Supervisors: Vladan Milovic Professor Per Artursson
Investigations of the integrity and transport characteristics of 2/4/A1 cells
have been done in this report. The cell line was isolated from rat fetal
intestinal epithelial cells and transfected with thermolabile SV40 large T
These cells proliferated at 33 C, but eliminated the antigen and ceased
proliferating at a non-permissive temperature (39C). At 39C 2/4/A1 cells
started to differentiate but simultaneously the cells also underwent massive
When cultured at 37C these cells formed confluent and tight monolayers that
seemed to have paracellular transport characteristics similar to that of the
human intestine. Transmission electron microscopy confirmed the development of
multilayers at 33C, monolayers at 37C and defects in the cell layer due to
apoptosis at 39C.
Different immunostainings of ZO-1, E-cadherin and vinculin confirmed formation
of tight and adherence junctions. Transepithelial resistance reached a plateau
of 25-35 Ohm.cm2, which was similar to the small intestine. In transport studies
2/4/A1 cell line monolayers selectively restricted the permeation of hydrophilic
permeability markers proportional to molecular weight and discriminated more
accurately between the molecules of intermediate molecular weight compared to
These results indicated that 2/4/A1 cells could be used as a model for
hydrophilic drug absorption.
The small intestine plays a crucial role in the absorption of drugs and
nutrients. Exogenous substances cross a series of barriers during the process
of intestinal absorption: (1) the aqueous boundary/mucus layer, (2) a single
layer of epithelial cells, and (3) the lamina propria, which contains the blood
and lymph vessels that then transport the absorbed drugs to other parts of the
body (Artursson 1991).
The cell monolayer is comprised of two parallel barriers: the cell membrane and
the tight junctions. Most drugs are absorbed by a passive diffusion across the
cell membrane by the transcellular route, or across the tight junctions between
the cells – the paracellular route. Drug transport can also be carrier mediated,
when the drug utilizes transporters located in the cellular membrane.
Transcytosis is another kind of active transport, in which macromolecules can be
transported across the intestinal epithelial cell in endocytosed vesicles.
The hydrophilic and charged drugs are absorbed after passing through the
paracellular route, the water-filled channels between the cells (Artursson
1991). Rates and extent of the paracellular transport are, therefore, highly
influenced by the structure and size of the tight junctions as well as by the
size of the molecules. Only small and hydrophilic drugs can pass between the
cells rapidly and completely; permeation of larger molecules can be limited
proportionally to their size and lipophilicity (Hillgren et al. 1995).
Simple assay methods are needed for drug absorption studies. Excised intestinal
tissue, isolated cells, membrane vesicles and in vivo models have distinct
limitations, which have been previously discussed in detail (Audus et al. 1990;
Artursson 1991; Hillgren et al. 1995). The most suitable method for the study of
drug intestinal transport appeared to be the use of cultured intestinal
epithelial cells. This model has several advantages over conventional drug
absorption models: (a) it is less time-consuming; (b) it enables rapid
evaluation of methods for improving drug absorption; (c) it allows an
opportunity to use human rather than animal tissues; (d) it can minimize
expensive and sometimes controversial animal studies.
Human colorectal carcinoma cell line Caco-2 is nowadays the most widely used and
the best explored model for drug intestinal transport (Hidalgo et al. 1989;
Artursson 1990; Artursson & Karlsson 1991). This cell line displays spontaneous
enterocytic differentiation in culture and forms a polarized monolayer with
apical brush borders and well differentiated tight junctions (Hidalgo, 1989).
