The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells |
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Authors: | Aisling M. Ross Darragh R. Walsh Rachel M. Cahalane Lynnette Marcar John J.E. Mulvihill |
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Affiliation: | 1. Bioscience and Bioengineering Research (BioSciBer), Bernal Institute, University of Limerick, Ireland;2. School of Engineering, University of Limerick, Ireland;3. Health Research Institute (HRI), University of Limerick, Ireland;4. Education and Health Sciences, University of Limerick, Ireland |
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Abstract: | 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 cm2 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 cm2, 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. |
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Keywords: | In vitro model Serum-free Transendothelial electrical resistance (TEER) Occludin Zonula occludens-1 (ZO-1) Drug delivery |
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