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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
K. Anselme, M. Bigerelle, Role of materials surface topography on mammalian cell response. Int. Mater. Rev. 56, 243–266 (2011)
C.K. Choi, M.T. Breckenridge, C.S. Chen, Engineered materials and the cellular microenvironment: a strengthening interface between cell biology and bioengineering. Trends Cell Biol. 20, 705–714 (2010)
M. Darnell, M. Ulvestad et al., In vitro evaluation of major in vivo drug metabolic pathways using primary human hepatocytes and HepaRG cells in suspension and a dynamic three-dimensional bioreactor system. J. Pharmacol. Exp. Ther. 343, 134–144 (2012)
V. Dinca, L.E. Sima et al., in Recent Advances in Biopolymers. Bio-interfaces engineering using laser based methods for controlled regulation of mesenchymal stem cells response in vitro, (InTech, Rijeka, 2016), p. 221–241
H.H. Gerets, K. Tilmant et al., Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol. Toxicol. 28, 69–87 (2012)
P. Gripon, S. Rumin et al., Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. U. S. A. 99, 15655–15660 (2002)
A. Guillouzoa, A. Corlub et al., The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem. Biol. Interact. 168, 66–73 (2007)
O. Hantz, R. Parent et al., Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J. Gen. Virol. 90, 127–135 (2009)
E.C. Harvey, P.T. Rumsby, M.C. Gower, and J.L. Remnant, in Proceedings of SPIE, Micromachining and Microfabrication Process Technology, Austin, TX. Microstructuring by excimer laser, (1995), p. 266–277
A.S. Holmes, Excimer laser micromachining with half-tone masks for the fabrication of 3-D microstructures. IEE Proc. Sci. Meas. Technol. 151, 85–92 (2004)
M.G. Kramer, M. Barajas et al., In vitro and in vivo comparative study of chimeric liver-specific promoters. Mol. Ther. 7, 375–385 (2003)
J. Lucifora, D. Durantel et al., Control of hepatitis B virus replication by innate response of HepaRG cells. Hepatology 51, 63–72 (2010)
A. Macovei, C. Petrareanu et al., Regulation of hepatitis B virus infection by Rab5, Rab7, and the endolysosomal compartment. J. Virol. 87, 6415–6427 (2013)
C. Petrareanu, A. Macovei et al., Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells. PLoS ONE 8, e71859 (2013)
A.M. Schmidt, S.R. Fagerer et al., Molecular phenotypic profiling of a Saccharomyces cerevisiae strain at the single-cell level. Analyst 139, 5709–5717 (2014)
B. Trappmann, J.E. Gautrot et al., Extracellular-matrix tethering regulates stem-cell fate. Nat. Mater. 11, 642–649 (2012)
S. Tsuji, F. Kawamura et al., Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells. PLoS ONE 9, e104123 (2014)
G.R. Wilkinson, Drug metabolism and variability among patients in drug response. N. Engl. J. Med. 352, 2211–2221 (2005)
H. Yan, G. Zhong et al., Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1, e00049 (2012)
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.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors have no conflict of interest to declare.
Rights and permissions
About this article
Cite this article
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
Published:
DOI: https://doi.org/10.1007/s10544-016-0146-z