Caco2 Cells and Others

Caco-2 cells have been the most extensively characterized and useful cell model in the field of drug permeability and absorption for over a decade now (Arturrson,

Table 5.2. Cell culture models commonly used for permeability assessment

Cell Line


Special characteristics


Human colon

Most well-established cell model, differentiates and


expresses some relevant efflux transporters, expression of influx transporters variable (lab-to-lab)


Mardin-Darby canine kidney

Polarized cells with low intrinsic expression of ABC

epithelial cells

transporters, ideal for transfections


Pig kidney epithelial cells

Polarized cells with very low intrinsic transporter expression, ideal for transfections


Rat fetal intestinal epithelial

Temperature-sensitive, ideal for paracellularly absorbed


compounds (leakier pores)


Caco-2 sub-clone

Similar to Caco-2


Human colon

Contains mucus producing goblet cells


Rat small intestine cell line

Provides a size selective barrier for paracellularly transported compounds

1991; Artursson et al., 1996; Rubas et al., 1996; Hidalgo, 2001; Balimane and Chong, 2005a). Caco-2 cells, a human colon adenocarcinoma, undergo spontaneous enterocytic differentiation in culture. When they reach confluence on a semi-permeable porous filter, the cell polarity and tight junctions are well established. In the last 10-15 years, there has been a tremendous growth in the use of Caco-2 cells for mechanistic studies and as a rapid in vitro screening tool in support of drug discovery within the pharmaceutical industry. The predictability and utility of this model to rank order a large number of compounds in terms of absorption potential have been demonstrated conclusively by several investigators (Artursson and Karlsson, 1991; Rubas et al., 1993). The recent trend in many pharmaceutical research laboratories is to completely automate Caco-2 cell permeability screen using robotics. A fully automated Caco-2 cell system allows much greater throughput in the order of 500-2,000 compounds studies per month without a proportional increase in resources. The use of 24-well monolayer (cell surface area ~0.33 cm2) coupled with the use of LC-MS/MS significantly reduced the amount of compound (no more than 50 |xg) required to perform permeability experiment in this model. A more in-depth discussion on the Caco-2 cell permeability model along with its strengths and weaknesses is discussed later in the chapter.

TC-7 is one of the sub-clones isolated from Caco-2 cells. Gres and colleagues (Gres etal., 1998) have reported comprehensive comparison of TC-7 to its parental Caco-2 cells. TC-7 clone had very similar cell morphology as seen in Caco-2 cells: presence of brush-border membrane and microvilli, and formation of tight junctions. Although permeability of mannitol and PEG-4000 was identical in both cell lines, TC-7 had a significantly higher transepithelial electrical resistance (TEER) value at 21 days in culture and beyond. Permeability values of passively absorbed drugs obtained in TC-7 clone correlated equally well as in parental Caco-2 cells to the extent of absorption in humans.

2/4/A1 cells is a modified cell line reported (Tavelin et al., 2003) to have been generated from fetal rat intestine. It is touted to better mimic the permeability of the human small intestine especially with regards to passive transcellular and paracellular drug transport. This immortalized cell line forms viable differentiated monolayers with tight junctions, brush-border membrane enzymes as well as the transporter proteins. Since the tight junctions in the Caco-2 cell line appear unreal-istically tighter than the tight junctions in the endothelial cells in human intestine, the 2/4/A1 cells were proposed as a better model to study passively transported compounds via paracellular route. The TEER value in the 2/4/A1 cells reached a plateau of 25 Q cm2 compared to a plateau of 234 Q cm2 in the Caco-2 cells. The transport rate of poorly permeable compounds (e.g., mannitol and creatinine) in 2/4/A1 monolayers was comparable to that in the human jejunum, and was up to 300 times faster than that in the Caco-2 cell monolayers, suggesting that a cell line like 2/4/A1 will be more predictive for compounds that are absorbed via paracellular route.

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