Permeability properties of monolayers of the human trophoblast
cell line BeWo
Fei
Liu1,
Michael J.
Soares2, and
Kenneth L.
Audus1
1 Department of Pharmaceutical
Chemistry, University of Kansas, Lawrence 66045; and
2 Department of Physiology,
University of Kansas Medical Center, Kansas City, Kansas 66160
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ABSTRACT |
The BeWo cell line (b30 clone) has been examined as a potential
in vitro system to study transplacental transport. At the light and
electron microscope level, the cells were observed to form confluent
monolayers on polycarbonate filters in ~5 days and morphologically
resembled the typical human trophoblast. BeWo monolayers developed a
modest transepithelial electrical resistance and a molecular
size-dependent permeability to hydrophilic passive diffusion markers,
fluorescein, and selected fluorescein-labeled dextrans. Linoleic acid
permeation across BeWo monolayers was asymmetric, saturable, and
inhibited by low temperature and excess competing fatty acid. Forskolin
and 8-bromoadenosine 3',5'-cyclic monophosphate treatments
stimulated morphological changes in BeWo cultures and enhanced the
asymmetric passage of linoleic acid across the BeWo monolayers while
having minimal effects on passive permeability, affirming that the
differentiation state of the cells can influence membrane transporters
and transmonolayer permeability. The basic permeability properties of
the BeWo monolayers suggest that the cells grown on permeable supports
may be examined as a convenient in vitro system to evaluate some
transplacental transport mechanisms.
placenta; passive diffusion; fatty acid transport
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INTRODUCTION |
THE POLARIZED EPITHELIAL-LIKE syncytiotrophoblasts
constitute the outermost layer of the placental villi and form a
physical and metabolic barrier between the fetal and maternal blood
circulations (25). Passive diffusion across this single layer of
epithelial cells is the principal mechanism of transfer from maternal
blood to fetal blood (10); however, several nutrient transport systems are also known to be present in these cells (8-11, 16, 17).
Human cytotrophoblasts have been successfully purified and cultured
(14). However, in primary culture, human cytotrophoblasts spontaneously
differentiate to a syncytiotrophoblast. The syncytialized trophoblasts
do not form a confluent, consistent monolayer and do not permit the
study of transcellular transport processes. Nutrient transport systems
have been investigated with use of the primary cultures of trophoblasts
but have been limited to unidirectional (maternal to fetal) uptake
studies (8, 13). The derivation of the BeWo cell line from a human
choriocarcinoma (19), a monolayer-forming trophoblast cell line, offers
a potential alternative culture system for studying transplacental
transport and metabolism in vitro. An in vitro system to study
transplacental transport could potentially provide a convenient system
to exploit in the investigation of those processes in the placenta
regulating the distribution of drugs, drugs of abuse, and pathogens
between maternal and fetal compartments. The BeWo cell line is
particularly attractive for this purpose because it is stable,
relatively easy to maintain by passage, and grows to a confluent
monolayer in a relatively short period of time. More importantly, the
BeWo cell displays morphological properties and biochemical marker enzymes common to normal trophoblasts (26) and has been shown to
exhibit polarized transcellular transport of transferrin (4, 5),
serotonin and monoamine uptake systems (6, 21), and relevant nutrient
uptake systems (17). Treated with adenosine 3',5'-cyclic
monophosphate (cAMP) or forskolin, BeWo cells further differentiate and
exhibit morphological characteristics similar to the fusion of
cytotrophoblasts to a syncytia in primary culture (28).
In this report, we used the BeWo cell line to produce confluent
monolayers on rat tail collagen-coated polycarbonate filters and in
Snapwell Transwell (Fisher Scientific, St. Louis, MO) inserts. Using
the Side-Bi-Side (Crown Glass, Somerville, NJ) cell diffusion systems,
we systematically tested the passive permeability and carrier-mediated
fatty acid transport characteristics of BeWo cell monolayers, features
not previously documented for BeWo monolayers grown on permeable
supports. The establishment and characterization of the basic
permeability properties of monolayers grown on a permeable support
establishes the basis for future application of this in vitro system to
investigate transplacental transport and metabolism of drugs and drugs
of abuse.
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METHODS |
Materials.
Translucent polycarbonate filters (13 mm diameter, 0.4 µm pore) and
Snapwell Transwell inserts (12 mm diameter, 0.4 µm pore size) were
purchased from Fisher Scientific. Dulbecco's modified Eagle's medium
(DMEM), DMEM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid modification, Hanks' balanced salt solution (HBSS) and Percoll were obtained from Sigma (St. Louis, MO). Heat-inactivated fetal bovine
serum (FBS) was from JRH Biosciences (Lenexa, KS).
