Transepithelial electrical resistance TEER

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. 

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 1. The correlation of transepithelial electrical resistance (TEER) with the transepithelial transport of 14C-sucrose in MDCK cell monolayers grown on microporous filters.

Evaluation of the epithelial integrity can be performed by measuring the transepithelial electrical resistance (TEER). TEER values ranging from 150 ohms.cm2 up to 600 ohms.cm2 have been reported. An alternative method for assessing the monolayer integrity is to monitor the flux of hydrophilic marker molecules that pass the monolayers by the paracellular route (e.g., mannitol, Na-fluorescein, or atenolol). 

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]).

The BRB is anatomically separated into an inner and outer blood barrier. The RPE is a tight, ion-transporting barrier and paracellular transport of polar solutes across the RPE from the choroid is restricted. This is reflected by the transepithelial electrical resistance (TEER) of the cell layers. It has been reported that the choroidal TEER ( 9 ohm cm2) is less than 10% the total resistance of isolated bovine RPE-choroid (100-150 ohm cm2). Passive RPE diffusion has been shown to be a function of lipophilicity. The endothelium of the retinal vessels represents the inner BRB and offers considerable resistance to systemic penetration of drugs. 

Generally, low molecular mass permeation enhancers can be divided into transcellular and paracellular permeation enhancers. On the one hand the potential of permeation enhancers to open the paracellular route of uptake can be determined by the reduction in the transepithelial electrical resistance (TEER) (enhancement potential = EP). On the other hand the potential of permeation enhancers to open the transcellular route of uptake can be determined by the lactate dehydrogenase (LDH) assay (LDH potential = LP). The parameter K = (EP—LP)/EP represents the relative contribution of the paracellular pathway. Consequently, a K value of 0 means predominantly transcellular and a K value of 1 means predominantly paracellular. Based on this classification system Whitehead and Mitragotri classified over 50 low molecular mass permeation enhancers showing that most of them are paracellular and only a few of them are transcellular permeation enhancers (2008). 

Sutton, S.C. Forbes, A.E. Cargill, R. Hochman, J.H. LeCluyse, E.L. Simultaneous in vitro measurement of intestinal tissue permeability and transepithelial electrical-resistance (teer) using sweetana grass diffusion cells. Pharm. Res. 1992, 9, 316-319. 

Table 3 summarizes different additives that have been proposed (1) as solubility enhancers, (2) to reduce adsorption or (3) to increase the biorelevance (pH, bile salts, sink conditions) in in ly r oabsorption models. The maximal tolerable concentration of each was determined by assessing the flux of a hydrophilic marker, the transepithelial electrical resistance (TEER) and/or the release of enzymes or the enzymatic status of the cell monolayers. 

This buffer is used in both the unidirectional (permeability) and the bidirectional (Efflux, Section 4.3.2). In the permeability assay, NCEs are placed into the apical compartment of the Transwell. Duplicate samples are taken immediately after compound addition from the apical compartment (zero time) and then after 2h from both the apical and basolateral compartments for LC-MS/MS analysis. The integrity of the monolayer is confirmed by the measurement of the transepithelial electrical resistance (TEER), which must be above a certain limit to be used for transport experiments. In addition, with each experiment a transcellular and a paracellular marker are included for quality control. 

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. 

These are just some examples that demonstrate the complexity of the processes involved in intestinal absorption other parameters that need to be taken into consideration when studying intestinal transport include transepithelial electrical resistance (TEER), active efflux pumps (such as P-gp), passive transcellular and paracellular diffusion. 

When using Caco-2 or TC7 cells, parameters such as transepithelial electrical resistance (TEER), differentiation marker, morphology, P-gp expression, and monolayer integrity need to be tested failure to do so is often the reason for wide interlaboratory variations. Caco-2 cell lines contain only a single cell type and do not form a mucus layer. As this can limit the absorption of lipophilic compounds in particular, the coculturing of Caco-2 cells and mucus-secreting lines such as HT29-MTX has been proposed to overcome this limitation [48]. 

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