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Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology
Primary hepatocytes are a major tool in biomedical research. However, obtaining high yields of variable hepatocytes is technically challenging. Most protocols rely on the two-step collagenase perfusion protocol introduced by Per Ottar Seglen in 1976. In this procedure, the liver is perfused in situ...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9496349/ https://www.ncbi.nlm.nih.gov/pubmed/36140299 http://dx.doi.org/10.3390/biomedicines10092198 |
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author | Poggel, Carsten Adams, Timo Janzen, Ronald Hofmann, Alexander Hardt, Olaf Roeb, Elke Schröder, Sarah K. Tag, Carmen G. Roderfeld, Martin Weiskirchen, Ralf |
author_facet | Poggel, Carsten Adams, Timo Janzen, Ronald Hofmann, Alexander Hardt, Olaf Roeb, Elke Schröder, Sarah K. Tag, Carmen G. Roderfeld, Martin Weiskirchen, Ralf |
author_sort | Poggel, Carsten |
collection | PubMed |
description | Primary hepatocytes are a major tool in biomedical research. However, obtaining high yields of variable hepatocytes is technically challenging. Most protocols rely on the two-step collagenase perfusion protocol introduced by Per Ottar Seglen in 1976. In this procedure, the liver is perfused in situ with a recirculating, constant volume of calcium-free buffer, which is maintained at 37 °C and continuously oxygenated. In a second step, the liver is removed from the carcass and perfused with a collagenase solution in order to dissociate the extracellular matrix of the liver and liberate individual cells. Finally, the dissected hepatocytes are further purified and concentrated by density-based centrifugation. However, failure in proper cannulation, incomplete enzymatic digestion or over-digestion can result in low cell yield and viability. Here we present a novel semi-automated perfusion device, which allows gentle, rapid and efficient generation of a single-cell suspension from rodent livers. In combination with prefabricated buffers, the system allows reliable and highly reproducible isolation of primary hepatocytes. |
format | Online Article Text |
id | pubmed-9496349 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-94963492022-09-23 Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology Poggel, Carsten Adams, Timo Janzen, Ronald Hofmann, Alexander Hardt, Olaf Roeb, Elke Schröder, Sarah K. Tag, Carmen G. Roderfeld, Martin Weiskirchen, Ralf Biomedicines Communication Primary hepatocytes are a major tool in biomedical research. However, obtaining high yields of variable hepatocytes is technically challenging. Most protocols rely on the two-step collagenase perfusion protocol introduced by Per Ottar Seglen in 1976. In this procedure, the liver is perfused in situ with a recirculating, constant volume of calcium-free buffer, which is maintained at 37 °C and continuously oxygenated. In a second step, the liver is removed from the carcass and perfused with a collagenase solution in order to dissociate the extracellular matrix of the liver and liberate individual cells. Finally, the dissected hepatocytes are further purified and concentrated by density-based centrifugation. However, failure in proper cannulation, incomplete enzymatic digestion or over-digestion can result in low cell yield and viability. Here we present a novel semi-automated perfusion device, which allows gentle, rapid and efficient generation of a single-cell suspension from rodent livers. In combination with prefabricated buffers, the system allows reliable and highly reproducible isolation of primary hepatocytes. MDPI 2022-09-06 /pmc/articles/PMC9496349/ /pubmed/36140299 http://dx.doi.org/10.3390/biomedicines10092198 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Communication Poggel, Carsten Adams, Timo Janzen, Ronald Hofmann, Alexander Hardt, Olaf Roeb, Elke Schröder, Sarah K. Tag, Carmen G. Roderfeld, Martin Weiskirchen, Ralf Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology |
title | Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology |
title_full | Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology |
title_fullStr | Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology |
title_full_unstemmed | Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology |
title_short | Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology |
title_sort | isolation of hepatocytes from liver tissue by a novel, semi-automated perfusion technology |
topic | Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9496349/ https://www.ncbi.nlm.nih.gov/pubmed/36140299 http://dx.doi.org/10.3390/biomedicines10092198 |
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