Methods and compositions for measuring cell permeability
Embodiments of the present invention provide an in vitro assay that measures cell monolayer permeability. The assay utilizes detection of a binding pair initially separated by the cell monolayer as a qualitative and quantitative measure of the permeability of the monolayer. In certain embodiments, the assay is performed in the presence of a test substance that may or may not affect cell permeability. In particular aspects, the assay is performed on a substrate that is elastic. One can also assay spatial resolution of local changes by visualizing regional variations of the cell monolayers.
1. An in vitro method for measuring the degree of permeability of an endothelial cell monolayer, comprising the following steps in the order listed:
providing a substrate having a matrix, the endothelial cell monolayer, and a plurality of first binding pair members associated with the matrix, wherein the matrix is contiguous with the substrate and the endothelial cell monolayer is in direct contact with the matrix and with the plurality of first binding pair members associated with the matrix, wherein the matrix and first binding pair members are positioned between the monolayer and the substrate;
subjecting the substrate to mechanical stimulation by stretching the substrate;
contacting the cell monolayer with a plurality of labeled second binding pair members; wherein the first and second binding pair members are separated by the cell monolayer; and
detecting the amount of binding of a plurality of first binding pair members to a plurality of second binding pair members by visualizing the label through the side of the substrate that is opposite the matrix, wherein the degree of permeability is determined from the amount of binding of the first binding pair members to the second binding pair members; and wherein the first binding pair members are biotin and the second binding pair members are streptavidin.
2. The method of claim 1 , wherein the amount of binding is detected in more than one region of the cell monolayer.
3. The method of claim 2 , further comprising detecting the presence or absence of differential permeability in different regions of the cell monolayer comprising comparing the amount of binding in the regions of the cell monolayer; wherein a difference in the amount of binding in the regions indicates a presence of differential permeability and wherein the same amount of binding in the regions indicates and absence of differential permeability.
4. The method of claim 1 , wherein the stretching of the substrate is performed cyclically.
5. The method of claim 1 , further comprising exposing the cell monolayer to a compound that detects a cell junction protein.
6. The method of claim 1 , wherein the permeability that is measured is intercellular permeability.
7. The method of claim 1 , wherein the permeability that is measured is transcellular permeability.
8. The method of claim 1 , wherein the substrate is impermeable.
9. The method of claim 1 , wherein the substrate is made of glass, metal, or a silicone-based material.
10. The method of claim 1 , wherein the substrate is a culture dish, slide, well, cover slip, stretch or flow chamber, or elastic substrate.
11. The method of claim 1 , further comprising the step of exposing a test substance to the cell monolayer before the amount of binding is detected.
12. The method of claim 11 , wherein binding is detected between five minutes and five days after exposing the test substance to the cell monolayer.