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 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. [Display omitted] •Serum starvation increases the intracellular resistance of Caco-2 cells.•Max TEER values of 4783 ± 610 Ω cm2 were achieved in serum free conditions.•A barrier of 3000–4000 Ω cm2 could be maintained for up to 18 days.•Serum starvation leads to a significant increase in occludin expression.•Occludin levels correlate significantly with corresponding TEER values.