Caco2 Cells

Caco-2 cell model has been the most popular and the most extensively characterized cell-based model in examining the permeability of drugs in both the pharmaceutical industry and academia. Caco-2 cells, a human colon adenocarcinoma, undergoes spontaneous enterocytic differentiation in culture and become polarized cells with well-established tight junctions, resembling intestinal epithelium in humans. It has also been demonstrated that the permeability of drugs across Caco-2 cell monolayers correlated very well with the extent of oral absorption in humans. In the last 10-15 years, the Caco-2 cells have been widely used as an in vitro tool for evaluating the permeability property of discovery compounds and for conducting in depth mechanistic studies (Chong et al., 1996; Aungst et al.,

2000; Braun et al., 2000; Horie et al., 2003; Ungell, 2004; Balimane and Chong, 2005b).

5.4.2.1 Caco-2 Cell Culture

Caco-2 cells (passage #17) were obtained from the American Type Culture Collection (Rockville, MD) and cultured as described below. The cells were seeded onto 24-well polycarbonate filter membrane (HTS-Transwell® inserts, surface area: 0.33 cm2) at a cell density of 80, 000 cells/cm2. The cells were grown in culture medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% L-glutamine, 100U/mL penicillin-G, and 100 |g/mL streptomycin. The culture medium was replaced every 2 days and the cells were maintained at 37 °C, 95% relative humidity, and 5% CO2. Permeability studies were conducted with the monolayers cultured for approximately 21 days with the cell passage numbers between 50 and 80. Caco-2 cell monolayers with TEER values greater than 400 Q cm2 were used.

5.4.2.2 Caco-2 cells Study Protocol

The transport medium used for the permeability studies was HBSS buffer containing 10 mM HEPES. Prior to all experiments, each monolayer was washed twice with buffer and TEER was measured to ensure the integrity of the monolayers. The concentration of test compounds ranged from 10 to 200 | M in this assay. The permeability studies were initiated by adding an appropriate volume of buffer containing test compound to either the apical (for apical to basolateral transport; A to B) or basolateral (for basolateral to apical transport; B to A) side of the monolayer. The monolayers were then placed in an incubator at 37 °C. Samples were taken from both the apical and basolateral compartment at the end of the incubation period (typically at 2 h) and the concentrations of test compound were analyzed by a high performance liquid chromatography method as described earlier (Chong et al., 1996). Permeability coefficient (Pc) was calculated according to the following equation:

Pc = dA/(dtSCo), where dA/dt is the flux of the test compound across the monolayer (nmol/s), S is the surface area of the cell monolayer (cm2), and Co is the initial concentration (|M) in the donor compartment. The Pc values were expressed as nm/s. All permeability data reported in this chapter were generated at the Bristol-Myers Squibb PRI. A minor variation in the experimental procedure often resulted in a significant difference in the permeability measurement making an interlaboratory data comparison very difficult. Therefore, the study protocol of PAMPA and Caco-2 cell permeability assay is also included as a point of reference.

Was this article helpful?

0 0
Natural Treatments For Dandruff

Natural Treatments For Dandruff

Stop Your Scalp From Snowing! Finally... A Real Cure for Dandruff! No medication! No weird cures! No messy creams, smelly solutions or strange diets! Are you sick to death of being sold products that could make the problem worse? Are you sick of the shampoos and conditioners that make your head smell like sulphur? If so then try a permanent revolutionary treatment for dandruff!

Get My Free Ebook


Post a comment