Oxygen Plasma Substrate and Specific Nanopattern Promote Early Differentiation of HepaRG Progenitors

Abstract

Fully differentiated HepaRG™ cells are the hepatic cell line of choice for in vitro work in toxicology and drug trials. They are derived from a hepatoblast-like progenitor (HepaRG™-P) that differentiates into a co-culture of hepatocyte-like and cholangiocyte-like cells. This process that requires 2 weeks of proliferation followed by 2 weeks of differentiation using dimethylsulfoxide (DMSO) can be time consuming and costly. Identifying a method to accelerate HepaRG™-Ps towards a mature lineage would save both time and money. The ability to do this in the absence of DMSO would remove the possibility of confounding toxicology results caused by DMSO induction of CYP pathways. Impact statementWe show significantly earlier differentiation and function of HepaRG progenitor cells when grown in DMSO-free medium on oxygen plasma substrates vs standard tissue culture plastic. Further investigation showed that nanopatterning of oxygen plasma substrates did not confer any additional advantage over smooth oxygen plasma, though one pattern (DSQ120) showed comparable early differentiation and function.

Advancements in stem cell-derived hepatocyte-like cell models for hepatotoxicity testing

“…A multiparametric screening assay showed that oxidative stress, mitochondrial damage, and disorders of neutral lipid metabolism were changed notably in HepaRG cells exposed to DILI-related drugs, which accounts for their high sensitivity as compared with other cell lines [ 54 ]. A high concentration of DMSO required in standard differentiation protocols limits the use of HepaRG cells [ 55 , 56 ], but studies have shown that 3D culture or a cocktail of soluble molecules can be used as an alternative to the DMSO-based differentiation protocol for HepaRG [ 57 , 58 ]. In addition, even though HepaRG cells express high functional levels of most phase I and II enzymes, the levels of some metabolic enzymes such as cytochromes P450 2A6 (CYP2A6), CYP2D6, and CYP2E1 still remain low [ 20 , 21 ].…”

Hepatic Differentiation of Stem Cells in 2D and 3D Biomaterial Systems

“…When ESC were cultured on the fibers of 400 nm and 1.1 µm in diameter, ectodermal commitment was stimulated whereas fibers of 200 nm in diameter promoted hepatic differentiation. A nanopatterned surface with 120 nm of pit spacing was fabricated by electron beam lithography and was shown to promote hepatic differentiation of HepaRG compared with tissue culture dish [ 95 ]. These results strongly suggest that tailored ECM-like substrates are capable of influencing the hepatic differentiation of stem cells.…”