Summary
An experimental model was optimised to assess functional modification of the permeability of cellular monolayers to calcium. Cells were cultured on transwell inserts in a multi-well plate format. Once monolayers had reached confluency, electrical resistance measurements (TEER) were used to determine barrier function. Apical chambers were subsequently incubated with test compounds and calcium. Barrier function was assessed by TEER to determine any changes in electrical resistance across the cell monolayer. Conditioned media was recovered from both apical and basal chambers of the transwell and calcium content determined by ICP-MS, in order to assess if test compounds could alter the specific permeability of the cell monolayer to calcium, resulting in a measurable increase in the basal chamber.
Assay principle
The basic assay principle is demonstrated in Figure 1. Cells grown on a transwell insert form an intact monolayer characterised by intercellular tight junction formation, associated with high electrical resistance (Ω) and low permeability. Should the monolayer be disrupted, e.g., through loss of cells or tight junctions, then the integrity of the monolayer is reduced, resulting in lower electrical resistance through increased passage of current. Furthermore, molecules (e.g., metal ions) may pass between the transwell apical and basal chambers. Liquid can be collected from each chamber and the presence of specific analytes detected by a range of techniques. These data reflect specific changes in permeability.
Figure 1: Schematic of the assay principle.
Case study
The following example data was generated as part of a study performed to assess the permeability of a monolayer of CaCo2 cells to calcium ions following treatment with a test compound. Cells were cultured in transwell chambers and an increase and then stabilisation of TEER resistance demonstrated the formation of a confluent monolayer of cells providing a non-permeable barrier. Media in both apical and basal chambers was replaced with a buffer solution. The buffer in the apical chamber was spiked with test compounds and calcium chloride. At pre-determined experimental timepoints, barrier function was assessed by TEER, and buffer solution from the apical and basal chambers was sampled. Chemical analysis by ICP-MS was used to determine calcium content in each sample; permeability to calcium was identified by a loss in the apical chamber and an increase in the basal chamber.
Example data are presented in Figure 2. Barrier function at t=0 h (reflecting ‘normal’ function in culture media) shows no difference between vehicle and test compounds. Following a 3 h incubation in HBSS spiked with calcium chloride and 1 % FBS, TEER demonstrates a significant loss in resistance induced by the test compound, characteristic of a loss of barrier function. Furthermore, analysis of calcium in HBSS recovered from the apical and basal chambers demonstrates that following incubation with the test compound, there is a small loss of calcium in the apical chamber. However, the reduction in barrier function (demonstrated by TEER) following incubation with the test compound is accompanied by a significant passage of calcium into the basal chamber of the transwell.
Figure 2 TEER analysis of barrier function and ICP-MS determination of calcium content in the apical and basal chambers of transwells. Bars represent the S.E.M., n=3 per condition, and significant differences were determined by t-test (*P=0.05-0.01).
Conclusion
At Cellomatics Biosciences, transwell format assays have been developed where barrier function of a confluent monolayer of cells reflecting a disease model of interest (e.g., CaCo-2 cells for inflammatory bowel disease) can be perturbed by treatment with test compounds. Barrier function can be assessed by means of electrical resistance (TEER). Furthermore, suitable analytical techniques can be used to assay specific molecules on the apical and basal sides of the cell monolayer, which correlates to specific changes in permeability hinted at by the barrier function.