Drug transport studies across the Caco-2 cell monolayers showed a satisfactory
correlation with other in vitro absorption models, e.g. rat intestinal segments
(Artursson et al. 1993) and in vivo drug absorption (Lennerns et al., 1995),
although a considerable variability has been reported, being related to
heterogenity, a number of sub populations, and number of passages (Walter &
Caco-2 cells however, form monolayers that resemble colonic rather than small
intestinal epithelial cells. Due to its well-formed tight junctions, Caco-2 cell
monolayers have a transepithelial electrical resistance of 260 Ohm.cm2 which is
similar to the transepithelial electrical resistance of the colon rather than of
the small intestine (Hillgren et al. 1995). Therefore, there is a need to
investigate drug intestinal transport in a model which has apparent transport
characteristics corresponding to the human intestine, and several studies have
been attempted to characterize a cell line that can be used for this purpose.
A novel intestinal epithelial cell line (2/4/A1) is derived from the rat fetal
intestinal epithelial cells conditionally immortalized with thermolabile SV40
large T antigen, pzipSVtsa58 (Paul et al. 1993). According to the original
report, these cells form more leaky monolayers, with paracellular transport
characteristics similar to that of the human intestine. When cultured at 32C
these cells continually proliferate and display few markers of intestinal
differentiation. However, after being switched to a non-permissive temperature
(39C), these cells cease proliferating and exhibit a more markedly
differentiated phenotype. They form a polarized monolayer covered with a few
microvilli; tight junctions are also present (Paul et al. 1993; Hochman,
The 2/4/A1 cell line has been preliminary investigated in this laboratory. It
appeared that cells grown at 39C underwent massive apoptotic cell death
simultaneously with differentiation, and that those grown at permissive
temperature continued proliferating and form multilayers. However, when grown at
an intermediate temperature (37C), the cells underwent apoptosis to a lesser
extent, but maintained their proliferative capacity sufficiently to form tight
and continuous monolayers.
The aim of this study was to investigate permeability of paracellular marker
molecules across the 2/4/A1 cell line monolayers and to look at the
characteristics of the cell line.
MATERIALS AND METHODS
2/4/A1 cells were expanded in flasks at 33C, in RPMI 1640 medium supplemented
with 2% fetal calf serum, 10 mM Hepes, 2 mM L-glutamine, 200 mg/ml geneticin, 1
mg/ml BSA, 2 mg/ml dexamethasone, 20 ng/ml EGF, 50 ng/ml IGF-I, 10 mg/ml insulin,
10 mg/ml transferrin and 10 ng/ml selenic acid (ITS premixTM, Collaborative
Research), with 5-6% CO2 and 95% humidity.
The cells were seeded on Transwell polycarbonate filter inserts ( 6.5 mm)
coated with ECL extracellular matrix (entactin-collagen IV-laminin; Promega,
Madison, Wisconsin, USA), at a density of 100,000 cm2 in a serum-free RPMI 1640
medium supplemented with 10 mM Hepes, 2 mM L-glutamine, 200 mg/ml geneticin, 1
mg/ml BSA, 2 mg/ml dexamethasone, 20 ng/ml EGF, 10 mg/ml insulin, 10 mg/ml
transferrin and 10 ng/ml selenic acid.
Paracellular markers of different size and molecular weight labelled with 14C or
fluorescein were used: mannitol (MW 182), fluorescein (MW 376), lucifer yellow
(MW 450), polyethylene-glycol 4000 (MW 4000), and dextran (MW 50,000). The
experiments were performed at 37C in Hank’s Balanced Salt Solution pH 7.2 under
“sink conditions”. When PEG 4000 was used unlabelled PEG 4000 was also added to
the donor solution to limit possible drug metabolism. The labelled marker
molecules, 250 ml, were added to the apical side of the monolayer and after 20,
40, 60 and 80 minutes the inserts were moved to new wells and 500 ml samples
taken from the basolateral solution. Prior to the experiments samples of 50 ml
were taken from the apical solutions for measurements of the initial
concentration (C0). All solutions were preheated to 37C, and a heating plate
was used when the wells were moved. Transport was measured over time (days 1-10)
and compared with the values obtained from Caco-2 monolayers used as standard.
The radioactivity of the samples was determined using a standard liquid
scintillation technique. The apparent permeability coefficient was calculated as
described before (Artursson 1990), using a Microsoft Excel 4.0 software package
(Macintosh Power PC computer and Microsoft Office software) and templates
modified by K. Palm.
