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Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas
The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDH-related PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9524698/ https://www.ncbi.nlm.nih.gov/pubmed/36178902 http://dx.doi.org/10.1371/journal.pone.0274478 |
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author | Bayley, Jean-Pierre Rebel, Heggert G. Scheurwater, Kimberly Duesman, Dominique Zhang, Juan Schiavi, Francesca Korpershoek, Esther Jansen, Jeroen C. Schepers, Abbey Devilee, Peter |
author_facet | Bayley, Jean-Pierre Rebel, Heggert G. Scheurwater, Kimberly Duesman, Dominique Zhang, Juan Schiavi, Francesca Korpershoek, Esther Jansen, Jeroen C. Schepers, Abbey Devilee, Peter |
author_sort | Bayley, Jean-Pierre |
collection | PubMed |
description | The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDH-related PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to the classical 2D-culture of SDH-related human paragangliomas and pheochromocytomas, with the ultimate goal of identifying a viable tumour-derived cell line. PPGL tumour tissue/cells (chromaffin cells) were cultured in a variety of media formulations and supplements. Tumour explants and dissociated primary tumour cells were cultured and stained with a range of antibodies to identify markers suitable for use in human PPGL culture. We cultured 62 PPGLs, including tumours with confirmed SDHB, SDHC and SDHD variants, as well as several metastatic tumours. Testing a wide range of basic cell culture media and supplements, we noted a marked decline in chromaffin cell numbers over a 4–8 week period but the persistence of small numbers of synaptophysin/tyrosine hydroxylase-positive chromaffin cells for up to 99 weeks. In cell culture, immunohistochemical staining for chromogranin A and neuron-specific enolase was generally negative in chromaffin cells, while staining for synaptophysin and tyrosine hydroxylase was generally positive. GFAP showed the most consistent staining of type II sustentacular cells. Of the media tested, low serum or serum-free media best sustained relative chromaffin cell numbers, while lactate enhanced the survival of synaptophysin-positive cells. Synaptophysin-positive PPGL tumour cells persist in culture for long periods but show little evidence of proliferation. Synaptophysin was the most consistent cell marker for chromaffin cells and GFAP the best marker for sustentacular cells in human PPGL cultures. |
format | Online Article Text |
id | pubmed-9524698 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-95246982022-10-01 Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas Bayley, Jean-Pierre Rebel, Heggert G. Scheurwater, Kimberly Duesman, Dominique Zhang, Juan Schiavi, Francesca Korpershoek, Esther Jansen, Jeroen C. Schepers, Abbey Devilee, Peter PLoS One Research Article The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDH-related PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to the classical 2D-culture of SDH-related human paragangliomas and pheochromocytomas, with the ultimate goal of identifying a viable tumour-derived cell line. PPGL tumour tissue/cells (chromaffin cells) were cultured in a variety of media formulations and supplements. Tumour explants and dissociated primary tumour cells were cultured and stained with a range of antibodies to identify markers suitable for use in human PPGL culture. We cultured 62 PPGLs, including tumours with confirmed SDHB, SDHC and SDHD variants, as well as several metastatic tumours. Testing a wide range of basic cell culture media and supplements, we noted a marked decline in chromaffin cell numbers over a 4–8 week period but the persistence of small numbers of synaptophysin/tyrosine hydroxylase-positive chromaffin cells for up to 99 weeks. In cell culture, immunohistochemical staining for chromogranin A and neuron-specific enolase was generally negative in chromaffin cells, while staining for synaptophysin and tyrosine hydroxylase was generally positive. GFAP showed the most consistent staining of type II sustentacular cells. Of the media tested, low serum or serum-free media best sustained relative chromaffin cell numbers, while lactate enhanced the survival of synaptophysin-positive cells. Synaptophysin-positive PPGL tumour cells persist in culture for long periods but show little evidence of proliferation. Synaptophysin was the most consistent cell marker for chromaffin cells and GFAP the best marker for sustentacular cells in human PPGL cultures. Public Library of Science 2022-09-30 /pmc/articles/PMC9524698/ /pubmed/36178902 http://dx.doi.org/10.1371/journal.pone.0274478 Text en © 2022 Bayley et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Bayley, Jean-Pierre Rebel, Heggert G. Scheurwater, Kimberly Duesman, Dominique Zhang, Juan Schiavi, Francesca Korpershoek, Esther Jansen, Jeroen C. Schepers, Abbey Devilee, Peter Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas |
title | Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas |
title_full | Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas |
title_fullStr | Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas |
title_full_unstemmed | Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas |
title_short | Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas |
title_sort | long-term in vitro 2d-culture of sdhb and sdhd-related human paragangliomas and pheochromocytomas |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9524698/ https://www.ncbi.nlm.nih.gov/pubmed/36178902 http://dx.doi.org/10.1371/journal.pone.0274478 |
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