A-1985A
APPLICATION INFORMATION
A D M E To x
CACO-2 BI-DIRECTIONAL TRANSPORT ASSAY USING
BECKMAN COULTER’S BIOMEK® AUTOMATED PLATFORMS
Yu Suen, Ph.D., Michael H. Simonian, Ph.D. and Graham Threadgill, Ph.D.
Beckman Coulter, Inc.
Introduction
Incorporating predictive ADME (absorption,
distribution, metabolism and elimination) assays
in earlier stages of drug discovery can help reject
molecules that lack drug-like properties as early as
possible.(1, 2) Drug bioavailability is influenced by
factors including absorption and metabolism. Based
on chemical properties, drugs are absorbed either by
passive diffusion or active transport mechanisms.
P-glycoprotein is an ATP-dependent, substratespecific, active carrier-mediated (efflux) transporter
that is responsible for the active transport of a large
number of drugs. Substrates of p-glycoprotein can
be transported via this efflux system, and this results
in a decreased intracellular drug concentration.
P-glycoprotein is located in human tissues that are
responsible for absorption and metabolism,
including liver, kidney, and gastrointestinal tract.(3)
Its function affects drug absorption,
pharmacokinetics, drug–drug interactions, and
contributes to the well-characterized mechanism of
multidrug resistance.(4)
The United States Food and Drug Administration
issued the Biopharmaceutics Classification System,
which is the guidance for using in vitro models for
assessing drug permeability and absorption. The
Caco-2 human colorectal adenocarcinoma cell line
forms monolayers of differentiated epithelial cells
joined by intercellular tight junctions. This cell line
model provides a selective barrier that can be used
to study structure-transport relationships for both
transcellular (from the apical to the basalateral
chamber) and carrier-mediated efflux transport
(from the basalateral to the apical chamber).
Differentiated Caco-2 cells express high levels of
P-glycoprotein; it is an excellent model for screening p-glycoprotein substrates through the efflux
transport mechanism(5) (see Appendix A for a diagrammatic representation of the Caco-2 transport
systems).
A high-throughput Caco-2 bi-directional
transport assay is needed for the drug discovery
industry. This bulletin describes two methods that
have been developed: one for the automation of
Caco-2 cell preparation and the other for the
bi-directional transport assay. The Biomek® 2000
Laboratory Automation Workstation in the biosafety
hood was used to automate Caco-2 cell preparation
and differentiation in a BD Falcon* HTS 96-Multiwell Insert System (BD Biosciences, Bedford, MA)
and MultiScreen* Caco-2 Assay System (Millipore,
Bedford, MA). Additionally, the Biomek FX liquid
handling system was used to automate the bi-directional transport assay and sample collection for
analysis in an ambient environment. A previously
published Application Information Bulletin
demonstrated the feasibility of using these Biomek
platforms for automating drug permeability assays(6)
with the 96-well insert systems and differentiated
Caco-2 cells (A-1984A). The intact cell monolayers
in each insert well functioned as a barrier, preventing paracellular diffusion of lucifer yellow from the
apical chamber to the basolateral chamber, but
allowing drug permeation through active transcelluar
or carrier-mediated transport mechanisms.
This study reports the functional efflux system
through p-glycoprotein with the bi-directional
transport system. With this system, rhodamine 123
and paclitaxel are used to demonstrate higher
permeation coefficients from the basolateral to
apical direction as compared to the permeation
coefficients from the apical to the basolateral
direction.
In this bulletin, we demonstrate the use of the
Biomek® 2000 Laboratory Automation Workstation
and Biomek FX Liquid Handling System for the
automated cell preparation and assay systems
described above. The use of these systems facilitated Caco-2 assay implementation, reduced the
chance of contamination, and minimized the intensive requirement of sterile skills. The automated
Caco-2 assay systems can be used for high-throughput screening of drug candidates for absorption
properties.
Biomek 2000 P200L (P/N 609022), MP200
(P/N 609025) and Gripper (P/N 609735) liquid
handling tools were used on the Biomek 2000
Workstation for cell preparation.
The Biomek FX 96-Channel Disposable Tip
Pipetting Head – 200 µL (P/N 719368), Biomek FX
Disposable Tip Loader (P/N 719356), and Standard
Three-Position ALP (P/N 719358) liquid handling
tools were used on the Biomek FX system.
The BMG FLUOstar system was used for
fluorescence signal detection of lucifer yellow and
rhodamine 123. The Beckman Coulter DU® 7000
Spectrophotometer was used for paclitaxel detection.