Transepithelial electrical resistance, short circuit current and
potential difference were measured by an in-house computer-based automatic
system using a single unit Transwell diffusion chamber (Grsj ; Karlsson,
unpublished results). Development of electrical parameters in 2/4/A1 cells was
studied over time (days 1-10). The data was processed using a Lab View software
package modified by Grsj et al.
2/4/A1 cells were routinely monitored under phase-contrast microscope each day.
At appropriate time points nuclei were stained with DAPI (4,6-diamidino-2-
phenylindolole, Molecular Probes, Leiden, Holland). The percentage of apoptotic
nuclei was quantified according to the method of Aharoni et al. (1995). Cells
grown on filters at different temperatures were examined by transmission
electron microscopy (TEM) after fixation in glutaraldehyde and dehydration with
1% osmium-tetroxide and 1% uranyl acetate. The presence of actin was assessed by
direct immunofluorescence with rhodamine-conjugated phalloidin. Development of
tight junctions were studied by indirect immunofluorescence to ZO-1 protein, and
adherence junctions by immunostaining to E-cadherin and vinculin.
Immunohistology slides were processed under laser scanning confocal microscope
(Leica, Heidelberg, Germany) and images were obtained by Silicon graphics
If not otherwise indicated, cell culture media and supplements were purchased
from Life Technologies AB, Tby, Sweden. Mouse monoclonal antibodies to SV40
large T antigen were from Oncogene Science, Uniondale, New York, USA, and
rhodamine-conjugated phalloidin from Molecular Probes, Leiden, Holland. Rabbit
polyclonal antibodies to ZO-1 were obtained from Zymed Laboratories Inc., San
Francisco, USA, and mouse monoclonal antibodies to human E-cadherin from
Transduction Laboratories, Lexington, Kentucky, USA. Mouse monoclonal antibodies
to human and rat vinculin were from Serotec, Oxford, UK.
Numerical data is expressed as the mean + SD of four to six experiments. One-way
ANOVA (corresponding to unpaired one-tailed Students t-test) was used to compare
means. A 95% probability was considered significant. RESULTS
Growth of 2/4/A1 cells
2/4/A1 cells seeded on ECL coated filter supports showed different growth rate
dependent on the temperature. At 33C 2/4/A1 cells proliferated rapidly, growing
exponentially until day 4 after seeding and forming multilayers consisting of
immature enterocytes. Growth was significantly reduced at 37C and the cells
formed monolayers. There was a decrease in cell number at 39C and 10 days after
seeding only 15% of the initial number of cells remained attached to the matrix.
Apoptosis, as calculated per 1000 cells, was present at 33C to a negligible
extent, although the proportion of apoptotic cells raised steadily at 39C.
After 10 days no nuclei without apoptotic morphology were noted at this
temperature. Number of apoptotic cells did not differ at the remaining two
temperatures (Figure 1).
As estimated qualitatively by the immunohistochemical detection of SV40 large T
antigen, the presence of the antigen was a prerequisite for growth in 2/4/A1
cells. SV40 large T antigen was present in the entire nuclei at 33C, less
prominent at 37C, and poorly stained in the nuclei at 39C (Figure 2).
Figure 2. Expression of SV40 large T antigen in 2/4/A1 cells seeded at 33C, 37
C and 39C. Bar indicates 10 mm.
Figure 3. ZO-1 (A,B,C), E-cadherin (D,E,F), and actin (G,H,I) in 2/4/A1 cells
seeded at 33C, 37C and 39C. Bar indicates 5 mm.
Figure 4. Vertical sections of 2/4/A1 cell layers seeded to 33C (A,C,E) and 37
C (B,D,F) stained to ZO-1 (A,B), E-cadherin (C,D) and vinculin (E,F). Bar
indicates 5 mm.
