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
| Autor: | Patrik Hoffmann, Valentina Dinca, Livia Elena Sima, Norica Branza-Nichita, Mihaela Uta |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Time Factors Cell Survival Cellular differentiation Population Cell Biomedical Engineering Cell Culture Techniques Drug Evaluation Preclinical Gene Expression 02 engineering and technology Biology 03 medical and health sciences Downregulation and upregulation Cell surface receptor Genes Reporter Cell Line Tumor medicine Humans education Molecular Biology Cell Proliferation Reporter gene education.field_of_study Cell growth Optical Imaging Cell Differentiation Cell sorting 021001 nanoscience & nanotechnology Cell biology Luminescent Proteins 030104 developmental biology medicine.anatomical_structure Hepatocytes 0210 nano-technology |
| Zdroj: | Biomedical microdevices. 19(1) |
| ISSN: | 1572-8781 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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