Transport MDCK cells

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. 

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).

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

Some laboratories have found an alternative to the short-term cultures by using cell lines other than Caco-2 cells. The most popular of these is Madin-Darby canine kidney (MDCK) cells, an epithelial cell line from the dog kidney. MDCK cells have been suggested to perform as well as Caco-2 cells in studies of passive drug permeability [56]. These cells have also been used to optimise the conditions for studies of low-solubility drugs [53]. However, as noted previously, the active transport processes of this cell line can be quite different to those of Caco-2 cells [28-30], Another cell line that only requires short-term culture is 2/4/A1, which is a conditionally immortalised rat intestinal epithelial cell line [86]. The 2/4/A1 cell line is discussed in Section 4.3.2.2 below. 

C. C. Paulusma, R. P. Oude Elferink et al. Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA, J. Clin. Invest. 1998, 101, 1310-1319... 

Cell lines, such as the Caco-2 and MDCK cells [27, 35, 47, 49, 57, 67, 128-133], have been used frequently to study different transporters in the GI tract. These cell lines have been evaluated for transport both in absorptive and secretory direction and in addition also been transfected with specified transporter systems of interests to yield new clones [23, 31, 72, 79, 80, 134] or co-cultures [135], Some of the uptake transporters belonging to the organic cation transporter (OCT) family have also been identified in cell lines such as the pig kidney cell line LLC-PK1, and MDCK [67, 136]. In fact, its presence in Caco-2 cells needs to be further elucidated as reports have shown both the absence and presence of transporters from this family of transporters [136-138],... 

MDCK Dog kidney epithelial cells Polarized cells with low intrinsic expression of transporters Suitable cell fine for transfections... 

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... 

Prydz K, Hansen SH, Sandvig K, van Deurs B. Effects of brefeldin A on endo-cytosis, transc5dosis and transport to the Golgi complex in polarized MDCK cells. J Cell Biol 1992 129 1241-1250. 

Usually, PAMPA does not have any aqueous pores and is therefore not suitable for examining paracellular transport. Some cell models, for example, Caco-2 and MDCK, have a narrower tight junction than the in vivo human intestine and may underestimate paracellular transport. However, the contribution of the paracellular pathway can be added using an in silico approach [76-78]. 

Further, drug transport through epithelial cells has been studied. For instance, Madin-Darby canine kidney (MDCK) cells have been grown into tight mono-layers on a permeable polycarbonate membrane. This membrane (0.4-p.m pores,... 

Mita S, Suzuki H, Akita H, et al. Vectorial transport of hile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump. Am J Physiol Gastrointest Liver Physiol 2005 288 G15943167. 

Although there is a widespread perception that wild-type MDCK cells contain insignificant levels of P-gp to affect substrate transport, it has been demonstrated that this is not the case. It was shown that the transport of vinblastine sulfate across MDCK monolayers was indeed apically polarized (203). These results were duplicated by Hirst et al. using the same test compound, verapamil, in two different strains of MDCK cells. The transport profiles of verapamil showed polarity in both a high-resistance strain [TEER 2000 Q-cm2 and a low-resistance strain (TEER < 200 Q-cm2] (365). Recently, parallel studies were performed measuring the transport of a novel peptide, K02, across both MDCK and Caco-2 cells (364). The results showed nearly identical profiles for the AP to BL and BL to AP transport of this agent in both cell types. Although it is unlikely that all P-gp substrates will behave identically in both cell lines, these studies indicate that there is sufficient P-gp expression in MDCK cells to affect transport studies. Thus, MDCK cells can be used to evaluate the transport of compounds that are suspected to be substrates of P-gp. 

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