| Assay Detail |
Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD). Cell culture medium (Dulbecco’s Modified Eagles Medium with 20% fetal bovine serum, 1% Non-essential amino acids, 1% Glutamax-1 and 0.08% gentamycin) and Hank’s balanced salt solution (HBSS) with 25 mM D-glucose monohydrate, 1.25 mM CaCl2 and 0.5 mM MgCl2; pH 7.4 or pH 6.5) obtained from Invitrogen (Grand Island, NY). Both protease inhibitor and albumin from bovine serum were obtained from Sigma Aldrich. Transwell permeable support 24-well cell culture plates (PET membrane, 0.33 cm2 growth area, 1.0 µm pore size) were obtained from BD Biosciences (Franklin Lakes, NJ). Transepithelial electrical resistance (TEER) was measured using EVOM from World Precision Instruments. Caco-2 cells were cultured at 37 °C with cell culture medium in an atmosphere of 10% CO2 and 90% relative humidity incubator. The cells were passaged upon reaching approximately 90% confluence from T-flasks using 0.25% trypsin-EDTA. Caco-2 cells were seeded onto polycarbonate membranes with 60,000 cells/well. The individual feeding tray wells received 1.0 mL of cell culture medium. The cell culture medium was changed bi-weekly. Caco-2 cells monolayers were used for experimentation 21-days post seeding. An apical→basolateral (A→B) assay was performed to assess the Papp of test compounds in the absorptive transport direction (Papp, A→B). Transport was determined under conditions in which HBSS (pH 6.5 with protease inhibitor) was used in A compartment and HBSS [pH 7.4 with 0.4% bovine serum albumin (BSA) and protease inhibitor] was used in B compartment. Protease inhibitor was added to both pH 6.5 and 7.4 HBSS at a ratio of 400 µL per 100 mL of buffer. These buffers are referred as “modified 6.5 HBSS” and “modified 7.4 HBSS”. Starting compound solutions were made from 10mM DMSO stock solution diluted to 10 µM in modified pH 6.5 HBSS. Silanized glass tubes were used for stock solutions to limit non-specific binding.
Experiments were initiated by removing the cell culture medium from the apical and basolateral sides of the cell monolayers. Then 0.3 mL of modified pH 6.5 HBSS was placed in the A compartment and 1.0 mL of modified pH 7.4 HBSS was placed in the B compartment, and the monolayers were preincubated for 15 min at 37°C . After 15 min, HBSS was removed from both compartments. To conduct an A→B assay, 0.3 mL of compound solution in modified pH6.5 HBSS was added to the A compartment and 1.0 mL of modified pH 7.4 HBSS was added to the B compartment. Experiments were carried out in triplicate. Monolayers were incubated for 2 hours 30 min at 37°C with continuous agitation (60 rpm), then the basolateral samples were collected. The assay samples collected were analyzed by LC-MS/MS. TEER was measured across the cell membranes at the beginning and the end of experiments to indicate the integrity of the Caco-2 monolayers (TEER >350Ω.cm2). The TEER (Ω.cm^2) values of the cell monolayers were calculated according to the following equation: Resistance of unit area = resistance (Ω) X effective membrane area (cm^2). Absorptive transport were represented as permeability values (Papp x 10^-6 cm/sec), calculated using the equation: Papp = {1 / (Area * C_D(0))} * {dM_r / dt} where Area is the surface area of the cell monolayer (0.33 cm^2), C_D(0) is the initial concentration of compound applied to the donor chamber, t is time, M_r is the mass of compound in the receiver compartment, and dM_r/dt is the flux of the compound across the cell monolayer.
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