HepaRG culture

The human cell line HepaRG  was purchased by Millipore. These cells were isolated for the first time from differentiated grade 1 Edmonson hepatoma associated with chronic C hepatitis (Cerec, 2006). Even though undifferentiated HepaRG cells exhibit a fibroblast-like morphology, the differentiation process induces both hepatocyte-like and biliary-like epithelial phenotypes at confluence, indicating bipotent progenitor features. HepaRG cells express a wide panel of liver genes, including drug metabolizing enzymes (e.g. cytochrome P450), similarly to human primary hepatocytes (Cerec, 2006; Aninat, 2006; Aninat, 2008).

HepaRG cells were seeded at 27000 cell/cm² confluence in a base medium composed by William’s E Medium + GlutaMAX (Gibco, 3255-020) supplemented with 10% FBS (Sigma, F7524), 100U/ml penicillin (Sigma), and 100ug/ml streptomycin (Sigma, P4333). To induce the trans-differentiation, cells were shifted to a Growth Medium composed by Base medium + 5ug/ml insulin (Actrapid®, Novo Nordisk), and 50uM hydrocortisone hemisuccinate (Pfizer) for two weeks, and then to the same medium supplemented with 2% DMSO (Differentiation Medium) (Sigma, 67-68-5). The medium was renewed every 2 or 3 days (Aninat, 2006; Cerec, 2006).

When seeded, HepaRG cells (control cells, CTRL) show an elongated morphology characterized by a clear cytoplasm. Then, they progressively form typical colonies of smaller epithelial cells with a granular and dark cytosol, and prominent nuclei, like hepatocytes surrounded by more flattened and clearer epithelial cells. Addition of DMSO induces the appearance of clusters of hepatocyte-like cells surrounded by biliary-like cells (Differentiated cells, DIFF) (Figures 1-3, 10X magnification).

References

Aninat, C., Piton, A., Glaise, D., Le Charpentier, T., Langouët, S., Morel, F., Guguen-Guillouzo, C., and Guillouzo, A. (2006). Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab. Dispos. 34, 75–83.

Aninat, C., Seguin, P., Descheemaeker, P.-N., Morel, F., Malledant, Y., and Guillouzo, A. (2008). Catecholamines induce an inflammatory response in human hepatocytes. Crit. Care Med. 36, 848–854.Cérec, V., Piquet-Pellorce, C., Aly, H.A.A., Touzalin, A.-M., Jégou, B., and Bauché, F. (2007). Multiple pathways for cationic amino acid transport in rat seminiferous tubule cells. Biol. Reprod. 76, 241–249.

Cérec, V., Piquet-Pellorce, C., Aly, H.A.A., Touzalin, A.-M., Jégou, B., and Bauché, F. (2007). Multiple pathways for cationic amino acid transport in rat seminiferous tubule cells. Biol. Reprod. 76, 241–249.