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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 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.

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Acknowledgements

We thank Dr. David Durantel (INSERM U871, Lyon, France), Prof. Marieta Costache (University of Bucharest, Romania) and Prof. Pedro Romero, M.D (University of Lausanne, Switzerland) for kindly providing the HepaRG, HeLa and Me290 cells, respectively. We thank Dr. Gabriela Kramer (University of Navarra, Pamplona, Spain), for sharing the pGL3 EIIPa1AT vector. We thank Dr. Alina Macovei (Institute of Biochemistry of the Romanian Academy, Bucharest, Romania) for technical assistance with initial steps of pLenti EIIPa1AT-DsRed cloning. This work was partially supported by the Romanian Academy PhD scholarship (SCOSAAR) awarded to Mihaela Uta. V.Dinca acknowledges financial support from Sciex project 12.313 and Romanian National Authority for Scientific Research (CNCS – UEFISCDI), under the projects PNII- PT-PCCA-2013-4-199 and PN-II-RU-TE-2014-4-2434. The authors would like to thank the assistance of Karl Boehlen and Erdem C. Siringil for the laser micromachining experiments, and to Vera Malheiro and K. Maniura-Weber for the topochip concept.

Author contributions

M.U. performed the plasmid cloning, generated and characterized the HepaRGDsRed stable cell line; V.D and PH prepared and characterized the PDMS microchips; L.E.S. performed cell sorting, assisted the set-up and analysis of TissueFAXS data, and assisted with preparing the manuscript; N.N. conceived the study and wrote the manuscript together with V.D.

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Correspondence to Valentina Dinca or Norica Branza-Nichita.

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Uta, M., Sima, L.E., Hoffmann, P. et al. 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 19, 3 (2017). https://doi.org/10.1007/s10544-016-0146-z

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  • DOI: https://doi.org/10.1007/s10544-016-0146-z

Keywords

  • HepaRG
  • Hepatocyte-like cells
  • HBV
  • Differentiation
  • Microstructured PDMS