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Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison

Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavio...

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Autores principales: Coronado, Ramon E., Somaraki-Cormier, Maria, Natesan, Shanmugasundaram, Christy, Robert J., Ong, Joo L., Halff, Glenn A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: SAGE Publications 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802637/
https://www.ncbi.nlm.nih.gov/pubmed/29390876
http://dx.doi.org/10.1177/0963689717742157
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author Coronado, Ramon E.
Somaraki-Cormier, Maria
Natesan, Shanmugasundaram
Christy, Robert J.
Ong, Joo L.
Halff, Glenn A.
author_facet Coronado, Ramon E.
Somaraki-Cormier, Maria
Natesan, Shanmugasundaram
Christy, Robert J.
Ong, Joo L.
Halff, Glenn A.
author_sort Coronado, Ramon E.
collection PubMed
description Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavior, adhesion, migration, differentiation, and proliferation that in turn affect homeostasis and tissue regeneration. Previous studies have shown that various processing methods have a distinctive way of affecting the composition of the decellularized ECM. In this study, we developed a bioactive substrate for hepatocytes in vitro, made of decellularized and solubilized liver tissue. The present work is a comparative approach of 2 different methods. First, we decellularized porcine liver tissue with ammonium hydroxide versus a sodium deoxycholate method, then characterized the decellularized tissue using various methods including double stranded DNA (dsDNA) content, DNA size, immunogenicity, and mass spectrometry. Second, we solubilized the decellularized porcine liver with hydrochloric acid versus acetic acid (AA) and characterized the resultant solubilized tissues using relevant methodologies including protein yield, immunogenicity, and bioactivity. Finally, we isolated primary porcine hepatocytes, cultured, and evaluated their bioactivity on the optimized decellularized–solubilized liver substrate. The decellularized porcine liver ECM processed by the ammonium hydroxide method and solubilized with AA displayed higher ECM integrity, low dsDNA, no evidence of intact nuclei, low human monocyte chemoattraction, and the presence of key molecules typically found in the native liver, a very important element for normal cell function. In addition, primary porcine hepatocytes showed enhanced functionality including albumin and urea production and bile canaliculi formation when cultured on the developed liver substrate compared to type I collagen.
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spelling pubmed-58026372018-02-12 Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison Coronado, Ramon E. Somaraki-Cormier, Maria Natesan, Shanmugasundaram Christy, Robert J. Ong, Joo L. Halff, Glenn A. Cell Transplant Original Articles Biologic substrates, prepared by decellularizing and solubilizing tissues, have been of great interest in the tissue engineering field because of the preservation of complex biochemical constituents found in the native extracellular matrix (ECM). The integrity of the ECM is critical for cell behavior, adhesion, migration, differentiation, and proliferation that in turn affect homeostasis and tissue regeneration. Previous studies have shown that various processing methods have a distinctive way of affecting the composition of the decellularized ECM. In this study, we developed a bioactive substrate for hepatocytes in vitro, made of decellularized and solubilized liver tissue. The present work is a comparative approach of 2 different methods. First, we decellularized porcine liver tissue with ammonium hydroxide versus a sodium deoxycholate method, then characterized the decellularized tissue using various methods including double stranded DNA (dsDNA) content, DNA size, immunogenicity, and mass spectrometry. Second, we solubilized the decellularized porcine liver with hydrochloric acid versus acetic acid (AA) and characterized the resultant solubilized tissues using relevant methodologies including protein yield, immunogenicity, and bioactivity. Finally, we isolated primary porcine hepatocytes, cultured, and evaluated their bioactivity on the optimized decellularized–solubilized liver substrate. The decellularized porcine liver ECM processed by the ammonium hydroxide method and solubilized with AA displayed higher ECM integrity, low dsDNA, no evidence of intact nuclei, low human monocyte chemoattraction, and the presence of key molecules typically found in the native liver, a very important element for normal cell function. In addition, primary porcine hepatocytes showed enhanced functionality including albumin and urea production and bile canaliculi formation when cultured on the developed liver substrate compared to type I collagen. SAGE Publications 2018-02-02 2017-12 /pmc/articles/PMC5802637/ /pubmed/29390876 http://dx.doi.org/10.1177/0963689717742157 Text en © The Author(s) 2018 http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
spellingShingle Original Articles
Coronado, Ramon E.
Somaraki-Cormier, Maria
Natesan, Shanmugasundaram
Christy, Robert J.
Ong, Joo L.
Halff, Glenn A.
Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison
title Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison
title_full Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison
title_fullStr Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison
title_full_unstemmed Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison
title_short Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture: Method Optimization and Comparison
title_sort decellularization and solubilization of porcine liver for use as a substrate for porcine hepatocyte culture: method optimization and comparison
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802637/
https://www.ncbi.nlm.nih.gov/pubmed/29390876
http://dx.doi.org/10.1177/0963689717742157
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