Cell culture barrier assays are used to model barrier tissues (kidney, lung, intestinal, etc.) in drug delivery, drug absorbance, wound healing, bacterial/viral infections, inflammation and other studies. These barriers can be formed in vitro when epithelial cells are seeded as a single cell suspension and grow together until fused into one tissue. Typically, this tissue will be one epithelial cell in thickness and is termed the epithelial monolayer. In standard barrier assays, cells are seeded onto porous membranes and observed noninvasively via trans-epithelial electrical resistance (TEER) measurements. TEER measurements are validated, label-free and fast techniques for indicating epithelial monolayer barrier formation before further evaluation and experimentation. Barrier formation is observed by the increase in resistance measure; a confluent epithelial monolayer will plateau at higher resistance values. For most barrier assays, an epithelial monolayer is the desired type of cell barrier because it models the single-cell thickness of lung-to-blood, kidney filtration, or intestinal absorption and can be used to analyze the paracellular space between epithelial cells. Once the integrity of the cellular barrier has been confirmed, these tissues can then be used in experiments that probe barrier permeability, disruption, or strength.
Tight junction pores are physiological gatekeepers of paracellular transport in epithelial tissues. Paracellular permeability assays assess monolayer barrier formation and tightness of paracellular tight junctions by measuring the amount of a molecule that passes through an epithelial cell monolayer in a given amount of time. This type of assay requires live cells and is usually non-destructive (e.g., Lucifer yellow paracellular permeability assay). Immunostaining and tissue immunocytochemistry (ICC) are commonly used for visualization, pre-empting the need for electron microscopy. Many of these analyses are focused on barrier pass-through tracked as dye, drug conjugates, or amount of target protein transferred. Alternative analyses involve visualizing the membrane using stains such as crystal violet, fluorescent antibody staining, and ICC.
Figure 1. Barrier assay principle using Millicell® hanging cell culture inserts. A) On day 0, epithelial cells are seeded in single-cell suspension onto the Millicell® hanging cell culture insert membrane. B) After several days (~3 days after seeding for MDCK cells) the cells will be confluent enough to begin taking TEER measurements, assessing the epithelial cell monolayer formation based on the resistance value. C) TEER measurements can be taken daily or every other day to track monolayer barrier formation until cells are seen to plateau at a consistent resistance value (~7 days after seeding for MDCK cells). D) At this point the media is exchanged for barrier assay liquid (Hank’s balanced salt solution) and permeability assay dye/molecule (e.g., Lucifer yellow dye) is added to the apical side of the membrane. E) The small molecules are incubated with the epithelial monolayer at 37°C for recommended protocol length (e.g., 1 hour), and then the liquid from the basolateral side of the membrane insert is evaluated for the amount of pass-through of the chosen molecule.
Barrier assays are performed on hanging inserts as porous membranes offer vastly improved characteristics over standard polystyrene surfaces for what is called 2.5D cell culturing. Membranes in these inserts differ in terms of pore size, which becomes important for different assay types. For example, migration assays will have the widest range of pore sizes based on the cell type involved in the experiment.
The Millicell® ERS-2 voltohmmeter uses low current and voltages to allow for non-destructive testing for epithelial monolayer confluence in cell cultures. TEER instruments produce a low AC current that avoids electrode metal deposits and adverse effects on tissues which can otherwise be caused by higher DC currents. Confluence of a cellular monolayer is determined by an increase or a plateau in tissue resistance detected using TEER readers.
MDCK culture media
Seeding cells onto a 24-well Millicell® hanging cell culture insert
Millicell® ERS-2 meter functionality check
Figure 2. During the meter functionality check, the Millicell® ERS-2 can be adjusted with a flathead screwdriver to the known value of the test electrode.
Measuring cell resistance
Figure 3. All membranes depicted are high-density (1×108) 0.4 µm pore size PET 24-well membranes. These results compare Millicell® hanging cell culture inserts (PTHT24H48) to two comparable brands of inserts. MDCK cells were seeded at the same seeding density in parallel across all three manufacturers. The three brands of inserts are not significantly different from each other on day 7 after seeding and follow a similar kinetic for the formation of a monolayer.
Figure 4. Lucifer yellow permeability assays test the formation of the cellular monolayer and tight junctions between cells. All membranes depicted are high-density (1 x 108 pores) 0.4 µm pore size PET membranes in 24-well formats. These results compare the Millicell® hanging cell culture insert (PTHT24H48) to two other brands of inserts. This assay was performed on Day 7 of the assay in Figure 3. Barrier formation is deemed successful if there is less than 5% pass-through of the dye from the apical side of the membrane into the basolateral side of the membrane (pass-through depicted in the final panel in Figure 1). There were no statistical differences between these inserts.
Figure 5. TEER values are dependent on starting seeding densities, which correlate with the confluence of cells on the Millicell® hanging cell culture insert. Higher cell seeding densities will form a cellular monolayer faster. Depending on the experiment, it might be more beneficial to have higher or lower seeding densities depending on the study’s focus. In the example above, MDCK cells were seeded from the same stock and were measured for resistance on the days indicated. All measurements were taken on the Millicell® ERS-2 voltohmmeter using the STX01 electrode with Millicell® hanging cell culture inserts (PTHT24H48).
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