Automated Method 1: Cell Preparation
Using the Biomek 2000 Workstation
Caco-2 cells were maintained in Dulbecco’s
Modified Eagle Medium (DMEM) (Invitrogen,
catalog number: 10569010) complete medium with
10% fetal calf serum (Invitrogen, catalog number
16000036), non-essential amino acids (Invitrogen,
catalog number: 11140050), and penicillin-streptomycin (Invitrogen, catalog number: 15140-148) in
tissue culture flasks. Cells were maintained between
pass 19 and 45 for the assay. Cells were prepared in
the same media at 2.5 × 105 cells/mL in a reservoir.
A volume of 50 µL was dispensed gently by the
Biomek 2000 Workstation into each insert well
(12,500 cells/50 µL, or 1.55 × 105 cells/cm2) of a
BD Falcon HTS 96-Multiwell Insert System (BD
Biosciences, catalog number: 351130), and the
MultiScreen Caco-2 Assay System (Millipore,
catalog number: MACA CO2S2).
The wells in rows A and E were filled with
media only as control wells to monitor the diffusion
of lucifer yellow without the Caco-2 cell monolayers as barriers. A volume of 36 mL of pre-warmed
complete media was added to the feeder tray of the
BD Falcon HTS 96-Multiwell Insert System. A volume of 250 µL of pre-warmed complete media was
added to each well of the 96-well receiver plate of
the Millipore MultiScreen Caco-2 Assay System.
Cells were then incubated at 37°C in 5% CO2 for
21 days of differentiation. Medium was changed in
both the insert wells and the feeder tray or receiver
plate for both systems every 48 to 72 hours. The run
time of this automated cell seeding and feeding
method for one plate was 15 minutes for the BD
Falcon HTS 96-Multiwell Insert System and 25
minutes for the Millipore MultiScreen Caco-2
Assay System.
Materials and Methods
Reagents
A Caco-2 cell line obtained from ATCC (Manassas,
VA) was used for the tissue culture model for compound permeability testing. Lucifer yellow
(Sigma-Aldrich, St. Louis, MO, catalog number:
L0259) at 100 µM concentration was used for
integrity testing of the Caco-2 cell monolayer.
Rhodamine 123 (Sigma-Aldrich, catalog number:
R8004) and paclitaxel (Sigma-Aldrich, catalog
number: T7191) at 10 µM concentration in transport
buffer were used for bi-directional transport studies.
Hank’s Balanced Salt Solution (Invitrogen,
Rockville, MD, catalog number: 14175095) was
used as the transport buffer for the bi-directional
transport assays.
Software
BioWorks™ version 3.2 was the liquid handling controlling system for the Biomek 2000 Laboratory
Automation Workstation. Biomek FX version 2.2
was the liquid handling controlling system for the
Biomek FX Liquid Handling System. FLUOstar
Galaxy Software version 4.30 was the fluorometer
controlling system for the BMG FLUOstar system.
Hardware
The Biomek 2000 Workstation in a Baker Company
class II biological safety cabinet provided a sterile
environment for tissue culture preparation. The
2
The Biomek® 2000 Workstation worksurface
layout for the BD Falcon HTS 96-Multiwell Insert
System is shown in Figure 1. The Biomek 2000
Workstation worksurface layout for the Millipore
MultiScreen Caco-2 Assay System is shown in
Figure 2. Two assay plates may be loaded on the
Biomek 2000 Workstation at one time, and multiple
runs can be performed to accommodate different
levels of throughput needs. (See Appendix B for a
detailed description of the Biomek 2000 methods
used for cell culture and differentiation preparation.)
dispensed in rows A to D of the Angled Bottom
Plate or receiver plate of both systems. The insert
plates were then placed onto the BD Angled Bottom
Plate or Millipore receiver plate using the FX
Gripper. Lucifer yellow at final concentration of
100 µM, rhodamine 123 at final concentration of
10 µM, and paclitaxel at final concentration of
10 µM were prepared in transport buffer and dispensed in rows A to D of the Drug Standard plate.
Transport buffer was dispensed into rows E to H of
the Drug Standard plate. A volume of 50 µL paclitaxel, rhodamine 123, or lucifer yellow was added
to each insert well. Both Caco-2 assay systems were
incubated at 37°C in 5% CO2 for 2 hours. After
2 hours, 50 µL of sample were collected in black,
clear-bottom 96-well plates from both the insert
plate wells and the bottom plate wells for analysis
(see Appendix C for details of the Biomek FX
transport assay method). The diffusion of lucifer
yellow through the insert membrane without a cell
barrier and the lack of lucifer yellow permeation
through Caco-2 cell monolayers were detected
using a BMG FLUOstar reader with excitation at
428 nm and emission at 540 nm. The bi-directional
transport of drug standards through Caco-2 cell
monolayers was detected using the BMG FLUOstar
reader and the Beckman Coulter DU® 7500
Spectrophotomer.
