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.
Highlights
IntroductionThe solubility and permeability of drug compounds are considered major factors contributing to drug bioavail ability and efficacy [1]
During drug development, the solubility and permeability of drug compounds are considered major factors contributing to drug bioavail ability and efficacy [1]
We investigated the effects of serum starvation on Caco-2 cell barrier formation
Summary
The solubility and permeability of drug compounds are considered major factors contributing to drug bioavail ability and efficacy [1]. Transendothelial electrical resistance (TEER) is commonly employed to assess the resistance of these models to para cellular diffusion The accuracy of these models relies on faithful rep resentation of tight junctional complexes. At tempts to improve TEER values in models include co-culturing [13,14,15,16,17,18,19,20,21], treatment with extracellular matrix components [20,21,22,23] and applica tion of fluid flow [20,21,24,25] These methods are costly and labor intensive, and still may not recapitulate in vivo barrier resistance
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