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 to the fact that patients with fatty liver are exposed to therapeutic drugs, contaminants, among other conditions that might be affected by steatosis. Thus, this protocol has potential applications in toxicology, pharmacology, and the identification of therapeutic targets for treatment of the disease.
On development of this protocol, one of the most critical steps is the preparation of the steatogenic mix: 1 part of palmitate: 2 parts of oleate, which induces steatosis from the day 2 (Figure 3, Figure 4), allowing hepatocytes to proliferate-despite a modest decrease in viability rate and increased mortality rate (Figure 2). However, decreased viability should not exceed 30%-40%, since that might represent a toxic effect rather than one that might be followed in the long-term. Hepatic steatosis is the result of a long-term overexposure to lipids. In this sense, lipids accumulate, at first, with mild affections on the hepatocytes, as observed in this model. Another feature is the lipid droplet profile. In mild steatosis, an increased size in lipid droplets is observed during the progression of the culture (Figure 4E-H). In severe steatosis, droplet size is considerably higher compared to mild steatosis (Figure 4I-L), whereas controls do not show changes in lipid droplet size (Figure 4A-D).
It is preferable to store both the mild and severe steatogenic media at 4 &#176;C for up to a week. Afterward, it is recommended to prepare fresh steatogenic medium. However, the OA stock can be preserved at -20 &#176;C for up to a month, whereas the PA stock can be stored at 4 &#176;C for up to a month. Using these stock solutions after the suggested time might represent a risk of degradation of the fatty acids. To ensure the proper concentration of the solutions before every use, it is recommended to measure fatty acid concentrations by a non-esterified fatty acids (NEFA) assay kit.
PA and OA, as well as their respective salts, have been used separately to induce lipid accumulation; however, differences are observed for every fatty acid16,20. On one hand, palmitate used alone is a good inductor of steatosis. It induces cell death, hepatic insulin resistance, mitochondrial dysfunction, reticular stress16,34,35,36. However, palmitate is highly toxic16,34, and the outcomes expected of using it alone in culture include lower viability and higher mortality compared with the mixture of PA and OA16,20. On the other hand, oleate also induces steatosis. It induces de novo lipogenesis, insulin resistance37,38, and hyperproliferation38. However, the outcomes observed with oleate are often milder compared with palmitate and the mixture16,20. This might be related to its protective role. Oleate is the major component in olive oil, a key ingredient in the Mediterranean diet, one of the well-known successful strategies against MAFLD39.
This protocol might be considered as a nice tool to study steatosis due to its reproducibility and the short time it takes to obtain results. This is in comparison with using experimental models of MAFLD, adding the fact that it does not imply the ethical issues inherent to using rodent models. This protocol allows being followed for several days, provided that steatogenic medium is refreshed every 24 h. This model is also cost-effective and possesses a wide range of applications. It can also be adjusted to other cell lines, not only hepatocytes, but a wide range of cells affected by lipid overexposure during obesity. One of the limitations of this protocol is the use of HepG2 cells. Since this is a carcinogenic cell line, it might conceal or increase some outcomes. However, the application of HepG2 cells in these types of studies is widely accepted due to its resemblance in lipid metabolism to healthy hepatocytes40. The use of the mixture PA:2OA might also prove to be controversial since it does not fully resemble the profiles of NEFA observed in the blood of NAFLD/MAFLD patients41. Other fatty acids, including linolenic or stearic acids, might be included in further modifications and improvements of this protocol. Another limitation is the fact that only one cell type, hepatocytes, is studied, lacking the interaction with other hepatic cells present in the liver, including sinusoidal endothelial, Kupffer, hepatic stellate cells, etc., that are engaged in the progression of MAFLD. Moreover, this is a model to induce steatosis exclusively, with no progression to steatohepatitis and fibrosis.
In conclusion, the study provides an in vitro protocol of hepatocyte steatosis that is easy to implement, reproducible, and with a wide range of applications in the study of steatosis as well as the hepatocyte function in the context of fatty liver.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 Severe, compared with the control conditions. In fact, viability progressively diminished as time of the culture increased, reaching the lowest of 60% at 4 days in severe steatosis (Figure 2A). Accordingly, the mortality rate was higher in hepatocytes cultured in the steatogenic conditions, and it progressively increased with the time of exposure to lipids (Figure 2B). Cell numbers progressively increased as a result of proliferation (Figure 2C). However, proliferation rate was lower in Mild steatosis at 3 days and 4 days. In contrast, Severe steatosis was associated with lower proliferation from 24 h.
HepG2 cells cultured in the proposed protocol show the most important feature of steatosis, intracellular lipid accumulation. Staining cells with Oil Red O allowed to observe at least a two-fold increase of lipid droplets in cells cultured under steatogenic conditions as shown in Figure 3 and Figure 4. Intracellular fat increased according to the time of exposure of culture in the steatogenic medium (Figure 3). In Mild steatosis, lipid contents were increased from day 2, whereas in Severe steatosis, they were significantly high from 24 h.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62543/62543fig1v2.jpg" /> 
Figure 1: Cell growth. HepG2 hepatocytes culture in control (Figure 1A-D) and mild steatogenic conditions (Figure 1E-H). Photographs show growth from 1-4 days of culture. Scale bar = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62543/62543fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62543/62543fig2v2.jpg" /> 
Figure 2: Viability and mortality rates. (A) Viability. (B) Mortality. (C) Cell number. HepG2 hepatocytes culture in control and steatogenic conditions were assessed for viability and mortality rates by trypan blue staining. Mean &#177; SD. Two independent experiments in triplicate per time of culture. Circles: control conditions; Squares: mild steatosis; Triangles: severe steatosis. One-way ANOVA was used to compare among conditions and time of culture for the same condition. p &#60; 0.05 was considered significant: &#34;*&#34;- control vs severe steatosis; &#34;&#167;&#34;- control vs mild steatosis; &#34;&#182;&#34;- mild vs severe steatosis. <a href="https://www.jove.com/files/ftp_upload/62543/62543fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62543/62543fig3v2.jpg" /> 
Figure 3: Lipid accumulation. HepG2 hepatocytes culture in control and steatogenic conditions were assessed for lipid contents by oil red O staining followed by a morphometric analysis using ImageJ software (NIH, USA). Percentage of lipids refers to the percentage of area stained by Oil-Red O (lipids) considering 100% as the complete area of each optical field analyzed. Mean &#177; SD. Two independent experiments in triplicate per time of culture. Circles: control conditions; Squares: mild steatosis; Triangles: severe steatosis. One-way ANOVA was used to compare among conditions and time of culture for the same condition. p &#60; 0.05 was considered significant: &#34;*&#34;- control vs severe steatosis; &#34;&#167;&#34;- control vs mild steatosis; &#34;&#182;&#34;- mild vs severe steatosis. <a href="https://www.jove.com/files/ftp_upload/62543/62543fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62543/62543fig4v2.jpg" /> 
Figure 4: Steatosis in vitro. HepG2 hepatocytes culture in control (Figure 4A-D), mild steatogenic (Figure 4E-H), and severe steatogenic (Figure 4I-L) conditions were assessed for lipid contents by oil red O staining. Photographs show hepatocyte lipid droplets from 24 h to 4 days. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62543/62543fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Universidad San Sebastian

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5.9K 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

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Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

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 ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.