Penicillin-streptomycin as a mixture was from GIBCO (Gaithersburg, MD).
Fluorescein isothiocyanate conjugated dextrans (FITCDs),
fluorescein, forskolin, and 8-bromoadenosine 3',5'-cyclic
monophosphate (8-BrcAMP) were purchased from Sigma. [14C]linoleic acid was
purchased from Amersham Life Science (Little, CA).
[3H]dihydroalprenolol
hydrochloride was purchased from DuPont-NEN Research (Boston, MA). All
other standard chemicals were purchased from either Fisher Scientific
or Sigma.
BeWo cell culture.
The BeWo cell line was originally derived from a human choriocarcinoma
(19). The BeWo clone (b30) was obtained from Dr. Alan Schwartz
(Washington University, St. Louis, MO). The cells were continuously
cultured in DMEM with 10% heat-inactivated FBS containing 0.37%
sodium bicarbonate and 1% antibiotics (10,000 U/ml penicillin and 10 mg/ml streptomycin). The cells were routinely maintained in
175-cm2 Falcon flasks at pH 7.4 under 5% CO2 and 95% humidity at
37°C. The cells were usually ready to be passaged after ~4 days
in culture. The cells were harvested by exposure (1-2 min) to a
trypsin-EDTA solution (0.25% trypsin and 0.02% EDTA in HBSS) and
passed onto polycarbonate membranes coated with rat tail collagen in
60- or 100-mm Corning culture dishes for the monolayer cultures or into 12-mm Snapwell Transwell permeable polycarbonate filters. With the
typical seeding density of 100,000 cells/cm2, the cells formed
monolayers between 4 and 6 days. The culture medium was changed every
other day. All cells used in this study were from
passages 10-30.
Forskolin was prepared in 95% ethanol and was added to the culture
medium with the final concentration of 100 µM. Control cultures
received the ethanol vehicle (0.6% final) in the same concentration as
the forskolin-treated cultures. The 8-BrcAMP was dissolved in the
culture medium and was sterilized by filtration through a 0.2-µm pore
sterile filter.
Electron microscopy.
BeWo cells grown on both polycarbonate filters and Snapwell Transwell
insert filters were washed three times for 10 min with phosphate-buffered saline [PBS; (in mM) 129 NaCl, 2.5 KCl, 7.4 Na2HPO4,
1.3 KH2PO4,
0.63 CaCl2, 0.74 MgSO4, 5.3 glucose, and 0.1 ascorbic acid, pH 7.4] and fixed with 2.5% glutaraldehyde solution in PBS for 4 h at 4°C. After being fixed, membranes were washed three times for 10 min with PBS and the samples were postfixed in 1% OsO4, dehydrated in a
series of ethanol and acetone solutions, and embedded in EmBed 812. The
sample was sectioned at 80 nm on a Sorvall Mt 500 Ultratome with a
diamond knife. The sections were stained (10 min each) with uranyl
acetate and lead citrate and examined under a Jeol 1200 EX II
transmission electron microscope.
Cross sections.
BeWo cells grown on both polycarbonate filters and Transwell insert
filters were washed three times with PBS and were fixed in buffered
10% Formalin solution at room temperature. After fixation in 10%
Formalin, tissue specimens were placed in a tissue processor for
dehydration and infusion with paraffin. The specimens were then
embedded in a small paraffin block to provide support for sectioning.
The paraffin blocks were placed on a microtome, and very thin tissue
sections were cut (~6 µm thick). Those tissue sections were then
placed on a glass microscope slide. Through a series of steps, the
paraffin was dissolved from around the tissue on the slide, leaving
only the tissue section on the slide. The slides were then stained with
the routine hematoxylin and eosin staining process.
Transmonolayer permeability.
