MDCK cell

Nelson, W.J. Hammerton, R.W. (1989). A membrane-cytoskeletal complex containing Na, K -AT-Pase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK cells) Implications for the biogenesis of epithelial cell polarity. J. Cell Biol. 108, 893-902. 

Large conductance CP-channels were described for renal epithelial cells such as MDCK-cells, urinary bladder, collecting duct and A6-cells [51-54] and in pulmonary alveolar cells [55]. 

Very few of the CP-channels described above have been purified, and the amino acid sequence is only known for the GABAA-receptor channel, the glycine-receptor channel and the Torpedo CP-channel [3-5], a CP-channel from skeletal muscle [125] and a CP-channel from MDCK cells [128]. In the following the current status will be briefly summarized. 

Kramer, S. D., Wunderli-Allenspach, H. The pH-dependence in the partitioning behaviour of (JlS)-[ H]propranolol between MDCK cell lipid vesicles and buffer. Pharm. Res. 1996, 13,... 

Recombinant CYP450s (also for safety assessment of drug-drug interactions) Permeability/absorption Caco-2 cells MDCK cells... 

Typical early in vitro permeability assessments measure the rate of flux of a compound from one side of a barrier to another [54, 55]. The barrier has historically been derived from a cell line, most commonly Caco-2 or Madin-Darby canine kidney (MDCK) cells. In the last several years, there has been substantial work and significant progress in the development of parallel artificial membrane permeability... 

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.

Figure 3. Transcellular transport of HRP-S-PLL in filter-grown MDCK cell monolayers. Confluent MDCK monolayers in Transwells were treated at the basal compartment (closedsquares)or the apical compartment (open squares) with 3 pg/mL HRP-S-PLL conjugate.

As shown in Table I, free HRP is poorly transported across MDCK cells but, when conjugated to a PLL carrier, HRP transport is increased considerably. The existence of a proteolytic compartment involved in the transcytotic digestion of HRP-S-PLL conjugate was further confirmed by the finding that when PLL was replaced by PDL, the transport of HRP was completely abolished (Table I) (8). In addition, when protease inhibitors such as leupeptin were added to the basal medium, the transcytosis of HRP was also significantly decreased (Table I). We have previously reported that the partial degradation of HRP-S-PLL was not inhibited by lysosomotropic amines (<8), indicating that this proteolytic process does not occur in lysosomes. 

Table I. Basal-to-Apical Transcytosis of HRP-S-PDL in Cultured MDCK Cell Monolayers8...

Figure 6. Transcellular transport of HRP-SS-PDL in a filter-grown MDCK cell monolayer. HRP-SS-PDL was added to the apical medium (closed squares) or to the basal medium (open squares).

Table H Release of HRP in HRP-SS-PDL and HRP-S-PDL from the Apical and the Basal Surface of MDCK Cell Monolayers8...

Culture-cell assays are also subject to sample retention by the monolayer. Sawada et al. [574] studied the transport of chlorpromazine across MDCK cell... 

Figure 11 Change in paracellular permeation of sucrose and mannitol across MDCK cell monolayers following addition of 1 pg/mL cytochalasin D.

Table 7 Permeability Coefficients of the Paracellular Route of Unperturbed and Cytochalasin D-Perturbed MDCK Cell Monolayers at 25°C... 

Figure 12 Molecular restriction factor as a function of the ratio of molecular radius to pore radius for mannitol and sucrose flux across MDCK cell monolayers that were untreated or treated with 1 pg/mL cytochalasin D (see Fig. 11).

To estimate the relative importance of the tight junction and the lateral space composing the paracellular route, let us consider the permeability of mannitol across Caco-2 and MDCK cell monolayers. The results taken from earlier examples are presented below ... 

Interestingly, the permeability coefficients of mannitol in the two cell types are identical, most probably for different reasons, since the physical dimensions of the Caco-2 and MDCK monolayers (Table 8) are markedly different. Compared to the MDCK cell monolayer, the Caco-2 cell monolayer has a taller cell height, a shorter length in tight junctions, longer tortuous path lengths, and smaller slit width in lateral space. One recognizes that... 

Table 8 Physical Dimensions of Caco-2 and MDCK Cell Monolayers...

Table 9 Theoretical Assessment of Lateral Space and Tight Junction Contributions to the Paracellular Permeability of Mannitol in Caco-2 and MDCK Cell Monolayers3...

Figure 15 An increase in transepithelial electrical resistance (TER) of MDCK cell monolayers with time in culture reflects the gradual formation of a continuous sheet of epithelia with restrictive tight junctions. [Redrawn from Cho et al. (1989) with permission from the publisher.]...

In contrast to cases 1 and 2, where the influence of opening tight junctions on Pe was assessed in a hypothetical context, this present case has been demonstrated in MDCK cell transmonolayer kinetic studies employing extracellular permeants, such as mannitol and sucrose, in the presence and absence of cytochalasin D (see Figs. 11 and 12). Here,... 

The flux of 3H-labeled PNU-78,517 across MDCK cell monolayers shows the characteristic disparity between the kinetics of disappearance from the donor solution and appearance in the receiver sink (Fig. 32). Drug uptake is rapid and exponential with time and approaches a quasi-equilibrium state in contrast, the concomitant efflux of drug into the receiver is slow and linear. While maintaining a 3% bovine serum albumin (BSA) concentration in the donor and varying the BSA concentration between 0.5 and 5% in the receiver, the results show that the... 

Figure 32 Disappearance and appearance kinetics of transcellular flux of the lipophilic antioxidant PNU-78,517 (pKa 6.5) across MDCK cell monolayers in Transwell systems at 37°C. Donor solutions contained 3% bovine serum albumin (BSA), and receiver solutions contained 0.5-5% BSA at pH 7.4. [Redrawn from Raub et al. (1993) with permission from the publisher.]... 

Given the low permeability of the antioxidant across MDCK cell monolayers and its large membrane partition coefficient, efflux kinetic studies using drug-loaded cell monolayers cultured on plastic dishes could yield useful information when coupled with the following biophysical model. The steady-state flux of drug from the cell monolayer is equal to the appearance rate in the receiver solution ... 

Table 19 Comparison Between Permeability Coefficients of the Apical and Basolateral Membranes from Independent Efflux Kinetics from Drug-Loaded MDCK Cell Monolayers...

---