Steatosis Assays

The steatosis assay provides a means to measure the accumulation of neutral lipids, mainly triglycerides, into intracytoplasmic macrovesicles and microvesicles due to the toxic effect of a test compound. We provide steatosis assays for all small molecule formulations such as pharmaceuticals, industrial chemicals and consumer products.

Our steatosis assay uses a genetically modified version of the immortalized human liver cell line HepaRG™, created with our proprietary CompoZr® zinc finger nuclease (ZFN) technology. Apart from fresh human hepatocytes, HepaRG cells are the most metabolically active liver cell line described to date and have the potential for use as a viable surrogate in many functional liver assays, including general toxicity testing, with none of the drawbacks of limited availability and donor-to-donor variation.

Steatosis is a pathological process characterized by abnormal accumulation of lipid within cells and can be induced by several factors, including prolonged exposure to certain drugs. Liver steatosis is characterized by excessive accumulation of neutral lipids, mainly triglycerides, into intracytoplasmic macrovesicles and microvesicles.  Fat accumulation is believed to result from an imbalance between hepatic fatty acid inflow and oxidation versus triglyceride synthesis and excretion. While fat accumulation is not necessarily a pathological condition, the condition may progress and lead to more serious complications including non-alcoholic steatohepatitis, cirrhosis and even liver failure.

Steatosis is assessed by cytometry using lipid-sensitive dyes following exposure of cells to a test substance. In the steatosis assay, Oil Red O is used to stain neutral lipids in the metabolically active human HepaRG cell line, clone 5F. Lipid accumulation can also be quantified using a plate reader after the dye is extracted from the lipid droplets. Tetracycline and oleic acid are used as positive controls.
 

 Steatosis Assay Protocol

 
Test System HepaRG liver cell, clone 5F (human)
Test Compound Concentration 1, 10 and 100 µM
Replicates 3
Assay Length 48 hours
Controls Vehicle (0.1% DMSO)
Tetracycline, 50 µM
Oleic acid, 500 µM
Analysis Method Oil Red O staining of neutral lipids
Data Delivery Photomicrographs
Plate reader-based quantitation

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References

  • Anthérieu S et al. (2011) Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in HepaRG cells, Hepatology 53, 1895-1905
  • Donato MT and Gómez-Lechón MJ (2012) Drug-induced liver steatosis and phospholipidosis: Cell-based assays for early screening of drug candidates, Curr Drug Metab 13, 1160-1173