A horizontal Side-Bi-Side diffusion apparatus (Crown Glass) was used to
measure the transmonolayer permeability of cells grown on the surface
of 0.4-µm pored polycarbonate filters, as previous described (20,
21). PBS was added in each of the donor and receiver chambers of either
the Side-Bi-Side diffusion apparatus or the Corning Costar diffusion
system. The cells faced the donor chamber as the apical side, and the
polycarbonate membrane faced the receiver chamber as the basolateral
side. The monolayer cultures always faced the donor chamber of the
diffusion systems. Polycarbonate filters treated with rat tail collagen
but without cell monolayers were used as a control. The transport area
was 0.636 cm2 in the Side-Bi-Side
diffusion system. The donor-side final concentration of the FITCDs was
10 µM for all transmonolayer permeability studies, and the donor-side
final concentration of fluorescein was 0.5 µM. The water jacket
surrounding the donor and the receiver chambers was thermostated at
37°C. The contents of each chamber were continuously stirred at 600 revolutions/min with magnetic stir bars in the Side-Bi-Side diffusion
system, and 5% CO2 and 95%
O2 were slowly and continuously
bubbled into the chambers. The monolayer cultures were equilibrated for
30 min at 37°C before they were undertaken in the transport
studies. Either fluorescein-labeled or radiolabeled compounds at
different final concentrations were added into the donor chambers. An
aliquot of 100 ml from the receiver chamber was taken at the scheduled
time points (0-120 or 0-180 min), and the same
volume (100 µl) of fresh PBS was replaced after each sample. The
samples were diluted with 0.9 ml of PBS in microcuvettes and were
measured by fluorescence spectroscopy (SLA Aminco, Urbana, IL) with an
excitation wavelength of 490 nm and an emission wavelength of 520 nm.
For concentration- and temperature-dependent studies of
[14C]linoleic acid
transport, studies were carried out as described above for fluorescein
and the FITCDs, except that the concentration of fatty acid was varied
and the buffer in the diffusion system contained bovine serum albumin
in a ratio of 6:1 (fatty acid to albumin), according to Lafond et al.
(15). To observe the effect of nonalbumin bound, unlabeled fatty acid
on [14C]linoleic acid
transport, we added increasing concentrations of linoleic acid
containing no albumin to the donor chamber of the diffusion cell just
before pulsing the chamber with a 1.7-µM pulse of
[14C]linoleic acid.
Radioisotope samples were added to 10 ml of ScintiVerse BD and were
assayed by liquid scintillation spectrometry in an LS 6800 Beckman
scintillation counter.
In other studies, 7 nM
[3H]dihydroalprenolol
hydrochloride was added either to the donor or to the receiver side of
the diffusion apparatus. After 30 min, the buffer in the donor and
receiver sides was aspirated out and the monolayers on the
polycarbonate filter were removed from the diffusion apparatus and
washed three times in 100 ml of ice-cold buffer (3). The monolayers
were added to 10 ml of ScintiVerse containing ScintiGest and were
assayed by liquid scintillation spectrometry.
Determination of transepithelial electrical resistance values.
The transepithelial electrical resistance (TEER) values were measured
in a chamber with planar electrodes (Endohm-Snap, World Precision
Instruments, Sarasota, FL). The TEER was determined with BeWo cells
grown on Snapwell Transwell filters. After three washes with PBS at
room temperature, the measurement was carried out and corrected for
resistance of the collagen-coated filters in the absence of BeWo cells.
In other experiments, the solutions of 10 mM EDTA and 10 mM amiloride
in PBS replaced the regular PBS. The cells were incubated 30 min for
EDTA and 10 min for amiloride at room temperature.
Calculations and data analysis.
Apparent permeability coefficients were estimated by the following
equation
where
P is the apparent permeability
coefficient, x is the amount of
substance (mol) in the receiver chamber at time
t (s), A is the diffusion area, and
Cd is the concentration of
substance in the donor chamber
(mol/cm3).
Cd remains >90% of the initial
value over the time course of the experiments. The transport of the
compounds studied, expressed as the flux
(mol · cm
2 · s
1),
was determined as the mean ± SD from three to six different monolayers.
The apparent permeability coefficient for the BeWo monolayers,
Pe, was
calculated from the following relationship
where
Pt is the
apparent permeability coefficient for the collagen-coated polycarbonate
membranes in the presence of BeWo cell monolayers, and
Pc is the
apparent permeability coefficient for collagen-coated polycarbonate
membranes alone (1). Michaelis-Menten parameters were estimated by
Sigma Plot (Version 2.0).
In all experiments, data were expressed as means ± SD for at least
four replicates. A one-way analysis of variance followed by Dunnett's
multiple-comparison test was applied to analyze the significance of
differences between treatment and control groups of raw data at the
0.05 level of significance.
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RESULTS |
BeWo cells (b30) have been continuously subcultured and were routinely
maintained in the 175-cm2 flasks.
In the presence of 10% FBS, the BeWo cells consistently developed
confluent monolayers between days 4 and 6. A typical confluent monolayer
grown on a rat tail collagen-coated polycarbonate filter is shown in
Fig. 1A.
