Cargando…

Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins

Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are h...

Descripción completa

Detalles Bibliográficos
Autores principales: Watanabe, Masaaki, Zemack, Helen, Johansson, Helene, Hagbard, Louise, Jorns, Carl, Li, Meng, Ellis, Ewa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012698/
https://www.ncbi.nlm.nih.gov/pubmed/27598296
http://dx.doi.org/10.1371/journal.pone.0161383
_version_ 1782452033472692224
author Watanabe, Masaaki
Zemack, Helen
Johansson, Helene
Hagbard, Louise
Jorns, Carl
Li, Meng
Ellis, Ewa
author_facet Watanabe, Masaaki
Zemack, Helen
Johansson, Helene
Hagbard, Louise
Jorns, Carl
Li, Meng
Ellis, Ewa
author_sort Watanabe, Masaaki
collection PubMed
description Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human hepatocytes, and that recombinant laminin is a promising xeno-free and chemical defined strategy for preservation of hepatocyte specific function in vitro.
format Online
Article
Text
id pubmed-5012698
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-50126982016-09-27 Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins Watanabe, Masaaki Zemack, Helen Johansson, Helene Hagbard, Louise Jorns, Carl Li, Meng Ellis, Ewa PLoS One Research Article Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human hepatocytes, and that recombinant laminin is a promising xeno-free and chemical defined strategy for preservation of hepatocyte specific function in vitro. Public Library of Science 2016-09-06 /pmc/articles/PMC5012698/ /pubmed/27598296 http://dx.doi.org/10.1371/journal.pone.0161383 Text en © 2016 Watanabe et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://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
Watanabe, Masaaki
Zemack, Helen
Johansson, Helene
Hagbard, Louise
Jorns, Carl
Li, Meng
Ellis, Ewa
Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
title Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
title_full Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
title_fullStr Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
title_full_unstemmed Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
title_short Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins
title_sort maintenance of hepatic functions in primary human hepatocytes cultured on xeno-free and chemical defined human recombinant laminins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012698/
https://www.ncbi.nlm.nih.gov/pubmed/27598296
http://dx.doi.org/10.1371/journal.pone.0161383
work_keys_str_mv AT watanabemasaaki maintenanceofhepaticfunctionsinprimaryhumanhepatocytesculturedonxenofreeandchemicaldefinedhumanrecombinantlaminins
AT zemackhelen maintenanceofhepaticfunctionsinprimaryhumanhepatocytesculturedonxenofreeandchemicaldefinedhumanrecombinantlaminins
AT johanssonhelene maintenanceofhepaticfunctionsinprimaryhumanhepatocytesculturedonxenofreeandchemicaldefinedhumanrecombinantlaminins
AT hagbardlouise maintenanceofhepaticfunctionsinprimaryhumanhepatocytesculturedonxenofreeandchemicaldefinedhumanrecombinantlaminins
AT jornscarl maintenanceofhepaticfunctionsinprimaryhumanhepatocytesculturedonxenofreeandchemicaldefinedhumanrecombinantlaminins
AT limeng maintenanceofhepaticfunctionsinprimaryhumanhepatocytesculturedonxenofreeandchemicaldefinedhumanrecombinantlaminins
AT ellisewa maintenanceofhepaticfunctionsinprimaryhumanhepatocytesculturedonxenofreeandchemicaldefinedhumanrecombinantlaminins