optimizing caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis
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Brief communication
Optimizing Caco-2 cell monolayers to increase throughput in drug
intestinal absorption analysis
Maria Markowskaa,*, Rebecca Oberlea, Steve Juzwina, Cheng-Pang Hsua,Margaret Gryszkiewiczb, Anthony J. Streetera
aDepartment of Drug Metabolism, The R.W. Johnson Pharmaceutical Research Institute, Route 202, P.O. BOX 300, Raritan, NJ 08869, USAbRutgers, The State University of New Jersey College of Pharmacy, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA
Received 11 October 2001; accepted 19 November 2001
Abstract
Introduction: The aim of this investigation was to evaluate methods for increasing Caco-2 cell throughput for assessing drug intestinal
absorption. The use of 6-, 12-, and 24-well membranes and the effect of membrane size on permeability and the integrity of the Caco-2 cell
monolayer were assessed. In an effort to optimize the assessment of drug permeability, increased throughput was investigated by testing
compounds singly or as mixtures of analytes. Method: The transepithelial electrical resistance (TEER) of cell monolayers was measured on
0.33, 1.0, and 4.7 cm2 polycarbonate membranes using EVOM, over a 25-day period. Absorptive transport was determined on all compounds
tested using LC-MS/MS assays, or liquid scintillation spectrometry. Results: The effect of multiple compounds in one well compared to
single compounds was assessed with atenolol, nadolol, metoprolol, and propranolol for mixtures of four compounds and with RWJ-53308,
atenolol, terbutaline, propranolol, naproxen, piroxicam, topiramate, and furosemide for mixtures of eight compounds. The apparent
permeability (Papp) values correlated well between single analytes and mixtures of four and eight analytes in each well. Drug permeability
decreased slightly with an increase in well size. The TEER value increased with the number of days in culture for each of the 6-, 12-, and 24-
well sizes. Discussion: It was demonstrated that the 24-well format system is ideal for high-throughput assessment. Furthermore, the
approach of mixing four or eight analytes in each well to further increase throughput was also demonstrated to be valid. D 2001 Elsevier
Science Inc. All rights reserved.
Keywords: Caco-2 monolayers; High-throughput screening; Intestinal transport; LC-MS/MS analysis; Methods; Permeability; Polycarbonate membrane
1. Introduction
Today, Caco-2 cell monolayers have gained enormous
popularity as a reliable and high-throughput in vitro model
system for the evaluation of a large number of drug candi-
dates for their intestinal absorption potential (Artursson &
Karlsson, 1991; Chong, Dando, Soucek, & Morrison, 1996).
For orally administered compounds, permeability through
Caco-2 cell monolayers correlates well with in vivo absorp-
tion in man (Artursson & Karlsson, 1991; Artursson, Palm,
& Luthman, 1996). Experimentally, the Caco-2 screening
assay includes two consecutive procedures, a transport ele-
ment and a subsequent sample analysis. The transport
experiment for new compounds is extensive and requires
optimization. The preparation of a fully differentiated con-
fluent Caco-2 cell monolayer, however, generally requires a
3-week cell culture period with 9–10 intensive cell feedings.
In order to expedite the discovery of orally available drugs,
several approaches to increase the efficiency of this tech-
nique for drug screening have been attempted. Our study
describes unique approaches for increasing productivity by
evaluating Caco-2 performance under the following condi-
tions: (i) single analytes and mixtures of four and eight
analytes per well tested, (ii) permeability values determined
in 6-, 12-, and 24-well formats, (iii) the integrity of cells
evaluated in 6-, 12-, and 24-well formats by the measure-
ments of transepithelial electrical resistance (TEER), and (iv)
the permeability properties evaluated in 6-, 12-, and 24-well
formats with the paracellular marker, mannitol, and several
passively transported compounds. Results from this study
demonstrate that these approaches can be used to optimize
1056-8719/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved.
PII: S1056 -8719 (01 )00161 -7
* Corresponding author. Tel. +1-908-704-4765; fax: +1-908-704-8412.
