MDCK

Production of the vims in a bioreactor reactor, using a continuous cell line, has also been studied (85,86). This will reduce production costs and side effects. Both Madin-Darby canine kedney (MDCK) and Vero cell lines are being developed for production of the vaccine. 

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. 

Figure 9. Domes in MDCK monolayers. Domes are the groups of cells that are slightly out of focus under the microscope when focusing on the monolayer (a). The cells in the monolayer are out of focus when focusing on the cells in the domes (b). 100 x magnification.

A good example of the problems encountered with the use of serum with primary cultures is illustrated by the case with cultured kidney cells. The kidney epithelial cell line MDCK grows in serum-free medium supplemented with five supplements ... 

Taub, M., Chuman, L., Saier, M.H., Sato, G. (1979). Growth of a kidney epithelial cell line (MDCK) in hormonally defined serum-free medium. Proc. Natl. Acad. Sci. USA 76, 3338-3342. 

Irvine JD, Takahashi L, Lockhart K, Cheong J, Tolan JW, Selick HE, et al. MDCK (Madin-Darby canine kidney) cells a tool for membrane permeability screening. J Pharm Sci 1999 88 28-33. 

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. 

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

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... 

Figure 8 shows PAMPA data for a subset of compounds from the two series in Fig. 7. The potential concern about low permeability for Series D is confirmed between the MDCK and PAMPA data. Series C, with the exception of one compound, also demonstrates good correlation between MDCK and PAMPA permeability the compound with low PAMPA permeability should be further analyzed for relevant structure-permeability information.

Fig. 7 MDCK A to B apparent permeability vs clogD. Filled squares and open triangles represent two different chemical series (series C and D, respectively)...

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...

Lipid6 [Ref.] BBMC [30] MDCK [38] BBBe [37] BBM ModeF 20% Extract Lecithin 60% Extract Lecithin ... 

The %w/w values in this table for BBB and MDCK are conversions from the originally reported %mol/ mol units. 

MDCK = Madin-Darby canine kidney cultured epithelial cells [563]. 

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