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 this stock will be sufficient. Sterilize the stock solution by using 0.22 &#181;m filters. Store at 4 &#176;C protected from light for up to 1 month.</li>
	<li>To prepare oleate stock solution, prepare a 50 mM solution of oleate in the standard RPMI 1640 previously supplemented with 1% of bovine serum albumin (lipid free). A volume of 10 mL will be sufficient. Sterilize the stock solution by using 0.22 &#181;m filters. Store at -20 &#176;C protected from light for up to 1 month.</li>
	<li>To prepare steatogenic medium from the previously prepared stocks, prepare a 1-part palmitate: 2-part oleate steatogenic medium at two possible levels: mild and severe steatosis.
	<ol>
		<li>Mild Steatosis: Prepare 100 mL of a 1-part palmitate: 2-part oleate (50 &#181;M) mix in standard RPMI 1640. Sterilize by using 0.22 &#181;m filters. Store at 4 &#176;C for up to 1 week.</li>
		<li>Severe Steatosis: Prepare 100 mL of a 1-part palmitate: 2-part oleate (500 &#181;M) mix in standard RPMI 1640. Sterilize by using 0.22 &#181;m filters. Store at 4 &#176;C for up to 1 week.</li>
		<li>Alternative preparation for the stock solutions.
		<ol>
			<li>Prepare both stock solutions using the respective fatty acids by using free lipid albumin as indicated above.</li>
			<li>When lacking free lipid albumin, use palmitate and oleate salts.
			<ol>
				<li>Dissolve either palmitate or oleate in 2 mL of absolute ethanol and then mix in the final volume of standard RPMI 1640 (5-10 mL). Dissolve oleate directly by stirring in standard RPMI 1640 culture medium.</li>
				<li>Allow evaporation of ethanol by incubating in a water bath at 70 &#176;C; mix thoroughly.</li>
			</ol>
			</li>
			<li>In every case, sterilize both stock solutions using 0.22 &#181;m filters. Store palmitate stock solution at 4 &#176;C and oleate stock solution at - 20 &#176;C. Protect both solutions from light. These solutions are stable for 1 month.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Pre-culture
<ol>
	<li>Seed 100,000 HepG2 cells per well in a 24-well plate. Add 1 mL of standard RPMI 1640.</li>
	<li>Pre-incubate at 37 &#176;C and 5% CO2 for 24 h, allowing cell attachment.</li>
</ol>
3. Steatogenic culture
<ol>
	<li>After pre-culture, discard the standard RPMI 1640 medium and add the steatogenic medium accordingly.</li>
	<li>Discard the supernatant and add fresh steatogenic medium every 24 h.</li>
</ol>
4. Viability and mortality assessment
<ol>
	<li>Seed 100,000 HepG2 cells per well in a 24-well plate. Add 1 mL of standard RPMI 1640.</li>
	<li>Pre-incubate for 24 h at 37 &#176;C and 5% CO2.</li>
	<li>Change standard RPMI 1640 medium for the steatogenic medium.</li>
	<li>Incubate for 24 h, 2 days, 3 days, and 4 days refreshing the steatogenic medium every 24 h.</li>
	<li>After the appropriate time, discard the supernatant.</li>
	<li>Detach cells from the well by adding 500 &#181;L of 0.05% Trypsin-EDTA. Incubate for 5 min at 37 &#176;C and 5% CO2.</li>
	<li>Collect the resuspended cells in a microtube.</li>
	<li>Centrifuge at 300 x g and discard the supernatant.</li>
	<li>Add 200 &#181;L of standard RPMI 1640 and resuspend the cells.</li>
	<li>Add 15 &#181;L of 0.4% Trypan blue solution&#160;in a fresh microtube. Mix with 15 &#181;L of the previous cell suspension.</li>
	<li>Count the stained and non-stained cells in a hemocytometer.</li>
	<li>Calculate the viability and mortality rates accordingly. 
	Viability = 
	<img alt="Equation 1" src="/files/ftp_upload/62543/62543eq01.jpg" /> 
	Mortality = 
	<img alt="Equation 2" src="/files/ftp_upload/62543/62543eq02.jpg" /></li>
</ol>
5. Lipid staining with Oil-Red O
<ol>
	<li>Put a cell culture coverslip in every well in a 24-well plate.</li>
	<li>Seed 100,000 HepG2 cells per well. Add 1 mL of standard RPMI 1640.</li>
	<li>Pre-incubate at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Change standard RPMI 1640 medium for the steatogenic medium.</li>
	<li>Incubate for 24 h, 2 days, 3 days, and 4 days, refreshing the steatogenic medium every 24 h.</li>
	<li>After the appropriate time, discard the supernatant.</li>
	<li>Wash with 1 mL of phosphate buffered saline (PBS). Discard the supernatant.</li>
	<li>Fix with 1 mL of 4% paraformaldehyde in PBS.</li>
	<li>Incubate for 1 h at room temperature.</li>
	<li>Discard the excess of paraformaldehyde.</li>
	<li>Rinse the cells with 1 mL of distilled water.</li>
	<li>Add 1 mL of 70% isopropanol and incubate for 5 min.</li>
	<li>Discard the excess of isopropanol. A PBS wash is not needed at this point.</li>
	<li>Add 1 mL of Oil-red O solution and incubate for 30 min.</li>
	<li>Discard the excess of the Oil-red O solution.</li>
	<li>Rinse with 1 mL of distilled water.</li>
	<li>Add 500 &#181;L of hematoxylin solution. Incubate for 3 min.</li>
	<li>Discard the excess of hematoxylin solution.</li>
	<li>Rinse with 1 mL of distilled water.</li>
	<li>Observe under the microscope at a magnification of 400x (Objective 40x, Ocular 10x).</li>
</ol>
6. Morphometric assessment of lipid contents
<ol>
	<li>Randomly select and capture photographs of 10 optical fields from the complete area of the well. Repeat for every well.</li>
	<li>Assess the percentage of red stained area using the Color Threshold tool in the ImageJ software according to Ferreira and Rasband33.</li>
	<li>Compare the stained area with the complete area of the optical field using the Analyze Particles tool in the ImageJ software according to Ferreira and Rasband33.</li>
	<li>Calculate the average percentage of every well.</li>
</ol>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>Biosafety cabinet</td><td>ESCO Airstream</td><td>AC2-452+C2:C26</td><td>Class II&amp;nbsp;Type A2&amp;nbsp;Biological Safety Cabinet</td></tr><tr><td>Bottle top filter</td><td>Corning, US</td><td>430513</td><td>&amp;nbsp;Non-pyrogenic, polystyrene, sterile. 1 filter/Bag. 0.22 &amp;mu;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 &amp;deg;C &amp;plusmn; 1 &amp;deg;C), humidity (90% &amp;plusmn; 5%) , CO&lt;sub&gt;2&lt;/sub&gt; (5% &amp;plusmn; 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 &amp;mu;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>&amp;nbsp;Non-pyrogenic, sterile, 5 mL</td></tr><tr><td>Serological Pipettes</td><td>Sarstedt, AUS</td><td>&amp;nbsp;86.1254.001</td><td>&amp;nbsp;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>&amp;nbsp;Non-pyrogenic, sterile, 28 mm, 0.2 &amp;mu;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 ...

