Development of a DsRed-expressing HepaRG cell line for real-time monitoring of hepatocyte-like cell differentiation by fluorescence imaging, with application in screening of novel geometric microstructured cell growth substrates

Biomed Microdevices. 2017 Mar;19(1):3. doi: 10.1007/s10544-016-0146-z.


The bipotent nature of the HepaRG cell line is a unique property among human hepatoma-derived cells. Cell treatment with specific differentiation inducers results in a mixture of hepatocyte- and biliary-like cells, accompanied by upregulation of liver-specific proteins, drug metabolizing enzymes, transcription regulators, membrane receptors or innate immune response effectors. These features make the HepaRG cells a suitable and handy replacement for primary hepatocytes, to study hepatic functions in vitro. However, cell differentiation is a long, variable process, requiring special culture conditions, while the resulting mixed cell populations is usually a major drawback. This process can potentially be controlled by interface characteristics, such as substrate topography. To screen for such novel substrates, we have first developed a new HepaRG cell line, designated as HepaRGDsRed, expressing the reporter gene DsRed. The fluorescent protein was expressed in hepatocyte- and not biliary-like cells, in a differentiation dependent-manner. We have further used replicated microstructured gradients of polydimethylsiloxane (PDMS) that allow three-dimensional manipulation in vitro, to monitor HepaRGDsRed differentiation in real time. We demonstrate that this approach enables the controlled assembly of viable hepatocyte-like cells for functional studies, which can be maintained in culture without loss of differentiation. The regulated expression of the DsRed reporter proved a valuable tool not only for rapid screening of novel cell growth substrates favoring cell differentiation, but also, to enrich the hepatocyte-like cell population by fluorescence-activated cell sorting to investigate liver-specific processes in vitro.

Keywords: Differentiation; HBV; HepaRG; Hepatocyte-like cells; Microstructured PDMS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods*
  • Cell Differentiation* / drug effects
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Drug Evaluation, Preclinical / methods*
  • Gene Expression
  • Genes, Reporter / genetics
  • Hepatocytes / cytology*
  • Hepatocytes / drug effects
  • Humans
  • Luminescent Proteins / genetics*
  • Optical Imaging*
  • Time Factors


  • Luminescent Proteins
  • fluorescent protein 583