EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 541

18.4.3. Established Cell Lines
In organs and tissues relevant to drug behavior, drugs are transported by polar cells
such as intestinal and renal epithelial cells bearing directional drug transport systems.
Therefore, established model cell lines with polarity provide useful model systems to
study directional transport of drugs. Representative lines include Caco-2 and LLC-
PK1 cells. Furthermore, conditionally immortalized cell lines are also available as
new in vitro models for the study of transporters. 36
Caco-2 Cells Caco-2 cells derived from human colon carcinoma are often used as
a model for intestinal epithelial cells to predict drug behavior in the intestine. 37 For
cultivation of Caco-2 cells, we adopt Dulbecco's modified Eagle's medium, containing
10% fetal calf serum, 1% nonessential amino acids, 2 mM L-glutamine, penicillin,
and streptomycin in a humidified incubator at 37 ◦ C under an atmosphere of 5% CO 2
in air. For transport experiments, Caco-2 cells are grown on dishes and on Transwell
microporous polycarbonate membranes. Caco-2 cells are cultured for 21 to 23 days
before assays. 25 , 38 , 39
LLC-PK1 Cells LLC-PK1 cells lines derived from pig kidney are often used for
studies of MDR1 (ABCB1), since human MDR1 is expressed on the apical membrane
of LLC-GA5-COL150 cells and confers upon them resistance to colchicines and other
P-glycoprotein substrates. In particular, comparative studies between LLC-PK1 and
LLC-GA5-COL150 cells can be performed to examine whether a test compound is
a substrate of MDR1. Ueda et al. used this approach to show that steroidal hormones
could be transported directionally by MDR1. 40
LLC-PK1 cells are grown in M199 medium containing 10% fetal calf serum,
14.3 mM NaHCO 3 and 3% L-glutamine. For cultivation of LLC-GA5-COL150 cells,
150 ng/mL colchicine is added to the medium. For transport experiments, the cells
are grown on Transwell microporous polycarbonate membranes and are cultured for
3 or 4 days before use. 25 , 41
18.4.4. Transfected Cell Lines and Xenopus laevis Oocytes
For evaluation of the transport activity of drug transporters, gene expression systems,
such as cultured cells and Xenopus laevis oocytes, provide simple experimental tools
to evaluate substrate specificity and the driving force of transporters. Here we describe
transport assay in transporter-expressing HEK293 cells and X. laevis oocytes.
Expression of Drug Transporters Expressed in HEK293 Cells For evaluation of
the transport activity of drug transporters, several types of cultured mammalian
cell expression systems can be used. We have adopted HEK293 cells derived from
human embryonic kidney. HEK293 cells are routinely grown in Dulbecco's modi-
fied Eagle's medium containing 10% fetal calf serum, penicillin, and streptomycin
in a humidified incubator at 37 ◦ C under an atmosphere of 5% CO 2 in air. Af-
ter cloning or purchase of cDNAs encoding transporters, they are inserted into