BeWo cells were also grown on Snapwell Transwell inserts to facilitate
TEER measures. Because the cell cultures on the Snapwell Transwell
inserts were not visible under the light microscope, culture samples
were observed in cross section to verify formation of monolayers.
Figure 1B is a representative cross
section of BeWo cells grown on the Transwell filter at
day 5 in culture. The importance of
monitoring growth of BeWo cells on the Transwell filter is illustrated
in Fig. 1C, in which higher seeding
densities and longer growth times allow for formation of multiple
layers of cells. Figure 2 shows a scanning
electron photomicrograph of BeWo cell cultures on polycarbonate
filters. The cells generally exhibit a flattened polygonal shape with
microvilli and close apposition with adjacent cells. Transmission
electron micrographs confirmed the presence of apical microvilli, close cell apposition, and the presence of a single layer of cells (not shown).

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Fig. 1.
A: 5-day-old confluent BeWo monolayer
grown on a rat tail collagen-coated polycarbonate in 60-mm dish
(×200). B: 5-day-old confluent
BeWo monolayer grown on a rat tail collagen-coated Snapwell Transwell
insert (×400). C: 7-day-old BeWo
cell culture of multiple layers grown on rat tail collagen-coated
Snapwell Transwell insert (×400).
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The cell seeding density was found to directly affect BeWo monolayer
permeability. The relationship between different seeding densities and
permeability to a FITCD (FD-04, average mol wt 4,400) is shown in Fig.
3. By day
5 in culture, seeding densities of 100,000 cells/cm2 and 200,000 cells/cm2 provided for lowest
dextran permeability. Because the higher seeding density resulted in
formation of multiple layers (similar to the condition shown in Fig.
1C), usually within 24 h and most readily in the Transwell insert, the seeding density of 100,000 cells/cm2 was chosen to be the
optimal density for reproducibly establishing BeWo monolayer cultures.

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Fig. 3.
Effect of cell seeding density on permeation of fluorescein
isothiocyanate-conjugated dextran (FITCD) FD-04 (average mol wt 4,400)
across 5-day-old cultures of BeWo monolayers at 37°C. Control represents FD-04 permeation across collagen-coated polycarbonate membrane in absence of BeWo cells. Data represent means ± SD from quadruplicate samples.
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At the established seeding density of 100,000 cells/cm2, the passage of selected
passive permeability markers across the BeWo monolayers was determined
for cells grown on polycarbonate filters. These filters were placed in
the side-by-side diffusion apparatus to facilitate permeability studies
with fluorescein (mol wt 376) and FITCDs varying in molecular weight
(average mol wt 4,400, 20,000, and 70,000). Figure
4 shows the day-to-day change in BeWo monolayer permeability to fluorescein and the different FITCDs. The
permeation of the hydrophilic fluorescein and FITCDs across BeWo
monolayers was dependent on the molecular weight and the age of the
cultures. The lower-molecular weight marker, fluorescein, was the most
sensitive to development of confluent monolayers but reached the
minimum permeability at day 5 in
culture or approximately the same time as the higher-molecular weight
markers. The largest dextran (FD-70) showed very little change in
permeability with day in culture. Figure 5
illustrates the relationship between the decreased permeation of the
selected markers across the BeWo monolayers and an increase in
molecular size. These results generally illustrated that passive
permeation across BeWo monolayers was sensitive to molecular size.

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Fig. 4.
BeWo monolayer permeability to fluorescein (Fluore, mol wt 376) and
FITCDs (average mol wt: FD-04, 4,400; FD-20, 20,000; FD-70, 70,000) at
selected days postseeding in culture at 37°C. Data represent means ± SD from quadruplicate samples.
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Fig. 5.
Relationship between 5-day-old BeWo monolayer permeability to
fluorescein (mol wt 376) and FITCDs (average mol wt: FD-04, 4,400;
FD-20, 20,000; FD-70, 70,000) and average mol wt of markers at
37°C. Apparent permeability coefficients
(Pe) have been
adjusted for effects of collagen and polycarbonate filter as described in METHODS. Data represent means ± SD from quadruplicate samples.
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The TEER developed by the BeWo monolayer was observed to be a maximum
of
~70
· cm2.
Figure 6 shows the development of
electrical resistance as a function of day in the culture postseeding.
The maximal TEER in the Snapwell Transwell filters developed at
approximately day 5 in culture and was
consistent with the overall development of sieving properties of the
monolayers with respect to fluorescein and the FITCDs described above.
