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