E-mail address: mmarkows@prius.jnj.com (M. Markowska).
Journal of Pharmacological and Toxicological Methods 46 (2001) 51–55
permeability assessment throughput of certain types of drugs
in the early phases of drug discovery.
2. Materials and methods
2.1. Materials
14C-Mannitol, 3H-propranolol, 14C-testosterone were ob-
tained from NEN (Boston, MA). 3H-Cimetidine and 14C-
PEG 4000were obtained fromAmersham Pharmacia Biotech
(Little Chalfont, Buckinghamshire, England).
RWJ-53308 ([S-(R*,S*)]-b-[[[1-[1-oxo-3-(4-piperidinyl)-propyl]-3-piperidinyl]carbonyl]amino]-3-pyridinepropanoic
acid and topiramate were obtained from The R.W. Johnson
Pharmaceutical Research Institute (RWJPRI), Raritan, NJ.
Atenolol, nadolol, furosemide,metoprolol, propranolol, cime-
tidine, naproxen, piroxicam, and terbutaline were obtained
from Sigma (St. Louis, MO).
All tissue culture reagents and media were obtained from
Gibco BRL Life Technology (Grand Island, NY). All flasks
were obtained from VWR Scientific Products (Bridgewater,
NJ). The transwell polycarbonate membranes inserts with
areas of 4.7 cm2, 1.0 cm2, and 0.33 cm2 (0.4 mm pore size)
were obtained from Corning Science Product Division
(King of Prussia, PA). The epithelial tissue voltohmmeter
(EVOM) was obtained from World Precision Instruments
(Sarasota, FL).
2.2. Cell culture
The Caco-2 cell line was obtained from American Type
Culture Collection (Manassas, VA). Cells were seeded at
6.3� 104 cells/cm2 and grown in a medium comprised of
Dulbecco’s Modified Eagle’s Medium (DMEM) containing
4.5 g/l glucose and supplemented with 10% (v/v) fetal bovine
serum (FBS), 1% (v/v) glutamine, penicillin (100 U/ml),
streptomycin (100 mg/ml), and 1% Minimum Essential
Medium (MEM) nonessential amino acids. Cultures were
maintained at 37 �C in an atmosphere of 95% air and 5%CO2.
The medium was changed every 2–3 days. Routine passing
of cell stocks was carried out in 75-cm2 flasks. Studies were
conducted on Caco-2, passages #20–35.
2.3. TEER measurements during growth
The TEER of the cell monolayers was measured over a
25-day period. Cells were seeded at 6.3� 104 cells/cm2 on
0.33 cm2, 1.0 cm2, and 4.7 cm2 polycarbonate membranes
with a pore size of 0.4 mm. Prior to TEER measurements,
Hanks’ Balanced Salt Solution (HBSS) with 25 mM HEPES
pH 7.4 was added to the apical and basolateral membranes
and allowed to equilibrate to 37 �C for 25 min. The TEER
of each monolayer was measured at Days 4, 8, 10, 16, 21,
and 25 using an EVOM. The intrinsic resistance of the
system (insert alone) was subtracted from the total resist-
ance (cell monolayer plus insert) to yield the monolayer
resistance. The resistance was expressed as V� cm2.
2.4. Transport study
All transport studies were conducted at 37 �C, in a
medium of HBSS, pH 7.4, containing 25 mM HEPES. Prior
to the transport study, cell monolayers were washed with the
transport buffer and preincubated for 25 min. Test com-
pounds at final concentrations of 10 mM in HBSS were
added to the apical compartment. The basolateral compart-
ment contained only HBSS. All transport studies were
conducted in the apical (A) to basolateral (B) direction. At
selected times, ranging from 10 to 60 min, 100–500 ml ofsamples were removed from the basolateral compartment
for analysis and replaced with an equal volume of HBSS.
