This protocol describes how to induce liver vesicular steatosis in differentiated HepaRG cells and methods for detection and quantification of lipid accumulation. This in vitro human cell model represents a valuable alternative to in vivo mice models and to primary human hepatocytes. Thaw a nitrogen cryopreserved low-passage batch of HepaRG cells by immersing them in a 37 degree Celsius bath until defrosted.
Rapidly transfer the cells into a 15-milliliter tube containing 10 milliliters of proliferating medium. After centrifuging for five minutes, discard the supernatant and resuspend the cells in five milliliters of proliferating medium. Count the cells and dilute in order to plate 250, 000 cells per square centimeter.
Plate the cells on 100-millimeter cell culture dishes and renew the medium every two or three days. Cells proliferate with a doubling time of around 24 hours. Once the HepaRG cells reach 80%confluency, detach them through a three-to five-minute incubation with 0.05%trypsin solution.
Collect the cells in proliferating medium and centrifuge for five minutes as before. Discard the supernatant and resuspend the cells with five milliliters of proliferating medium. Again, count the cells and dilute in order to plate 250, 000 cells per square centimeter.
At this stage, amplify the cells by repeating these steps to reach an appropriate number of cells to start experiments. To cryopreserve HepaRG cells, detach the cells 24 hours after plating through a three-to five-minute incubation with 0.05%trypsin solution. Collect the cells in proliferating medium and centrifuge for five minutes as before.
Discard the supernatant, resuspend the cells in one milliliter per batch of freezing medium, and cryopreserve the batches in liquid nitrogen. On day zero, seed HepaRG cells at 250, 000 cells per square centimeter into culture-treated dishes with an appropriate volume of proliferation medium. Two dishes need to be plated for each assay, one for control cells and one for oleate-treated cells.
On day two and day four, change the medium with an appropriate volume of proliferation medium and let the cells grow until they reach confluence. On day seven, the cells must be 100%confluent. Wash the cells once with 1X PBS.
Then, remove the 1X PBS and add an appropriate volume of differentiation medium. On days eight, nine, 12, 15, and 18, wash the cells once with 1X PBS before adding an appropriate volume of differentiation medium. On day 21, observe HepaRG cells under a microscope and ensure that the cells are confluent differentiated cultures.
As a control, harvest one control dish to perform gene expression analysis of differentiation marker genes. At day 21, differentiated HepaRG cells can be cryopreserved in liquid nitrogen. On day 21, dilute sodium oleate with an appropriate volume of complete differentiation medium to a final concentration of 250 micromolar.
Add 99%methanol at a ratio of one to 400 into another aliquot of differentiation medium to make the vehicle control treatment. Wash the cells once with 1X PBS and add vehicle or sodium oleate medium. On day 23 and day 25, change the medium with the appropriate volume of freshly-prepared medium.
On day 26, observe the cells by optical microscopy. Lipid droplets accumulate in the sodium oleate treated cells and are easily visible as translucent droplets in the cytoplasm. To perform Oil Red O staining, first wash the cells once with 1X PBS and remove the 1X PBS completely.
Add 4%paraformaldehyde and incubate for 15 minutes at room temperature. Remove the paraformaldehyde and wash the cells twice with 1X PBS. After removing the PBS, incubate the cells with 60%isopropanol for five minutes at room temperature.
Remove the isopropanol and let the cells dry completely at room temperature. Now add Oil Red O working solution to the cells and incubate at room temperature for 30 minutes. The volume of working solution required for each sample corresponds to the volume of media used for culturing the cells.
Remove the Oil Red O solution and immediately add double-distilled water. After washing the cells four times with double-distilled water, acquire images under the microscope for analysis. To elute the Oil Red O dye, remove all the water and allow to dry.
Then, add one milliliter of 100%isopropanol and incubate for 10 minutes with gentle shaking at room temperature. Pipette the isopropanol with eluted Oil Red O dye up and down several times, ensuring that all the Oil Red O is in the solution. Transfer the solution to a cuvette.
Measure the optical density at 500 nanometers by spectrophotometry and use 100%isopropanol as the blank. Wash the cells once with 1X PBS. Then, incubate with 100 nanomolar of Bodipy diluted in 1X PBS in the dark for 40 minutes at 37 degrees Celsius.
An unstained control should be included in the flow cytometry measurements. Remove the staining solution and wash the cells once with 1X PBS. Then, wash the cells again and remove the PBS completely.
Add 4%paraformaldehyde and incubate for 15 minutes at room temperature. Remove the paraformaldehyde and wash the samples three times for five minutes in PBS. Cells can be kept in 1X PBS at four degrees Celsius or imaged immediately.
For storage, wrap with Parafilm and cover with aluminum foil to prevent the cells from drying. To verify efficient induction of steatosis, oleate-treated and control HepaRG cells were stained with Oil Red O dye. After staining, lipid droplets are easily visible as red droplets.
Staining can be quantified by spectrophotometric measurement of Oil R O dye eluted with isopropanol. Absorbance of eluted Oil Red O from the cells is directly proportional to cytoplasmic lipid droplet accumulation. Sodium oleate concentration and exposure time were determined by MTT colorimetric cell viability assay.
The Formosan product is quantified by its absorbance at 490 nanometers and is directly proportional to the number of living cells in culture. Sodium oleate treatment was compared to sodium palmitate treatment. The apoptotic drug doxorubicin was used as a control.
To quantify the increase of cellular lipid content after sodium oleate treatment, staining with Bodipy dye was used to label lipid droplets. Using cytofluorometric analysis, it is possible to quantify the triglyceride content by Bodipy mean fluorescent intensity, which is higher in oleate-treated cells as compared to control cells. This indicates efficient fat accumulation after oleate treatment.
Indeed, images of Bodipy-stained cells show bright green fluorescent lipid droplets in oleate-treated cells that are not visible in the control cells. To further characterize the accumulation of lipid droplets after sodium oleate treatment, CARS microscopy can be performed as described in the text protocol which enables lipid droplets quantification without labeling. This cell-based model may increase knowledge of the molecular mechanisms involved in non-oncotic fatty liver disease and could lead to the development of new markers for early diagnosis and new treatment strategies.
