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Fig. 2. Microphotograph of differentiated Caco-2 cells on a polycarbonate filter. Caco-2 cells grown on a filter and differentiated for 19 d were embedded in metacrylate resin (JB4; Polysciences Inc., Warrington, PA) and cut into 3-^M-thick sections, stained with hematoxylin-eosin and observed by light microscopy. The cells appear well polarized with basal nuclei and a regular brush border on the AP surface. Bar = 8 |J,m.

2-3 wk and leads to the formation of a monolayer of cells coupled by functional tight junctions. The establishment of tight junctions can be followed during the differentiation process by measuring the transepithelial electrical resistance (TEER) of the cell monolayer (21,32) or the transepithelial passage of radioactively labeled mannitol (33). In addition to mannitol, other molecules of different molecular weight (MW) and Stoke radius have been utilized as extracellular markers of paracellular flow, including PEG 4000 (17,34,35), inulin (27,36,37), and phenol red (22,38,39).

2.3.1. Measurement of Transepithelial Electrical Resistance

Analysis of TEER to passive ion flow relies on a simplified equivalent circuit model viewing the epithelium as a parallel circuit consisting of a paracellular pathway (composed of tight-junction resistance in series with subjunctional lateral space resistance) and a transcellular pathway (as the AP and BL membranes in series). Because the tight junction is often rate limiting to paracellular solute movement, alterations in TEER are commonly used as an index of tight-junction permeability (5). It has, however, to be noted that activation of membrane channels may lead to large changes in TEER that are independent of altered paracellular solute transport, as described in ref. 5. In addition, TEER measure is influenced by the ionic composition of the medium, such that in particular experimental conditions (i.e., changes in pH or different composition of AP and BL media), the TEER may change independently of paracellular solute permeability. Therefore, TEER measures alone cannot be considered as accurate indicators of paracellular permeability. It is therefore advisable to couple the TEER assay with an analysis of transepithelial flux of an extracellular marker (see Section 2.3.2.).

The permeability of the tight junctions was determined by measuring the TEER of filter-grown cell monolayers in a complete culture medium, unless otherwise stated, using a commercial apparatus (Millicell ERS; Millipore Co., Bedford, MA) employing Ag-AgCl electrodes, according to the manufacturer's instructions (21). Briefly, the resistance system was calibrated and the electrical resistance of the cell monolayer was measured by placing one electrode on either side of the polycarbonate filter. Because TEER is influenced by temperature (17), it is important to control the temperature at which TEER is measured. In the present study TEER was always measured at 37°C in a water bath. TEER was expressed as Ohm ■ centimeter squared (Q ■ cm2) after subtracting from the reading the resistance of the supporting filter and multiplying it by the surface area of the monolayer.

2.3.2. Permeability to Extracellular Markers

The analysis of transepithelial flux of inert solutes such as mannitol (MW 182) or inulin (MW 4000) has often been used to determine the permeability characteristics of the tight junctions (32,33,40). Whereas the detection of mannitol and inulin relies on their radioactive label (either 3H or 14C), a nonradioactive alternative for flux measurement is phenol red (MW 354) (38,39). Analysis of solute movement across epithelial tight junctions is often reported as a percentage of the tracer, which moves from one side of the epithelium to the other per unit time (32,33,40). Alternatively, solute passage across cell monolayers can be expressed as the apparent permeability coefficient (Papp), which is reported in units of mass per area per unit time, thus normalizing for filters of different size. Flux measurements, expressed as Papp (cm/s), were calculated using the following equation:

where K is the steady-state rate of change in concentration in the receiver chamber (Ct/C0) vs time (s); Ct is the concentration in the receiver compartment at the end of each time interval, C0 is the initial concentration in the AP chamber at each time interval (mol/mL), Vt is the volume of the receiver chamber (mL), and A is the surface area of the filter membrane (cm2) (41).

When using extracellular flux markers labeled with 3H, the production of 3H2O upon spontaneous degradation of the compound with time has to be considered, as water can move across paracellular and transcellular pathways (42,43). This problem can be overcome by using fresh reagents or 14C-labeled compounds. Phenol red has the advantages of a nonradioactive marker, but it exhibits some disadvantages: The phenol red in the cell growth medium has to be well washed away before the assay and the colorimetric assay is subject to interference from other colored substances that may be used to treat the cell monolayer. In addition, because the passage of phenol red in differentiated "tight" Caco-2 cell monolayers is very low, the influence of the blank value may be high, thus increasing the variability of the measurement.