The test compound (50–500 ml) (donor solution) was re-
moved for analysis. Samples were analyzed using liquid
chromatography mass spectrometry (LC-MS/MS) and liquid
scintillation spectroscopy.
The initial flux of the drug ( J ) was determined from the
slope of the linear plot of the cumulative amount of drug
transported versus time. Permeability was estimated by
calculating the apparent permeability coefficient (Papp)
according to (Artursson & Karlsson, 1991):
Papp ¼ J=ASC0
where C0 is the initial concentration in the donor compart-
ment and AS is the surface area of the monolayer.
Potential depletion of the donor drug concentration during
the course of the study was monitored by analysis of the
donor solution at the termination of each study. Two con-
ditions were required to be satisfied for the equation to be
accurately applied: (i) sink conditions in the receiver com-
partment, i.e., the accumulated concentration must be less
than 10% of the donor concentration at all times within one
hour after experiment initiation, and (ii) the donor concen-
tration must remain within ± 10% of the initial donor con-
centration throughout the experiment.
Standards were prepared in HBSS containing 25 mM
HEPES. To minimize ion suppression due to the matrix
effects from HBSS, and to eliminate manual preparation, an
on-line extraction method was developed involving column
switching. This served to remove the high salt concentra-
tions in the biological buffer. After a 1-min desalting period,
the LC column eluate was switched to an analytical column.
Valve switching prevented the early eluting salts from
entering and contaminating the LC/MS interface. Detection
was accomplished on a PE Sciex API-365 mass spectro-
meter (Toronto, Ontario, Canada). The data collected from
the analytes were monitored in the multiple reaction mode
(MRM) using turboionspray in the positive and negative ion
mode. All standards were assayed in duplicate. Analyte
concentrations were calculated from standard curves of the
peak area versus standard concentration. Radiolabeled com-
M. Markowska et al. / Journal of Pharmacological and Toxicological Methods 46 (2001) 51–5552
pounds tested, containing either tritium or carbon-14, were
analyzed with a Packard liquid scintillation spectrometer
(Downers Grove, IL). After correction for background, dpm
values were converted to nanograms per milliliter.
2.5. Statistical analysis
The Student’s t test was used to compare the data of the
single analytes and mixture of analytes. P values < .05 were
considered to be statistically significant. Results are ex-
pressed as mean values ± S.D. The value of n is defined as
the number of wells per compound.
3. Results
3.1. Single and multiple compound assessment
The Papp values across Caco-2 cell monolayers of het-
erologous series of single analytes and mixtures of analytes
per well using 1 cm2 membrane (Table 1) correlated closely.
Two control markers, mannitol and propranolol, for para-
cellular and transcellular transport, respectively, were
included in all of the studies (although mannitol data were
not included in Table 1). In this study, the Papp values
determined for a single compound per well are consistent
with values determined for mixtures of four analytes as well
as with literature values (Artursson & Karlsson, 1991;
Chong et al., 1996; Fagerholm et al., 1996; Gres & Julian,
1998). Based on rank-order comparison of permeabilities
obtained for several reference compounds (Table 2), these
compounds fall within the same rank order (low, medium,
and high permeability of compounds).
Due to the sensitivity and selectivity provided by the LC-
MS/MS analytical method, inclusion of more analytes in a
mixture was studied. To minimize the cost and enhance HTS
using Caco-2 cells, a mixture of eight analytes was utilized to
perform the transport of drugs across the Caco-2 cell mono-
layers. Similarly, the Papp values determined for a single
compounds per well using 1 cm2 membrane were compar-
able with values determined from the mixture of eight
compounds per well (Table 1), and are also in agreement
with literature values (Artursson & Karlsson, 1991; Chong et
al., 1996; Fagerholm et al., 1996; Gres & Julian, 1998; Irvine
et al., 1999; Lennernaes et al., 1996; Polli & Ginski, 1998).