Development of tight and adherence junctions
As estimated by the appropriate antibodies, ZO-1 protein was present in 2/4/A1
cells grown at all temperatures. Its distribution, however was uneven in the
multilayers at 33C, reaching an intensively stained network at 37C. At the
non-permissive temperature the ZO-1 pattern was discontinuous, indicating
loosening of cell-to-cell contact preceding cell death (Figure 3, A-C).
Adherence junctions were also present at all temperatures. E-cadherin formed a
dotted network distributed diffusely in the cytoplasm at both 33 and 39C; the
pattern was located more closely near the cellular membrane at 37C (Figure 3,
D-F). Actin filaments were well developed at all three temperatures, showing
stress fibers at 33C and being distributed evenly at 37C in the cell membrane.
At 39C the actin network indicated broadening of extracellular spaces and
defects in the monolayer (Figure 3, G-I).
ZO-1 protein was located diffusely across the membrane at 33C. On the contrary,
at 37C ZO-1 was located exclusively in the upper pole of the cell-to-cell
junctions, indicating that normal tight junctions are formed at 37C. At 39C
the ZO-1 formed a discontinuous pattern located at the upper pole of the
monolayer, but with clear defects in the staining pattern indicating defects in
the cellular layer. E-cadherin and vinculin were located below the ZO-1 band,
forming a dotted network of filaments accumulated around the cell membrane
(Figure 4). Transmission electron microscopy confirmed the development of
multilayers at 33C, monolayers at 37C, and defects in the layer due to
apoptosis at 39C (Figure 5). Tight junctions occurred at all temperatures,
although those at 37C were longer and appeared tighter than those at 33C. At
all temperatures, at least within the time interval studied, the brush border
membrane surface remained undifferentiated, with few microvilli and without
visible brush borders. These data imply that 2/4/A1 cells may be presumably used
as a model of paracellular transport, in which the influence of brush border
enzymes and transcellular transport systems does not interfere with the
This data indicates that well developed tight and adherence junctions occur when
2/4/A1 cells are grown at 37C. We therefore decided to evaluate 2/4/A1 cells
grown at 37C as a model for paracellular transport of hydrophilic drugs across
the small intestine.
TEER reached a plateau of 25-35 Ohm.cm2 after four days in culture. Resting
potential and short circuit current were low throughout the time studied, and
were consistent with the cellular morphology (Figure 6).
Figure 6. Transepithelial resistance, resting potential and short circuit
current of 2/4/A1 cell line monolayers seeded at 37C. Experiments were
performed in Hanks balanced salt solution at 37C. N=6.
Figure 5. Transmission electron microscopy of 2/4/A1 cells seeded at (a) 33C,
(b) 37C and (c) 39C. Bar indicates 5 mm.
Transport experiments were studied 1, 2, 4, 6 and 10 days after seeding. 2/4/A1
cell line discriminated well between the paracellular markers of increasing
molecular weight, maintaining such a selective permeability throughout the
investigated period. Papp values for molecules with molecular weight around 400
were about 4.5×10-6 cm/s and correlated well to the human intestine (Figure 7).
When compared to Caco-2 cell line, 2/4/A1 cells had 40 to 250 times higher Papp
values and discriminated more accurately between the molecules of intermediate
molecular weight (Figure 8). Transport of mannitol and PEG-4000 in a calcium-
free medium showed a two-fold increase in comparison to normal values (Figure 9).
Since the adherence junctions can not function properly without calcium, this
data indicates that the permeation of the markers is restricted mainly to the
Figure 7. Permeability of hydrophilic marker molecules across 2/4/A1 cell line
Figure 8. Permeability of hydrophilic marker molecules across 2/4/A1 cell line
monolayers (A) and Caco-2 cell line monolayers (B). Note that Papp values differ
aprox. 100-fold. N=6.
Figure 9. Permeability of mannitol (MW 182) and PEG-4000 across 2/4/A1 cell line
monolayers in Hanks balanced salt solution with (left) and without calcium
(right). N=4. *, p