The transepithelial passage of the radiolabeled extracellular marker D-1[3H(N)]-mannitol (specific activity 706.7 GBq/mmol) (NEN Life Sciences Products, Zaventem, Belgium) across the cell monolayer

How Often Passage Caco Cell

Fig. 3. Establishment of functional tight junctions during differentiation of Caco-2 cells. After seeding Caco-2 cells on the filters (d 0) TEER and transepithelial flux of 3H-mannitol and of phenol red were measured during the process of spontaneous differentiation (from 3 to 24 d) as described in Sections 2.3.1. and 2.3.2. TEER is expressed as Ohm • centimeter squared and the AP to BL flux of the two extracellular markers 3H-mannitol and phenol red as Papp, calculated as in Eq. 1. Data are the means ± SD from a representative experiment performed in triplicate.

Fig. 3. Establishment of functional tight junctions during differentiation of Caco-2 cells. After seeding Caco-2 cells on the filters (d 0) TEER and transepithelial flux of 3H-mannitol and of phenol red were measured during the process of spontaneous differentiation (from 3 to 24 d) as described in Sections 2.3.1. and 2.3.2. TEER is expressed as Ohm • centimeter squared and the AP to BL flux of the two extracellular markers 3H-mannitol and phenol red as Papp, calculated as in Eq. 1. Data are the means ± SD from a representative experiment performed in triplicate.

was determined by adding approx 40 pM 3H-mannitol (1/1000 of stock solution) in serum-free DMEM to the AP compartment, and after 60 min of incubation at 37°C, the radioactivity in the BL compartment was measured in the liquid scintillation counter (LS1801; Beckmann Instruments, Inc., Irvine, CA) (32,40).

The transepithelial passage of phenol red was measured after washing cells in Hank's balanced salt solution (137 mM NaCl, 5.36 mM KCl, 0.44 mM KH2PO4, 0.34 mM Na2PO4, 1 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose [HBSS]) additioned with 10 mM N-2-hydroxyethyl piperazine-N-4-butanesulfonic acid (HEPES) at pH 7.4. 1 mM phenol red in HBSS/HEPES (pH 7.4) was added to the AP compartment, and after incubation for 60 min at 37°C, the BL medium was collected. After the addition of 100 pL 0.1 N NaOH to 1 mL of BL medium, the absorbance at 560 nm was measured in the spectrophotometer (DU-70, Beckmann Instruments) and the concentration of phenol red calculated from the absorbance of a standard curve after subtraction of the blank obtained by incubating control cells in HBSS/HEPES in the absence of phenol red (39). The transepithelial flux of 3H-mannitol and phenol red were expressed as Papp according to Eq. 1.

2.3.3. Establishment of Functional Tight Junctions During Differentiation of Caco-2 Cells

As shown in Fig. 3, after seeding Caco-2 cells on filters (d 0) and measuring TEER and transepithelial flux of mannitol and phenol red during the process of spontaneous differentiation (from 3 to 24 d), a progressive increase in TEER was observed, accompanied by a decrease in the AP to BL permeability to mannitol and phenol red. After 14 d from seeding, all parameters reached a plateau that remained stable up to 24 d, corresponding to a TEER of 1133 ± 88 Q • cm2 and a Papp for mannitol of 4.2 x 10-7 ± 6.5 x 10-8 cm/s and of phenol red of 1.0 x 10-7 ± 4.8 x 10-8 cm/s. The Papp of the two flux markers, mannitol and phenol red, decreased in parallel during the process of differentiation, exhibiting differences in absolute values that arise from the different size of the two molecules.

The Papp of mannitol in differentiated Caco-2 cells is of the same order of magnitude as that previously reported by other authors (44,45), although higher Papp (10-6 cm/s) have also been reported (17,46). These differences probably reflect differences in permeability of Caco-2 cells from various sources or passage number, often reported in the literature (17,47,48).

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