Although increases in the Papp value were observed with
atenolol (0.7 ± 0.1 vs. 1.4 ± 0.5, P < .05) and with piroxicam
(38.1 ± 0.5 vs. 53.1 ± 1.9, P < .05, Table 1), nevertheless,
based on rank-order comparison of permeabilities obtained
for several reference compounds (Table 2), these compounds
also fall within the same rank order (low, medium, and high
permeability of compounds).
3.2. Single compound using different size membrane
Permeability values of PEG-4000, mannitol, cimetidine,
propranolol, and testosterone, conducted in 6-, 12-, and 24-
Table 1
Papp values for compounds conducted either singly or in mixtures of four or eight analytesa across Caco-2 monolayers
Papp� 10� 6 (cm/s)
Compound Single Mixture-4 Single Mixture-8 Fa%b References
Atenolol 1.4 ± 0.4c 1.1 ± 0.2 50 Artursson & Karlsson, 1991
Nadolol 0.7 ± 0.1 0.8 ± 0.1 35 Chong et al., 1996
Metoprolol 32.3 ± 0.1 34.1 ± 1.4 95 Artursson & Karlsson, 1991; Fagerholm, Johansson, & Lennernaes, 1996
Propranolol 32.5 ± 0.3c 42.4 ± 0.8 90 Artursson & Karlsson, 1991; Gres & Julian, 1998
RWJ-53308 0.6 ± 0.3 0.6 ± 0.1
Atenolol 0.7 ± 0.1c 1.4 ± 0.5* 50 Artursson & Karlsson, 1991; Chong et al., 1996
Terbutaline 1.4 ± 0.8 0.9 ± 0.2 73 Artursson & Karlsson, 1991; Chong et al., 1996
Propranolol 23.9 ± 0.5c 29.3 ± 0.9 90 Artursson & Karlsson, 1991; Gres & Julian, 1998
Naproxen 54.2 ± 2.3 52.9 ± 3.6 100 Lennernaes, Palm, Fagerholm, & Artursson, 1996
Piroxicam 38.1 ± 0.5 53.1 ± 1.9* 100 Polli & Ginski, 1998
Topiramate 28.4 ± 2.2 30.8 ± 1.2 81 Wu et al., 1994
Furosemide 0.6 ± 0.1 0.6 ± 0.1 61 Fagerholm et al., 1996; Irvine et al., 1999
a The apparent permeability ( Papp) was conducted at 10 mM donor drug concentration in the A to B direction. This experiment was performed at pH value
of 7.4 across Caco-2 cell monolayers using a 1.0 cm2 membrane. Results are the means ± S.D. (n= 4).b Fraction of dose absorbed in humans is the absorption of the drugs after oral administration in humans (Artursson & Karlsson, 1991).c P < .05 compared in two settings of single determination.
* P < .05 compared to a single analyte.
Table 2
Papp values for reference compounds across Caco-2 cell monolayers using
three differently sized membranesa
Papp� 10 � 6 (cm/s)
Compound
0.33 cm2
membrane
1.0 cm2
membrane
4.7 cm2
membrane
[14C] PEG-4000 0.2 ± 0.1 0.13 ± 0.02 0.14 ± 0.01
[14C]-Mannitol 1.4 ± 0.2 1.3 ± 0.1 0.90 ± 0.1
[3H]-Cimetidine 1.4 ± 0.1 1.3 ± 0.1 1.04 ± 0.1
[3H]-Propranolol 38.9 ± 1.9 36.6 ± 0.9 24.2 ± 0.3
[14C]-Testosterone 53.5 ± 1.0 48.1 ± 1.6 36.4 ± 2.3
a The apparent permeability ( Papp) was conducted at 10 mM donor drug
concentration in the A to B direction. This experiment was performed at a
pH value of 7.4 across Caco-2 cell monolayers. Results are the
means ± S.D. (n= 4).
M. Markowska et al. / Journal of Pharmacological and Toxicological Methods 46 (2001) 51–55 53
well format (Table 2), showed slight declines in drug
permeability with an increase in well size (Table 2).