The run time of the automated Biomek FX
transport assay method before the two hours of
incubation was 20 minutes. The run time for the
final sample collection after the two hours of incubation was 10 minutes. One assay plate could be
processed if all the tips and reagents labware were
loaded on the deck. Automated methods for handling multiple assay plates can be accomplished
using a stacker carousel to supply plates and tips to
the workstation. Assays can be staggered during the
two hours of incubation time to accommodate different levels of throughput needs.
Automated Method 2: Bi-Directional
Transport Assay Using the Biomek FX
System
Caco-2 cell monolayers grown in the apical compartments (insert wells) were washed three times
with fresh transport buffer using the Biomek FX
System and washed twice with fresh transport
buffer in basal compartments (feeder tray for the
BD Falcon HTS 96-Multiwell Insert System;
receiver plate for the Millipore MultiScreen Caco-2
Assay System) (Figures 3, 4, and 5). Volumes of
250 µL of rhodamine 123 at a final concentration of
10 µM and paclitaxel at a final concentration of 10
µM were prepared in transport buffer and dispensed
in rows E to H of the BD Falcon 96-Square-Well
Angled Bottom Plate, or in rows E to H of the
Millipore MultiScreen Caco-2 Assay System receiver plate. A volume of 250 µL transport buffer was
Figure 1. Biomek 2000 Workstation worksurface
layout for media change in cell preparation for the
BD Falcon HTS 96-Multiwell Insert System.
Figure 2. Biomek 2000 Workstation worksurface
layout for media change in cell preparation for the
Millipore MultiScreen Caco-2 Assay System.
Figure 3. Biomek FX Liquid Handling System for
Caco-2 bi-directional transport assay.
3
Results
Summary
Lucifer yellow was used as the reference compound
for verifying Caco-2 cell monolayer integrity. Data
shown in Table 1 demonstrate that lucifer yellow
diffused from the insert wells to the receiver wells
in the absence of a Caco-2 cell barrier. However, in
the presence of a Caco-2 cell barrier, the permeability coefficient of lucifer yellow was insignificant in
both Caco-2 assay systems. The presence of an
intact Caco-2 monolayer in both assay systems
demonstrates that both Biomek® platforms provided
gentle cell handling, medium exchange, and cell
washing for Caco-2 monolayer differentiation.
Rhodamine 123 was used as a reference compound for evaluating the bi-directional transport
efflux across the Caco-2 cell monolayer. Data
shown in Table 2 demonstrate that rhodamine
123 was transported from the apical chamber to
the basolateral chamber by transcellular transport
(A to B) in both systems. Rhodamine 123 was
also transported from the basolateral chamber to
the apical chamber by carrier-mediated efflux transport (B to A). The ratio of efflux to transcelluar
transport was greater than 1.5 in both systems,
which shows effective p-glycoprotein function.(5)
Paclitaxel was used as a sample drug with
known efflux transport through p-glycoprotein for
testing the bi-directional transport efflux across
the Caco-2 monolayer. Data shown in Table 3
demonstrate that paclitaxel was transported from
the apical chamber to the basolateral chamber by
transcelluar transport (A to B) in both systems.
Paclitaxel was also transported from the basolateral
chamber to the apical chamber by carrier-mediated
efflux transport (B to A). The ratio of efflux to transcellular transport matched published data,(7) which
further supports the effective p-glycoprotein function in Caco-2 monolayers in both systems.
Caco-2 cell monolayers function as barriers preventing paracellular diffusion of lucifer yellow,
while permitting carrier-mediated active and efflux
transport of drug standards. The Caco-2 cells prepared using the Biomek 2000 Workstation differentiated into tight monolayers in insert wells within 21
days of culture. The Biomek FX system was then
used to prepare monolayers of Caco-2 cells for a
bi-directional transport assay. An intact cell monolayer is required for this in vitro cellular model.
Lucifer yellow was used as a control to assess the
integrity of the Caco-2 cells. Data presented in
Table 1 show that the Biomek 2000 methods for
Caco-2 cell culture and Biomek FX method for bidirectional transport assay maintained excellent cell
integrity. Tables 2 and 3 document the transcellular
transport of rhodamine 123 and paclitaxel from the
apical chamber to the basolateral chamber (A to B
permeability coefficient) and efflux transport of
these compounds from the basolateral chamber to
the apical chamber (B to A permeability coefficient)
respectively. Polarized efflux transport of rhodamine 123 and paclitaxel through p-glycoprotein is
demonstrated by the ratio of B to A compared to
A to B permeability coefficient which was above
1.5 for both rhodamine 123 and paclitaxel in both
systems. The automated cell preparation and assay
systems presented here using the Biomek 2000 and
Biomek FX Laboratory Automation Workstations
facilitated Caco-2 assay implementation, reduced
the chance of contamination and minimized the
intensive requirement of sterile skills. The automated
Caco-2 assay systems can be used for high-throughput screening of drug candidates for absorption
properties.
