Epithelial cells, transepithelial resistance

The in vitro system we have been using to study the transepithelial transport is cultured Madin-Darby canine kidney (MDCK) epithelial cells (11). When cultured on microporous polycarbonate filters (Transwell, Costar, Cambridge, MA), MDCK cells will develop into monolayers mimicking the mucosal epithelium (11). When these cells reach confluence, tight junctions will be established between the cells, and free diffusion of solutes across the cell monolayer will be markedly inhibited. Tight junction formation can be monitored by measuring the transepithelial electrical resistance (TEER) across the cell monolayers. In Figure 1, MDCK cells were seeded at 2 X 104 cells per well in Transwells (0.4 p pore size) as described previously. TEER and 14C-sucrose transport were measured daily. To determine 14C-sucrose... 

Figure 14 Observed permeability coefficients of urea and mannitol across monolayers of rat alveolar epithelial cells in primary culture in the Transwell system are correlated with transepithelial electrical resistance and days in culture.

Figure 16 Correlation of effective pore radius of the paracellular route of rat alveolar epithelial cell monolayers with transepithelial electrical resistance and time in culture. Pore radii were calculated from the data shown in Figure 14.

Figure 9.1 Relationship between the transepithelial electrical resistance (TEER) value of the passage-cultured human nasal epithelial cell layer and permeability of 14C-mannitol (o, passage-2 A, passage-3 , passage-4) and budesonide ( , passage-2 , passage-3 , passage-4). (Data from Ref. [40]).

Figure 9.3 Changes in transepithelial electrical resistance (TEER) of human nasal epithelial cell layers grown under LCC ( ) versus AIC (A) conditions. Each data point represents the mean SD of three determinations. (Data from Ref. [46]).

Figure 26.5 Immunofluorescent staining for transformed airway epithelial cells with antibodies specific for (A) keratin 18 (a marker of epithelial cells) and (B) ZO-1 (marker of tight junction formation). Both markers indicate that the cells have retained epithelial characteristics after transformation. The staining for the presence of ZO-1 at the periphery of the cells indicates that the cells have not lost their polarity and can form tight monolayers that will generate a transepithelial resistance. This is a particularly attractive feature for the analysis of ion transport and transcellular transport of macromolecules. ZO-1 is also found in the nucleus and can also be detected by the anti-ZO-1 antibody.

Because of their similarities to alveolar type II cells, A549 cells appear also as an attractive model of the alveolar epithelium. An early study reported that A549 cells form cell layers with a low transepithelial electrical resistance [37] reflecting the lack of barrier formation, which made these cells a poor epithelial cell model. The formation of epithelial barrier function has recently been improved, when A549 cells are cultivated at an air-liquid interface [14]. 

FIGURE 20.11 Effect of DHA-LPC itself on transepithelial electrical resistance (TEER) between the apical chamber and the basolateral chamber. When the TEER value decreases, it indicates that tight junctions between the epithelial cells are opened. 

The differences between the various types of epithelia do not seem to depend on the properties of the cell membranes themselves but rather on the variation in the properties of the tight junctions which join the epithelial cells together at their apical borders. When these tight junctions are not highly resistive, then the transepithelial potential is literally short-circuited through the tight junction. 

Under prevalidation (ECVAM) we find the transepithelial resistance (TER) and para-ceUular permeability (PCP) in two renal cell lines (LLC-PKl, epithelial proximal tubular cells and MDCK, epithelial distal cells). TER measuranent and the transepithelial transport of uncharged small molecules like FlTC-inulin (PCP) are reliable parameters for characterising the intactness of an epithelial barrier. Cells are seeded onto 24-weU polycarbonate filter plates and then exposed to test substances. Barrier damage is measured for TER assessment and with fluorescence measuranent in the base plate for PCP (Duffet al.,2002). 

The HCE-T Tissue Construct (Gillette model) uses a transfected human corneal epithelial cell line (Kahn et al., 1993) cultured on collagen-membrane cell culture inserts, which, at the air-liquid interface, stratify to form a four- to six-layer epithelium, known as the HCE-T model. Transepithelial permeabiUty (TEP) to sodium fluorescein and transepitheUal electrical resistance (TER) have been identified as physiologically relevant parameters to evaluate the barrier function of the corneal epithelium. Cell viability can be determined by the MTT assay, and histomorphology can also be used as an endpoint (Kruszewski et al., 1997). 

We used in vitro models of lung epithelial cell lines or primary cells to determine E25 permeability. Two different cell types were used to mimic the airway and alveolar epithelium of the lung to study transport. Calu-3, a human cell line derived from an airway carcinoma, when grown at an air/liquid interface, differentiate to form a secretory airway epithelium (17). Rat primary epithelial cells isolated as described by Cheek et al. (18) form a tight barrier similar in structure and function to the alveolar surface. Both cell types when grown to confluence form tight junctions and differentiate and polarize so that the apical or air surface has different characteristics than the basolateral or blood side. The typical transepithelial resistance observed was 350 or >1000 ohms-cm for Calu-3 cells or primary rat alveolar cells, respectively. Once an acceptable resistance was achieved, E25 (2 mg/mL) was placed in either the apical or the basolateral chamber. Cell monolayers were incubated at 37°C for up to 3 hours and ELISA measured the amount of E25 that translocated the epithelial layer and appeared in the receiver well. The apparent permeability (Papp) of the epithelium for E25 was calculated as ... 

Cell cultures. MDCK cells were seeded in the Transwells at a density of 2.2 x 104 cells/cm. Cells were fed by changing medium in both upper (apical) and lower (basal) compartments periodically. Confluent monolayers were obtained at 5-7 days post-inoculation, when the cell density reached 4.5-5.0 x 105 cells/cm2, and a transepithelial electrical resistance (TEER) of about 2,000 ohms cm2 was measured using an epithelial voltohmmeter (EVOM, World Precision Instruments, West Haven, CT). The amount of FBS in the cell culture medium could be decreased as the cells approached their maximum resistance, and could be maintained at that point for 2 days or longer in medium containing 1% FBS. 

---