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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...

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Autores principales: Poggel, Carsten, Adams, Timo, Janzen, Ronald, Hofmann, Alexander, Hardt, Olaf, Roeb, Elke, Schröder, Sarah K., Tag, Carmen G., Roderfeld, Martin, Weiskirchen, Ralf
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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