SUMMARY
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 antigen.
These cells proliferated at 33 ?C, but eliminated the antigen and ceased proliferating at
a non-permissive temperature (39?C). At 39?C 2/4/A1 cells started to differentiate but
simultaneously the cells also underwent massive cell death.
When cultured at 37?C 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 33?C,
monolayers at 37?C and defects in the cell layer due to apoptosis at 39?C.
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 Caco-2 cells.
These results indicated that 2/4/A1 cells could be used as a model for hydrophilic drug
absorption.
INTRODUCTION
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
(Lennernas et al., 1995), although a considerable variability has been reported, being
related to heterogenity, a number of sub populations, and number of passages (Walter &
Kissel, 1995).
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 32?C these cells continually proliferate and display
few markers of intestinal differentiation. However, after being switched to a
non-permissive temperature (39?C), 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, personal
communication).
The 2/4/A1 cell line has been preliminary investigated in this laboratory. It appeared
that cells grown at 39?C 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 (37?C), 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
Cell culture
2/4/A1 cells were expanded in flasks at 33?C, 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.
Transport studies
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 37?C 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 37?C, 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.
Electrophysiological measurements
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 (Grasjo & 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 Grasjo et al.
Cell morphology
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 software package.
Materials
If not otherwise indicated, cell culture media and supplements were purchased from Life
Technologies AB, Taby, 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.
Statistics
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 33?C 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 37?C and the cells formed monolayers. There was a
decrease in cell number at 39?C 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 33?C to a negligible extent, although the proportion of apoptotic cells raised
steadily at 39?C. 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 33?C, less prominent at 37?C, and poorly
stained in the nuclei at 39?C (Figure 2).
Figure 1. Cell number and apoptosis in 2/4/A1 cells seeded on ECL matrix at 33?C,
37?C and 39?C. Cells were counted after staining with DAPI and apoptosis estimated
in accordance to the accepted criteria. *, p<0.05; **, p<0.01.
Figure 2. Expression of SV40 large T antigen in 2/4/A1 cells seeded at 33?C, 37?C and
39?C. 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
33?C, 37?C and 39?C. Bar indicates 5 mm.
Figure 4. Vertical sections of 2/4/A1 cell layers seeded to 33?C (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
33?C, reaching an intensively stained network at 37?C. 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 39?C; the pattern was located more closely near the cellular membrane at
37?C (Figure 3, D-F). Actin filaments were well developed at all three temperatures,
showing stress fibers at 33?C and being distributed evenly at 37?C in the cell membrane.
At 39?C 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 33?C. On the contrary, at 37?C
ZO-1 was located exclusively in the upper pole of the cell-to-cell junctions, indicating
that normal tight junctions are formed at 37?C. At 39?C 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 33?C,
monolayers at 37?C, and defects in the layer due to apoptosis at 39?C (Figure 5). Tight
junctions occurred at all temperatures, although those at 37?C were longer and appeared
tighter than those at 33?C. 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
paracellular pathway.
This data indicates that well developed tight and adherence junctions occur when 2/4/A1
cells are grown at 37?C. We therefore decided to evaluate 2/4/A1 cells grown at 37?C as a
model for paracellular transport of hydrophilic drugs across the small intestine.
Transepithelial resistance
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 37?C. Experiments were performed in Hanks balanced
salt solution at 37?C. N=6.
Figure 5. Transmission electron microscopy of 2/4/A1 cells seeded at (a) 33?C, (b) 37?C
and (c) 39?C. Bar indicates 5 mm.
Transport studies
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 4.5x10-6 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
paracellular pathway
Figure 7. Permeability of hydrophilic marker molecules across 2/4/A1 cell line
monolayers. N=6.
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<0.05; **, p<0.01.
DISCUSSION
Cell cultures have been broadly used in the studies of drug intestinal transport. It has
been generally accepted that the data obtained from the cell culture models are easy to
interpret, since the influence of adjacent structures (submucosa, luminal enzymes,
intestinal transit) has been minimized or completely abolished. However, most of the cell
culture models used for the studies of drug intestinal transport are of cancer origin;
there is a possibility that their transport characteristics may differ from the normal
intestine. Furthermore, in Caco-2 cells the tight junctions are more similar to the colon
than to the small intestine; also, for instance, T84 cells correspond to crypt cells,
with a negligible role in the intestinal transport under in vivo conditions, and the
HT-29 cell line can be induced to mimic various cell types, but also showed a number of
phenotypic variations and appeared to be a poorly reproducible model for drug intestinal
transport (Artursson 1991; Hillgren et al., 1995). In addition, cells which undergo
spontaneous differentiation (e.g. Caco-2) require to be kept in culture for several
weeks, which obviously increases the costs of maintenance and experiments. Several
attempts to cultivate normal intestinal cells appeared to be unsuccessful.
In order to overcome disadvantages of the use of cancer cell lines in studying intestinal
transport, we evaluated a conditionally immortalized rat fetal intestinal epithelial cell
line, 2/4/A1, as a possible alternative model. Such cells can be isolated from transgenic
mice (Whitehead et al,1991), or, as was the case in our study, from rat fetal intestinal
cells conditionally immortalized with a thermolabile SV40 large T antigen (Paul et al,
1993). These cells proliferate continuously at a permissive temperature (33?C), but cease
proliferating and undergo apoptotic cell death at a non-permissive temperature (39?C). In
the preliminary part of our study, we found out that 2/4/A1 cells preserved certain
proliferative capacity when kept at an intermediate temperature (37?C), presumably due to
an incomplete elimination of SV40 large T antigen. Growth rate at the intermediate
temperature was significantly reduced in comparison to the growth at a permissive
temperature. Furthermore, we have shown that 2/4/A1 cells at 37?C developed well
differentiated tight and adherence junctions, which differed morphologically from the
less developed tight junctions observed at 33?C. Our data also showed that intestinal
transport systems located at the brush border membrane of the absorptive cell remained
undifferentiated when 2/4/A1 cells were cultured at an intermediate temperature.
Permeability characteristics of the model appeared to be more similar to human ileum than
to human colon, as was the case with Caco-2 model: TEER values were 25-35 Ohm.cm2,
similar to human ileum and slightly lower in comparison to the human jejunum (Lennernas
et al 1995). In spite of a great variability of TEER values in different clones and
passage numbers of Caco-2 cells (Walter and Kissel, 1995), TEER values in 2/4/A/1 cells,
according to our data, remained significantly lower than in any type or clone of the
Caco-2 cells studied.
2/4/A1 cell line monolayers also selectively restricted the permeation of hydrophilic
permeability markers proportional to molecular weight and size. Permeability of larger
markers (PEG-4000, dextran) was negligible. This effect can be attributed exclusively to
the selective nature of tight junctions in 2/4/A1 cells, since the permeability decreased
rapidly after a calcium-free medium was introduced. Morphologic examination of the tight
junctions (by immunostaining to ZO-1 protein, data not shown) clearly supported the
evidence that tight junctions in 2/4/A1 cells were sensitive to calcium depletion and
showed subsequent impairment in their function.
2/4/A1 cell line monolayers discriminated well between the molecules of an intermediate
molecular weight, i.e. between 180 and 4000. Since most of the drugs transported via the
paracellular route have molecular weight within this range, we conclude that 2/4/A1 cells
fulfil the functional criteria to be used as a model for the hydrophilic drugs that
utilize the paracellular pathway when absorbed in the human small intestine.
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