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MDCK (wild type) permeability assay

Assess wild type MDCK permeability alongside MDR1-MDCK permeability to confirm the role of human P-gp in the efflux of your compound.

Wild type MDCK permeability is one of our portfolio of in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Use of wild type MDCK permeability as a negative control in the identification of human P-gp substrates

  • Madin Darby canine kidney (MDCK) cells are an epithelial cell line of canine kidney origin.
  • MDCK cells have low expression of transporter proteins and low metabolic activity2.
  • MDCK cells are often transfected with transporter proteins to investigate drug efflux e.g., the MDR1-MDCK cell line which expresses human P-glycoprotein. This cell line is a useful model for the identification of P-gp substrates and inhibitors.
  • As MDCK cells endogenously express other transporters such as canine P-gp3, it is recommended that the compound is screened through the MDCK (Wild Type) Permeability assay to calculate a net flux ratio in order to confirm the role of human P-gp in the MDR1-MDCK studies.
The net flux ratio is calculated as ratio of efflux ratio in MDR1-MDCK cells to efflux ratio in wildtype MDCK cells.

1Li X, J Voorman R, de Morais SM, and Lao Y (2011) Drug Metab Dispos 39(7); 1161-1169


Wild type MDCK permeability protocol

Test Article Concentration 10 μM (different concentrations available)
Direction Apical to Basolateral and/or Basolateral to Apical
Number of Replicates 2
Incubation Time 60 min
Growth Period 4 days
Test Article Requirements 100 µL of 10 mM solution
Analysis Method LC-MS/MS quantification
Data Delivery Papp
Efflux ratio for Bidirectional Assessment
Net Flux Ratio
(if MDR1-MDCK data available)


Data from Cyprotex's Wild Type MDCK Permeability assay

20 compounds were screened in Cyprotex's (Wild Type) MDCK Permeability assay (pH 7.4 buffer in the apical and basolateral compartments) in quadruplicate on 3 separate occasions. These data are highly reproducible for both low and high permeability values.

Figure 1
Cyprotex's MDCK (Wild Type) Permeability validation for apical to basolateral transport.
Figure 2
Comparison of efflux ratio generated in the wild type and MDR1-MDCK assays.
Figure 3
Net flux ratio for a set of 20 compounds (calculated using the efflux ratios of the wild type and MDR1-MDCK bidirectional assays).


Questions and answers on wild type MDCK permeability

What are the main advantages and disadvantages of MDCK cells over Caco-2 cells for permeability studies?

MDCK (Madin-Darby Canine Kidney) cells are an established dog kidney epithelial cell line often used as an alternative to Caco-2 cells in permeability studies. Under standard culture conditions, MDCK cells develop tight junctions and form monolayers of polarized cells. Confluency is reached after 3-5 days which one of the main advantages over Caco-2 cells. In addition, compared to Caco-2 cells, MDCK cells have lower transepithelial electrical resistance (TEER) measurements which are more comparable with the TEER values of the small intestine in vivo4. However, one of the main advantages of the Caco-2 cells over MDCK cells is the fact that they are of human intestinal origin whereas MDCK cells originate from the canine kidney. Due to the low expression levels of transporters and low metabolic activity, often Madin Darby canine kidney (MDCK) cells are transfected with genes expressing individual transporters, one of the most popular of these being the human MDR1 gene encoding for the efflux protein, P-glycoprotein (P-gp)5. As the MDCK cells are of canine origin, it is important to correct for any underlying canine transporter activity by screening in the wild type cells if efflux is observed in the transfected-MDCK cells.

Please provide an overview of Cyprotex's Wild Type MDCK Permeability assay.

The cells are seeded on a Millicell™ plate (Millipore, MA, USA) and form a confluent monolayer over 4 days prior to the experiment. On day 4, the test compound is added to the apical side of the membrane and the transport of the compound across the monolayer is monitored over a 60 min time period. To study drug efflux, it is also necessary to investigate transport of the compound from the basolateral compartment to the apical compartment and calculate an efflux ratio.

The permeability coefficient (Papp) is calculated from the following equation:

Where dQ/dt is the rate of permeation of the drug across the cells, C0 is the dosing solution concentration at time zero and A is the area of the cell monolayer.

An efflux ratio is calculated from the mean apical to basolateral (A-B) Papp data and basolateral to apical (B-A) Papp data.

Providing data from the MDR1-MDCK bidirectional study is available, the net flux ratio can be calculated by relating the efflux ratio from the wild type and MDR1-MDCK studies.

The net flux ratio is derived from the following equation:

How do you know if the cells have formed a confluent monolayer?

Transepithelial electrical resistance (TEER) measurement is used to determine tight-junction formation between cells. In addition, lucifer yellow, a membrane integrity marker, is co-incubated with the test compound at the start of the experiment. If the Pappof the lucifer yellow exceeds 0.5 × 10-6 cm/s (MDR1-MDCK) or 1 × 10-6 cm/s (wild type MDCK) then it is assumed that the formation of the cell monolayer has been unsuccessful and the compound is re-screened. If both lucifer yellow Papp values fail for the same compound on 2 separate occasions then it is assumed that the compound exhibits cytotoxic effects against the MDCK cells.

Two control compounds, propranolol (passive transcellular transport) and prazosin (subject to efflux in the MDR1-MDCK assay but not the wild type MDCK assay) are screened alongside the test compounds.

How and why is the % recovery calculated?

The % recovery can be useful in interpreting the MDCK data. If the recovery is very low, this may indicate problems with binding of the compound to the plate or accumulation of the compound in the cell monolayer. However, poor solubility is the most common reason for unexpected recoveries in the MDCK screen.


1 Li X et al. (2011) Drug Metab Dispos 39(7); 1161-1169
2 Braun A et al. (2000) Eur J Pharmaceut Sci 11; (Suppl 2) S51-S60
3 Goh LB et al. (2002) Biochem Pharmacol 64; 1569-1578
4 Deferme S et al. (2008) in Drug absorption studies – In situ, in vitro and in  silico models Ed. Ehrhardt C and Kim K-J, 182-215
5 Pastan I et al. (1988) Proc Natl Acad Sci USA 85; 4486-4490

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