Monolayers Madin-Darby canine kidney cells

VolSurf was also successfully applied in the literature to predict absorption properties [156] from experimental drug permeability data of 55 compounds [165] in Caco-2 cells (human intestinal epithelial cell line derived from a colorectal carcinoma) and MDCK cell monolayers (Madin-Darby canine kidney). In this interesting case, it was shown that models including counterions for charged molecules clearly show significantly better quality and overall performance. The final model was also able to correctly predict, to a great extent, the relative ranking of molecules from another Caco-2 permeability study by Yazdanian et al. ]166]. 

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.]. 

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... 

Cho MJ, DP Thompson, CT Cramer, T Vidmar, JF Scieszka. (1989). The Madin-Darby canine kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier. Pharm Res 6 71-77. 

A confluent monolayer of Madin-Darby canine kidney (MDCK) cells was grown in 96-well plates. Serial tenfold dilutions in minimal essential medium were prepared from the aliquots of allantoic fluid taken from the irradiated specimen. These dilutions were applied to MDCK cells and incubated for 48 h at 36 °C in 5% C02. The cells were then washed two times for 5 min with phosphate buffered saline (PBS) and incubated for 1 h with 100 pi of 0.5 mg/ml solution of 3-(4,5-dimethyl-thiazolyl-2) 2,5-diphenyltetrazolium bromide (MTT, ICN Biochemicals Inc., Aurora, Ohio). After lh, the colored deposit was dissolved in 100 pi DMSO, and optical density in the wells was measured on plate reader Victor 1420 (Perkin Elmer, Finland). Based on the data obtained, the infectious titer of the vims was determined as a decimal logarithm of reciprocal to the dilution of the specimen causing destruction of 50% of cells. The inhibiting action of irradiation was evaluated by decreasing the vims titer. 

MDCK Madin-Darby canine kidney (MDCK) cells have received attention as an alternative to Caco-2 cells for permeability measurements. When grown under standard culture conditions, MDCK cells develop tight junctions and form monolayers of polarized cells. The main advantage over Caco-2 cells is the shorter culture time to confluence (3-5 days). The transep-ithelial electrical resistance of MDCK cells is lower than that of Caco-2 cells and thus, closer to the TEER of the small intestine in vivo. The permeability coefficients of hydrophilic compounds are usually lower in Caco-2 cells than in MDCK cells, which is consistent with the lower TEER values for MDCK cell monolayers. The nonhuman (canine) and nonintestinal (renal) origin of MDCK cells is considered as a disadvantage. They have low expression levels of transporter proteins and low metabolic activity [34], MDCK cells that are stably transfected with P-gp/MDRl are often proposed as an alternative for Caco-2 cells to study bidirectional transport of compounds and, more... 

Cho MJ, Adson A, Kezdy FJ. Transepithelial transport of aliphatic carboxylic acids studied in Madin Darby canine kidney (MDCK) cell monolayers. Pharm Res 1990 7(4) 325-331. 

Cho MJ et al (1989) The Madin Darby canine kidney (MDCK) epithelial cell monolayer as a... 

Modulation of the intercellular junctions by inhibiting the cadherin-cadherin interaction at the adherens junction has also been investigated. Peptides derived from the sequence of the extracellular domain of E-cadherin have been shown to modulate the intercellular junction of bovine brain microvessel endothelial cell (BBMEC) and Madin-Darby canine kidney (MDCK) cell monolayers. These peptides enhance the paracellular penetration of marker molecules such as 14C-mannitol and lower the transepithelial resistance of the monolayers.52 55 The use of these cadherin-derived peptides as adjuvants to enhance paracellular permeability of drugs is still under investigation. 

Drug absorption generally occurs either through passive transcellular or paracellu-lar diffusion, active carrier transport, or active efflux mechanisms. Several methods have been developed to aid in the understanding of the absorption of new lead compotmds. The most common ones use an immortalized cell line (e.g., Caco-2, Madin-Darby canine kidney, and the like) to mimic the intestinal epithelium. These in vitro models provide more predictive permeability information than the artificial membrane systems (i.e., PAMPA and permeability assays, described previously) based on the cells ability to promote (active transport) or resist (efflux) transport. Various in vitro methods are listed in the U.S. FDA guidelines. These are acceptable to evaluate the permeability of a drug substance, and includes a monolayer of suitable epithelial cells, and one such epithelial cell line that has been widely used as a model system of intestinal permeability is the Caco-2 cell line. 

M. J. Cho, J. F. Scieszka, C. T. Cramer, D. P. Thompson, and T. J. Vidmar. Neutrophil-mediated transport of liposomes across the Madin Darby canine kidney epithelial cell monolayer. Pharm. Res. 6 78 (1989). 

These models consist of cells grown on permeable inserts. Transport of compounds across the cell monolayer can be used to quantitate the permeability of a new chemical entity in a rapid manner. One of the most popular cell lines is Caco-2, derived from human colon adenocarcinoma cells. The monolayer exhibits ion conductance and possesses transepithellal electrical resistance indicative of fully formed tight junctions that restrict the paracellular transport of a chemical entity. Although Caco-2 cells are the most commonly used cells, Madin-Darby Canine Kidney (MDCK) cells are becoming more widespread in use, in part because of the shorter culture time (4-7 days versus 21-30 days for Caco-2 cells) needed for their use in permeability experiments. 

The use of a faster-growing cell line, MDCK (Madin-Darby canine kidney) cells, appears to be a good replacement for Caco-2 cells (Irvine et al. 1999). The parallel artificial membrane permeation assay (PAMPA) is a rapid in vitro assay, in which transcellular permeation is evaluated (Kansy et al. 1998). PAMPA may also be used to predict oral absorption, blood-brain barrier penetration, and human skin permeability (Fujikawa et al. 2007) by using QSAR models. To our knowledge, neither PAMPA, Caco-2 cell monolayers nor MDCK cells have been used to examine the absorption/permeability of the pyrethroids. The advantages and limitations of the Caco-2 model were reviewed by Artursson et al. (1996) and Delie and Rubas (1997). 

The Madin-Darby canine kidney (MDCK) cell model is another commonly used cell monolayer system for the assessment of human intestinal absorption. MDCK cell lines offer a couple of key advantages over Caco-2 cells, including the ability to reach full differentiation in 3-7 days (as opposed to 21 days for Caco-2) as well as the ability to be co-transfected with Pgp, the primary intestinal efflux transporter. Good correlation has been observed between MDCK permeability and human absorption. " The primary disadvantage of the MDCK ceU line is that it is derived from canine, rather than human, cells and, as such, expression of transporters and metaboUzing enzymes is not identical. 

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