| Assay Detail |
Caco-2 cells were kindly gifted to us by Associate Professor Jayashree Arcot from the School of Chemical Engineering at UNSW Sydney. Cell culture medium (Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum, 1% non-essential amino acids and 1% penicillin/streptomycin), Hank's balanced salt solution (HBSS) with CaCl2 and MgCl2, and trypsin-EDTA were purchased from Life Technologies. Transwell permeable support 12-well cell culture plates (PET membrane, 1.12 cm2 growth area, 0.4 µm pore size) were purchased from Corning (Sigma). Transepithelial electrical resistance (TEER) was measured using a Millicell ERS-2 voltohmmeter (Merck). Caco-2 cells were cultured at 37˚C with culture medium in a humidified atmosphere of 10% CO2 in air. The cells were passaged upon reaching 80-90% confluence from flasks using 0.25% trypsin-EDTA. Caco-2 cells seeded on to PET membranes at 56,000 cells/wellin 0.5 mL. The basolateral compartment was filled with 1 mL culture medium. The media was changed every 2 days for 14 days, then daily until 21 days. The Caco-2 cell monolayers were used for experimentation on days 21 or 22 post seeding. An apical to basolateral (A to B) assay was performed to evaluate the apparent permeability (Papp) of compounds. Transport was determined in transport buffer, which consisted of HBSS buffered with 25 mM HEPES to pH 7.4.
Starting compound solutions were made from 5 mM DMSO stocks diluted in transport buffer to 50 µM. The TEER was measured across the cell monolayers at the beginning of the experiment to evaluate the integrity of the Caco-2 monolayer (TEER > 350 Ω.cm^2). The TEER (Ω.cm^2) values of the monolayers were calculated by multiplying the resistance (Ω) by the cell growth area (1.12 cm^2). Assays were then initiated by aspirating the cell culture medium from the basolateral then the apical compartment and then replacing the media with transport buffer (1.5 mL in B and 0.5 mL in A). The monolayers were precincubated at 37˚C for 20 minutes, whilst agitating. After 20 minutes, the HBSS was removed from both compartments. Then 0.4 mL of compound dilutions were placed in the apical compartment and 1.2 mL of transport buffer was placed in the basolateral compartment. Monolayers were incubated for 60 minutes, whilst agitating, then the basolateral samples were collected. Assay samples were then analysed using LC/MS in selected ion monitoring (SIM) mode to detect the parent masses and quantified using a standard curve of known compound concentrations. The permeability values (Papp x 10^-6 cm/s) were calculated using the following equation: Papp = VB/(A*Ci) * CB/deltaT. Where VB = volume of basolateral compartment (1.2 cm^3), A = growth area (1.12 cm^2), Ci = concentration added to apical compartment (50 µM), CB = concentration determined by LC/MS in the basolateral compartment, and t = time (3,600 seconds). Compounds were tested in duplicate wells and in at least 2 independent experiments (minimum 4 data points collected). Compounds were compared to control compounds phenol red and atenolol, which had Papp values of 10.52 ± 1.30 and 1.61 ± 0.12, respectively.
|