Figure 4. Biomek FX Liquid Handling System
deck layout for bi-directional transport assay using
the BD Falcon HTS 96-Multiwell Insert System.
Figure 5. Biomek FX Liquid Handling System
deck layout for bi-directional transport assay using
the Millipore MultiScreen Caco-2 Assay System.
4
References
5. Schwab, D., Fischer, H., Tabatabaei, A., Poli,
S., and Huwyer, J. (2002) Comparison of in vitro Pglycoprotein Screening Assay: Recommendations
for their use in Drug Discovery. J Med Chem 46:
1716-1725.
6. Suen, Y., Bang, K., Simonian, MH.,
Threadgill, G. (2002). High-throughput Caco-2
Permeability Screening Assay Using Biomek
Automated Platforms from Beckman Coulter.
Application Information Bulletin A-1944A.
7. Walle, U.K., Walle, T. (1998) Taxol transport
by human intestinal epithelial Caco-2 cells. Drug
Metabolism and Disposition 26:343-346.
1. DePalma, A. (2002) Analyzing ADME
Absorption Values. Drug Discovery &
Development, volume 5 (7): 60-66.
2. Biganzoli, E., Cavenaghi, L., Rossi, R.,
Brunati, M., Nolli, M. (1999) Use of a Caco-2 cell
culture model for the characterization of intestinal
absorption of antibiotics. IL Farmaco 54: 594-599.
3. Thiebaut, F., Tsuruo, T., Hamada, H.,
Gottesman, M., Pastan, I., Willingham, M. (1987).
Cellular localization of the multidrug-resistance
gene product P-glycoprotein in normal human
tissue. Proc. Natl. Acad. Sci. 84: 7735-7738.
4. Fojo, A.T., Ueda, K., Slamon, D.J., Poplack,
D.G., Gottesman, M. M., Pastan, I. (1987)
Expression of a multidrug-resistance gene in human
tumors and tissues. Proc. Natl. Acad. Sci. U.S.A.
84:265-269.
Table 1.1 Papp A to B (× 10-6 cm/sec)
Caco-2 Cell Monolayer as a Barrier Prevents Leakage of Lucifer Yellow in Both
BD Falcon HTS 96-Multiwell Insert System and Millipore MultiScreen Caco-2
Assay System.
With Caco-2 cell barrier
Without Caco-2 cell barrier
BD Falcon HTS 96-Multiwell Insert System Millipore MultiScreen Caco-2 Assay System
0.24 ± 0.051
0.03 ± 0.011
200.78 ± 15.271
183.52 ± 9.161
Table 2.1 Papp A to B (× 10-6 cm/sec)
Caco-2 Cell Monolayer Has Active Functional Efflux Transport of Rhodamine 123
in Both BD Falcon HTS 96-Multiwell Insert System and Millipore MultiScreen
Caco-2 Assay System.
A to B
B to A
B to A / A to B
BD Falcon HTS 96-Multiwell Insert System
3.93 ± 0.921
7.39 ± 0.041
2.0
MultiScreen Caco-2 Assay System
3.46 ± 0.121
51.20 ± 5.271
14.8
Table 3.1 Papp A to B (× 10-6 cm/sec)
Caco-2 Monolayer Has Active Functional Efflux Transport of Paclitaxel in Both
BD Falcon HTS 96-Multiwell Insert System and Millipore MultiScreen Caco-2
Assay System.
A to B
B to A
B to A / A to B
BD Falcon HTS 96-Multiwell Insert System BD
Falcon HTS 96-Multiwell Insert System
7.24 ± 0.061
56.40 ± 1.081
7.7
5
MultiScreen Caco-2 Assay System MultiScreen
Caco-2 Assay System
4.36 ± 0.191
103.00 ± 4.431
22.6
Appendix A. Caco-2 Cell Monolayer Transport Systems
1. Transcellular Transport
2. Paracellular Transport
3. Carrier-Mediated Transport
4. Endocytosis
5. Active Transport (Efflux)
Appendix B. Biomek® 2000 method: Caco-2 Cell Culture and Differentiation
i
6
Appendix C. Biomek® FX Method: Caco-2 Drug Permeability Assay
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B2004-6250
© 2004 Beckman Coulter, Inc.