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In the European Union, in vitro methods will play a major role 10 in future legislation on testing chemicals and also in relation to the seventh amendment to the Cosmetic Directive. Both of these policies call for broad replacement, reduction and refinement of animal experiments. Therefore, an extensive interest has been shown by many pharmaceutical, food and cosmetic industries in applying these body-on-a-chip systems for studying drug, nutrient and xenobiotic absorption and possible toxic effects.

The development of chip-based systems with in vitro cell barrier models can be used to study parameters that control permeability and predict drug transport across these barriers in the early stages of drug discovery. The growing interest in body-on-a-chip systems is due to their potential for providing a high throughput, cost-effective and reliable method for predicting drug interactions in humans including transport phenomena.

These cell culture models also have an advantage of precisely controlling important transport parameters and experimental conditions. To perform permeability assessments on the cellular barriers, the complexity 11 of the in vitro models in these systems should reflect the variety of membrane transport systems, metabolic pathways involved and include a polarized cell layer. The in vitro models should also incorporate apical as well as basolateral compartments with appropriate composition of the aqueous medium on each side of the cell membrane.

It may not be possible to develop a single in vitro system that can simulate all the in vivo conditions, but use of various in vitro systems with more than one type of cell co-culture as decision making tools in early drug discovery 12 is a common practice. Numerous barrier systems 13 - 14 for predicting drug permeability, typically including cell cultures grown on permeable membranes, have been reported. The configuration in these systems is designed to allow access to both apical and basolateral compartments.

These models primarily include cells that grow in a monolayer when seeded on permeable membranes, and have physiologic characteristics similar to the barrier physiology and functionality.

The successful application of a system to predict drug absorption depends on how closely the in vitro model can mimic the characteristics of the in vivo barrier integrity.

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These in vitro models can be based on primary cells 15 or cell lines. A freeze-fracture electron microscopy of transmembrane fibrils and immunostaining for proteins characteristic of tight junctions occluding, ZO-1 and ZO-2 can provide qualitative insights into the barrier integrity of an endothelial or epithelial monolayer.

A simple assay that has been widely used is based on the permeability of the barrier to paracellular tracer compounds of various molecular weights. The first use of sucrose molecular weight: Da labeled with carbon for flux measurement on a brain endothelial monolayer has been reported.

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Radiolabeled markers have a short half-life and are not suitable for long term storage. Some of the other frequently used paracellular tracers include inulin molecular weight: 5 kDa and mannitol molecular weight: Da.

Albumin molecular weight: 67 kDa is another transendothelial permeability marker and has been observed in endothelial vesicles. An alternative approach that has been used is based on non-radioactive fluorescence labeled marker proteins such as fluorescein isothiocyanate FITC -labeled dextrans.

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