The Madin Darby Canine Kidney (MDCK) Epithelial Cell Monolayer as a Model Cellular Transport Barrier

Abstract

Two strains of Madin Darby canine kidney (MDCK) cells were grown on a polycarbonate membrane with 3-µm pores without any extracellular matrix treatment. The membrane, 2.45 cm in diameter, which is part of a commercially obtained presterilized culture insert, provides two chambers when placed in a regular six-well culture plate. This device was found to be convenient for investigating transport of a few selected fluid-phase markers across the MDCK cell monolayer. Both the strain from the American Type Culture Collection (ATCC) and the so-called highly resistant strain I, at a serial passage between 65 and 70, showed a seeding concentration-dependent lag phase followed by a growth phase with a 21-hr doubling time. When seeded at 5 × 10⁴ cells/cm², cell confluence was achieved in 5 days in a modified Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum under a 5% CO₂ atmosphere. Similarly, transepithelial electrical resistance (TEER) also reached a plateau value in 5 days. Both light and electron microscopic examinations revealed well-defined junctional structures. Transport of the fluid-phase markers, sucrose, lucifer yellow CH (LY), inulin, and dextran across the MDCK cell monolayers was studied primarily at 37°C following the apical-to-basolateral as well as the basolateral-to-apical direction. Large variations in the steady-state transport rate were observed for a given marker between the cell layer preparations. Thus, the present study proposes an “internal standard” procedure for meaningful comparisons of the transport rate. When normalized to the rate of sucrose, the rate ratio was 1.00:0.80:0.67:0.15 for sucrose:LY:inulin:dextran. This ratio was virtually independent of temperature, cell strain, direction of the marker migration, and TEER value, suggesting a common transport mechanism. The observed rate ratio appears to reflect molecular size and charge. The transport observed in the present study would consist, in theory, of both paracellular shunt and transcellular vesicular transport. Quantitative assessment of each transport mechanism in the overall transport has been difficult. The initial uptake of [³H]dextran estimated for the slowest transport observed in the present study was still 300-fold faster than a literature value. This appears to indicate that the transport observed in the present study is largely through the paracellular shunt pathway.