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The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells
Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 b...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358646/ https://www.ncbi.nlm.nih.gov/pubmed/34401532 http://dx.doi.org/10.1016/j.bbrep.2021.101096 |
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author | Ross, Aisling M. Walsh, Darragh R. Cahalane, Rachel M. Marcar, Lynnette Mulvihill, John J.E. |
author_facet | Ross, Aisling M. Walsh, Darragh R. Cahalane, Rachel M. Marcar, Lynnette Mulvihill, John J.E. |
author_sort | Ross, Aisling M. |
collection | PubMed |
description | Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 barrier stability and resistance. The effect of serum starvation on tight junction production was examined using transwell models; specifically, transendothelial electrical resistance (TEER), and the expression and localization of tight junction proteins, occludin and zonula occludens-1 (ZO-1), were studied using western blotting and immunofluorescence. Changing cells to serum-free media 2 days post-seeding resulted in TEER readings of nearly 5000 Ω cm(2) but the TEER rapidly declined subsequently. Meanwhile, exchanging cells to serum-free media 4–6 days post-seeding produced barriers with resistance readings between 3000 and 4000 Ω cm(2), which could be maintained for 18 days. This corresponded to an increase in occludin levels. Serum starvation as a means of barrier formation is simple, reproducible, and cost-effective. It could feasibly be implemented in a variety of pre-clinical pharmaceutical assessments of drug permeability across various biological barriers with the view to improving the clinical translation of novel therapeutics. |
format | Online Article Text |
id | pubmed-8358646 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-83586462021-08-15 The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells Ross, Aisling M. Walsh, Darragh R. Cahalane, Rachel M. Marcar, Lynnette Mulvihill, John J.E. Biochem Biophys Rep Research Article Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 barrier stability and resistance. The effect of serum starvation on tight junction production was examined using transwell models; specifically, transendothelial electrical resistance (TEER), and the expression and localization of tight junction proteins, occludin and zonula occludens-1 (ZO-1), were studied using western blotting and immunofluorescence. Changing cells to serum-free media 2 days post-seeding resulted in TEER readings of nearly 5000 Ω cm(2) but the TEER rapidly declined subsequently. Meanwhile, exchanging cells to serum-free media 4–6 days post-seeding produced barriers with resistance readings between 3000 and 4000 Ω cm(2), which could be maintained for 18 days. This corresponded to an increase in occludin levels. Serum starvation as a means of barrier formation is simple, reproducible, and cost-effective. It could feasibly be implemented in a variety of pre-clinical pharmaceutical assessments of drug permeability across various biological barriers with the view to improving the clinical translation of novel therapeutics. Elsevier 2021-08-07 /pmc/articles/PMC8358646/ /pubmed/34401532 http://dx.doi.org/10.1016/j.bbrep.2021.101096 Text en © 2021 The Authors. Published by Elsevier B.V. https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Ross, Aisling M. Walsh, Darragh R. Cahalane, Rachel M. Marcar, Lynnette Mulvihill, John J.E. The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells |
title | The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells |
title_full | The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells |
title_fullStr | The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells |
title_full_unstemmed | The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells |
title_short | The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells |
title_sort | effect of serum starvation on tight junctional proteins and barrier formation in caco-2 cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358646/ https://www.ncbi.nlm.nih.gov/pubmed/34401532 http://dx.doi.org/10.1016/j.bbrep.2021.101096 |
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