filters and store the supplement at four degrees Celsius.To prepare a palmitate stock solution prepare a 50 millimolar solution of palmitate in the standard RPMI 1640 supplemented with 1%of lipid free BSA. Sterilize about five to ten milliliters of the stock solution by using 0.22 micrometer filters and store at four degrees Celsius protected from light for up to one month. To prepare oleate stock solution prepare a 50 millimolar solution of oleate in the supplemented RPMI 1640.And sterilize the stock solution by using 0.22 micrometer filters. Store it at minus 20 degrees Celsius, protected from light for up to one month. To prepare steatogenic medium from the previously prepared stocks, prepare 100 millimeters of a 50 micromolar mix of one part palmitate and two part oleate.For mild levels, pour a 500 micromolar mix of one part palmitate and two part oleate for severe levels in standard RPMI 1640. And sterilize using 0.22 micrometer filters. Store at four degrees Celsius for up to one week.Alternatively, to prepare stock solutions with the respective fatty acids by using free lipid albumin, dissolve either palmitate or oleate in two milliliters of absolute ethanol and then mix in the final volume of standard RPMI 1640. Dissolve oleate directly by storing in standard RPMI 1640 culture medium. Allow evaporation of ethanol by incubating in a water bath at 70 degrees Celsius and mix thoroughly.Sterilize both stock solutions using 0.22 micrometer filters and store the palmitate stock solution at four degrees Celsius. And oleate stock solution at minus 20 degrees Celsius. Seat 0.1 million Hep G2 cells per well in a 24-well plate.Add one milliliter of standard RPMI 1640. Pre-incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours, allowing cell attachment. After pre culture, discard the standard RPMI 1640 medium.And add the steatogenic medium. Discard the supernatant and add a fresh steatogenic medium every 24 hours. To perform viability and morphology assessment, discard the supernatant and detach cells from the well by adding 500 microliters of 0.05%trypsin EDTA.Incubate for five minutes at 37 degrees Celsius and 5%carbon dioxide. Collect the resuspended cells in a micro tube. And centrifuge at 300 times G.Then discard the supernatant, add a 200 microliters of standard RPMI 1640, and resuspend the cells.Add 15 microliters of 0.4%of trypan blue solution in a fresh micro tube. Add and mix 15 microliters of the previous cell suspension. Count the stained and non stained cells in a hemocytometer and calculate the viability and mortality rates.Put a cell culture cover slip in every well in a 24-well plate and seed Hep G2 cells as described in the text manuscript. After the appropriate incubation, wash the cells with one milliliter of PBS and discard the supernatant. Mix them with one milliliter of 4%paraformaldehyde in PBS and incubate for one hour at room temperature.Discard the excess paraformaldehyde and rinse the cells with one milliliter of distilled water. Then add one milliliter of 70%isopropanol and incubate for five minutes. Discard the excess isopropanol and add one milliliter of Oil-Red O solution and incubate for 30 minutes.Discard the excess Oil-Red O solution, and then rinse with one milliliter of distilled water. Observe the cells under the microscope at a magnification of 400x. Randomly select and capture photographs of 10 optical fields from the complete area of the well.Repeat the imaging for every well. To assess the percentage of red stained area, open ImageJ software and import required files, then click on adjust and using the color threshold"tool, define the hue parameter for red color detection. Then adjust saturation and brightness for the image.Compare the stained area with the complete area of the optical field, using the analyze particles"tool and calculate the average percentage of every well. Seating 0.1 million hepatocytes per well in 24-well plates provides optimum confluence. Viability progressively diminished as time of the culture increased, reaching the lowest of 60%at four days in severe steatosis.Accordingly, the mortality rate was higher in hepatocytes cultured in the steatogenic conditions. And it progressively increased with the time of exposure to lipids. Cell numbers progressively increased as a result of proliferation.However, proliferation rate was lower in mild steatosis at three and four days. In contrast, severe steatosis was associated with lower proliferation by 24 hours. Staining cells with Oil-Red O demonstrated at least a twofold increase of lipid droplets in cells cultured under steatogenic conditions.Intracellular fat increased according to the time of exposure of culture in the steatogenic medium. In mild steatosis, lipid contents were increased from day two, whereas in severe steatosis they were significantly high starting at 24 hours. Cell culture requires previous experience.Culturing Hep G2 cells in a standard condition prior to using this protocol could be helpful. Preparation of the fatty-acid stock is also a crucial point. Palmitic acid is solid at room temperature, and it needs to be warmed before its management.This method should allow the molecular analysis of different pathways, including, but not limited to proliferation, apoptosis, autophagia, oxidative stress and pharmacologic and toxicologic analysis.

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