Influence of the Microporous Substratum and Hydrodynamics on Resistances to Drug Transport in Cell Culture Systems: Calculation of Intrinsic Transport Parameters

Abstract

Although cell culture models are increasingly used to study drug transport and metabolism, the influence of the substratum on the transport properties of the cell monolayer has not been studied in great detail. Furthermore, the use of effective (or apparent) permeabilities (Peff) assumes that the contribution of the microporous filter substratum and the aqueous boundary layer (ABL) to transport are negligible or are at least constant for a series of drugs. In the present study, the permeabilities of the substratum, ABL, and monolayer were obtained for a series of compounds at variable flow rates in side-by-side diffusion chambers. Comparisons of transport properties were made between cell monolayers grown on substrata made of polycarbonate (PC) and polyester (PE). All paracellular markers demonstrated a reduction in permeability and a corresponding increase in transepithelial electrical resistance (TEER) through PE-grown monolayers. The permeabilities of two carrier-mediated compounds, phenylalanine and proline, were 55% higher and 48% lower through PE-grown monolayers than through the PC-grown monolayers, respectively. The resistance to progesterone transport attributed to the PE and PC filters was large (71% and 27% of total resistance, respectively) at a flow rate of 20 mL/min, indicating that the monolayer was not the rate-limiting transport barrier. Therefore, for highly permeable compounds, reporting Peff has limited value since it is an indicator of the transport properties of the substratum rather than of the monolayer. These results demonstrate that substratum properties (e.g., membrane composition, pore size, etc.) significantly affect the barrier properties of the Caco-2 cell monolayer. The most probable mechanism is by the modulation of the functional expression of nutrient and ion transporters resulting in variable transcellular and paracellular transport properties. These results further demonstrate the importance of calculating intrinsic membrane transport parameters if the monolayer is not maintained as the rate-determining barrier in the transport experiment. Using higher flow rates and higher porosity substrata supports may help maintain the monolayer as the rate-limiting transport barrier.

Expression, Localization, and Functional Characteristics of Breast Cancer Resistance Protein in Caco-2 Cells

“…1, B and C). Although our findings are novel, they are supported by the previous reports that culture conditions and cell-growing matrices may affect efflux pump protein expression (Yu and Sinko, 1997). The BCRP-mediated E3S efflux in the day 12 p59 Caco-2 cells was much lower than that of day 12 p36 Caco-2 cells, with the B/A ratio of 2 and 30, respectively (Fig.…”

“…Similar to P-gp, MRP, and lung-resistant protein (Yu and Sinko, 1997), the expression of BCRP is passage-dependent. The expression of BCRP increased approximately 3-fold from the early passage to the late passage when Caco-2 cells were cultured in T-flasks, but decreased about 3-to 10-fold for monomer and dimer, respectively, from the early passage to the late passage when Caco-2 cells were grown on Transwell plates (Fig.…”

Role of PEPT2 in Peptide/Mimetic Trafficking at the Blood-Cerebrospinal Fluid Barrier: Studies in Rat Choroid Plexus Epithelial Cells in Primary Culture

“…The apparent permeability coefficient (P app ) of mannitol was calculated according to the following equation: P app ϭ Vr ⅐ dC/(A ⅐ Co ⅐ dt), where V r is the volume of the receiver chamber, A is the surface area of the filter, Co is the initial mannitol concentration in the donor chamber, and dC/dt is the change in mannitol concentration in the receiver chamber per unit of time. A correction was made for filter and aqueous boundary layer effects by determining mannitol transport in the absence of a cell monolayer and applying the equation (Yu and Sinko, 1997): 1/P mono ϭ 1/P app Ϫ 1/P corr , where P mono is the permeability of the cell monolayer and P corr is the measured permeability using blank filters (i.e., 1/P corr ϭ 1/P aqueous ϩ 1/P filter ).…”

The Asymmetry of the Unstirred Water Layer in Permeability Experiments

“…The permeabilities of total and apical UWLs were determined without and with vigorous basolateral stirring. The UWL thickness was studied by both stirring (1,9,23) and pH (4,22) methods. In addition, the permeabilities were studied also in a side-by-side (SbS) apparatus to compare the hydrodynamics under these two different experimental settings.…”