3.3. Membrane integrity
To optimize the high-throughput screening, assessment
of the integrity of the Caco-2 cell monolayers was deter-
mined by measuring the TEER in 6-, 12-, and 24-well
format for 25 days. These wells had membrane sizes of
0.33, 1.0, and 4.7 cm2, respectively. The TEER of Caco-2
monolayers in the three different size membrane formats
increased significantly with days in culture (Fig. 1), as
expected. Little increase in TEER was observed between
Days 16 and 25.
4. Discussion
To the best of our knowledge, this is the first report of an
in vitro system that compares the transport of single analytes
(atenolol, nadolol, metoprolol, or propranolol) with a mix-
ture of the four analytes across Caco-2 cell monolayers.
There have been a number of studies performed utilizing
high-throughput approaches for rapid pharmacokinetic
screening (Bayliss & Frick, 1999; Bu, Poglod, Micetich,
& Khan, 2000; Frick, Higton, Wring, & Wells-Knecht,
1998; Garberg, Eriksson, Schipper, & Sjostrom, 1999;
Liang, Chi, Wright, Timby, & Unger, 1999; Tabit & Ber-
man, 1998; Taylor, Gibbons, & Braeckman, 1997) and high-
throughput Caco-2 permeability screening (Bu et al., 2000),
but none of them differentiate the permeability of analytes
singly versus in a mixture of analytes.
To our knowledge, this is also the first report of an in
vitro system that compares the transport of single analytes
(RWJ-53308, atenolol, terbutaline, propranolol, naproxen,
piroxicam, topiramate, furosemide) with a mixture of the
eight analytes across Caco-2 monolayers. One should
always bear in mind the possibility of potential drug–drug
interactions involving transporters or metabolism when mix-
tures of compounds are used, and this may explain the
difference seen for atenolol and piroxicam. In our studies,
the permeability values measured by this approach (mix-
tures of four or eight analytes) suggest that using either
mixture of analytes is a reliable approach for assessing high-
throughput screening early in drug discovery. In the present
study, it has to be noted that Papp values of atenolol and
propranolol (P < .05, Table 1), assessed singly in separate
experiments, are different. However, these differences may
be due to the use of a heterogeneous cell line.
To enhance the HTS assessment and to lower the cost,
more studies were conducted to compare and select the best
conditions to study drug transport across Caco-2 monolayers.
Five reference compounds, PEG-4000, mannitol and cime-
tidine, propranolol, and testosterone with very low, low,
medium, and high permeability, respectively, were selected.
Transport was measured as described above, except that three
different membrane sizes were tested. The permeability
decline observed was attributable to the heterogeneous
Fig. 1. The TEER of Caco-2 monolayers in three differently sized transwell polycarbonate membranes measured over a 25-day period. Results are the
means ± S.D. (n= 4).
M. Markowska et al. / Journal of Pharmacological and Toxicological Methods 46 (2001) 51–5554
nature of the cell line in the Caco-2 monolayer. Studies have
shown that more permeable monolayers form poorly de-
veloped actin rings, suggesting differences in passive trans-
port caused by variable development of tight junctions
(Walter & Kissel, 1995), and our data are consistent with
this observation. This suggests that permeability of an
impermeable marker like mannitol is a more sensitive indic-
ator of the size of the tight junctions than the TEER assay
(Table 2 and Fig. 1). If studies were to be performed on a
cloned cell line, one ought not to see this discrepancy.
To use this model successfully, it is necessary to validate
the use of Caco-2 monolayers using marker compounds
under the defined culturing and experimental conditions that
will be used.
Caco-2 monolayers reached an optimal stable state for
study after 16–26 days, regardless of membrane size format.
However, a number of studies have reported a wide range of
TEER value measurements, indicating that in different
laboratories, under different culture conditions, Caco-2
monolayers can display electrical properties typical of either
small intestinal or colonic enterocytes (Yee, 1997). There-
fore, as stated above, an impermeable marker such as man-
nitol is more reliable and sensitive than the resistance
measurement as an indicator of tight junctions.
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