a-model-experimental-steatosis-vitro-hepatocyte-cell-culture-lipid

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Medicine

6.0K Views.  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.

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

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Immunology and Infection

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Biology

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

The occurrence of non-alcoholic fatty liver disease (NAFLD) has been increasing at an alarming rate worldwide. Platycodon grandiflorum is widely used as a traditional ethnomedicine for the treatment of various diseases and is a typical functional food that can be incorporated into the everyday diet. Studies have suggested that platycodin D (PD), one of the main active ingredients in Platycodon grandiflorum, has high bioavailability and significantly mitigates the progress of NAFLD, but the underlying mechanism of this is still unclear. This study aims to investigate the therapeutic effect of PD against NAFLD in vitro. AML-12 cells were pretreated with 300 &#181;M palmitic acid (PA) for 24 h to model NAFLD in vitro. Then, the cells were either treated with PD or received no PD treatment for 24 h. The levels of reactive oxygen species (ROS) were analyzed using 2&#8242;,7&#8242;-dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining, and the mitochondrial membrane potential was determined by the JC-1 staining method. Moreover, the protein expression levels of LC3-II/LC3-I and p62/SQSTM1 in the cell lysates were analyzed by western blotting. PD was found to significantly decrease the ROS and mitochondrial membrane potential levels in the PA-treated group compared to the control group. Meanwhile, PD increased the LC3-II/LC3-I levels and decreased the p62/SQSTM1 levels in the PA-treated group compared to the control group. The results indicated that PD ameliorated NAFLD in vitro by reducing oxidative stress and stimulating autophagy. This in vitro model is a useful tool for studying the role of PD in NAFLD.

Investigating the Protective Effects of Platycodin D on Non-Alcoholic Fatty Liver Disease in a Palmitic Acid-Induced In Vitro Model

This protocol investigates the protective effects of platycodin D on non-alcoholic fatty liver disease in a palmitic acid-induced in vitro model.

The occurrence of non-alcoholic fatty liver disease (NAFLD) has been increasing at an alarming rate worldwide. Platycodon grandiflorum is widely used as a ...

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...

Oleic Acid-Induced HepG2 Cells for Metabolic Disease Analysis 

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

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&#39;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 &#60; 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 &#60; 0.05). Additionally, triglyceride levels in the OA groups were significantly higher than those in the control group (P &#60; 0.05).

Author Spotlight: Establishing MASLD Cell Models for Investigating Disease Mechanisms and the Lipid-Lowering Effects of Koumiss

This article describes the use of oleic acid-induced HepG2 cells as a model for metabolic dysfunction-associated steatotic liver disease.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

Oil Red O Staining: A Technique for Staining Neutral Lipids in Hepatocytes for Detecting Hepatic Steatosis In Vitro

Put a cell culture coverslip in every well in a 24-well plate, and seed HepG2 cells, as described in the text manuscript. After the appropriate incubation, wash the cells with 1 milliliter of PBS, and discard the supernatant. Fix them with 1 milliliter of 4% paraformaldehyde in PBS, and incubate for 1 hour at room temperature. Discard the excess paraformaldehyde, and rinse the cells with 1 milliliter of distilled water.
Then, add 1 milliliter of 70% isopropanol, and incubate for 5 minutes. Discard the excess isopropanol, and add 1 milliliter of Oil-red O solution, and incubate for 30 minutes. Discard the excess Oil-red O solution, and then, rinse with 1 milliliter of distilled water. Observe the cells under the microscope at a magnification of 400x.

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

Summary

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Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

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

Oil Red O Staining: A Technique for Staining Neutral Lipids in Hepatocytes for Detecting Hepatic Steatosis In Vitro