title: 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.
abstract: 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 HepaRGDsReddifferentiation 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.
authors.affiliation: Department of Viral Glycoproteins, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Sector 6, Bucharest, Romania.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Uta
authors.affiliation: Department of Molecular Cell Biology, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Bucharest, Sector 6, Romania.
authors.forename: Livia E
authors.initials: LE
authors.lastname: Sima
authors.affiliation: Laboratory for Advanced Materials Processing, EMPA - Swiss Federal Laboratories for Materials Science and Technology, Feuerwerkerstrasse 39, 3602, Thun, Switzerland.
authors.forename: Patrik
authors.initials: P
authors.lastname: Hoffmann
authors.affiliation: Laboratory for Advanced Materials Processing, EMPA - Swiss Federal Laboratories for Materials Science and Technology, Feuerwerkerstrasse 39, 3602, Thun, Switzerland. dincavalentina@yahoo.com.|National Institute for Lasers, Plasma and Radiation Physics, Atomistilor 409, 077125, Magurele, Romania. dincavalentina@yahoo.com.
authors.forename: Valentina
authors.initials: V
authors.lastname: Dinca
authors.affiliation: Department of Viral Glycoproteins, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Sector 6, Bucharest, Romania. norica70@yahoo.co.uk.
authors.forename: Norica
authors.initials: N
authors.lastname: Branza-Nichita
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D061848:Optical Imaging; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; C413663:fluorescent protein 583
keywords: Differentiation; HBV; HepaRG; Hepatocyte-like cells; Microstructured PDMS