The BeWo monolayer TEER was substantially reduced when the cells were
subsequently exposed to EDTA (10 mM), whereas the resistance was only
partially attenuated in the presence of amiloride (10 µM), a sodium
channel blocker.

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Fig. 6.
Development of transepithelial electrical resistance (TEER) by BeWo
monolayer cultures and effects of amiloride and EDTA at room
temperature (~25°C). Data represent means ± SD from
quadruplicate samples.
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To further define some of the nutrient transport properties of the BeWo
monolayers, we examined whether the cell line retained a transporter
for fatty acids. The passage of linoleic acid across the cells was
found to be asymmetric, with the permeation rate greater in the
apical-to-basolateral direction, as shown in Fig. 7. As affirmed in Fig.
8A, the
passage of linoleic acid across the monolayers was saturable
[Michaelis constant
(Km) = 324 µM and maximum velocity = 14.6 fmol/min] in the
presence of albumin (6:1 fatty acid-to-albumin ratio), inhibited by low
temperature, and enhanced in the presence of albumin (data not shown).
The Km for the
fraction of the estimated free or unbound linoleic acid available for
transport (15) was 7.4 µM. Figure 8B
illustrates the inhibition of
[14C]linoleic acid
transport in the presence of increasing concentrations of albumin-free
linoleic acid (50% inhibitory concentration = 30 µM). The asymmetry
of the monolayers was further supported by an approximately sevenfold
greater binding of
[3H]dihydroalprenolol
hydrochloride on the basolateral surface (e.g., 1.6 ± 0.50 pmol/cm2 total bound on the
basolateral surface, 0.22 ± 0.03 pmol/cm2 total bound on the apical
surface) of the BeWo monolayers.

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Fig. 7.
Bidirectional passage of
[14C]linoleic acid
across 5-day-old BeWo monolayers at 37°C. Data represent means ± SD from quadruplicate samples.
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Fig. 8.
A: concentration and temperature
dependence of apical-to-basolateral
[14C]linoleic acid
transport across 5-day-old BeWo monolayers in presence of albumin.
Ratio of fatty acid to albumin was 6:1. Data represent means ± SD
from quadruplicate samples taken at 1 h. Dotted line represents
best-fit nonlinear regression through data points.
B: effect of increasing unlabeled
linoleic acid (LA) concentration (cold LA) on apical-to-basolateral
[14C]linoleic acid
transport across 5-day-old BeWo monolayers at 37°C in absence of
albumin. Data represent means ± SD from quadruplicate samples
taken at 1 h.
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The BeWo cells are not capable of spontaneous differentiation in
culture (28). Development of culture conditions that facilitate BeWo
cell differentiation and inhibit cell proliferation potentially may
make the monolayer system morphologically similar to the primary trophoblast cultures and to the in vivo placental barrier. Figure 9 shows the morphological changes of BeWo
cell cultures (5 days) after 4 days forskolin treatment. Addition of
8-BrcAMP has a similar effect on the cells (not shown). The morphology
of the treated BeWo cells was similar to the primary trophoblast
cultures (Fig. 9C), including the
presence of the large openings between the cells, making them unuseable
for transport studies.

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Fig. 9.
A: 4-day-old BeWo culture
(×200). B: 5-day-old BeWo
culture after 4 days of treatment with forskolin (×200).
C: 6-day-old primary culture of human
cytotrophoblasts that have aggregated and fused (×200).
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The effects of forskolin and 8-BrcAMP on BeWo cell growth and
morphology were significant and suggested that monolayer permeability might be altered in the presence of agents that stimulate formation of
a syncytia. Table 1 summarizes experiments
in which the permeability of the BeWo monolayers in the presence of
either forskolin or a cAMP was monitored with fluorescein before
formation of syncytia. Except for a small but statistically significant
increase with the 3-day treatment with forskolin, no substantial
changes in the permeability of the BeWo monolayers to fluorescein were
observed after a 2- or 3-day treatment with either forskolin or
8-BrcAMP. Table 2 summarizes similar data
for linoleic acid for a 3-day treatment with forskolin. Statistically
significant trends of increased linoleic acid transport were observed
after forskolin pretreatment, with the greater effects of cell
differentiation on the apical-to-basolateral transport. After a 4-day
exposure to forskolin, the permeability of the treated monolayers
dramatically increased and was quite variable (not shown), and this was
likely because of the sloughing and aggregation of cells, which created large intercellular openings (see Fig. 9).
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Table 1.
Effects of 10 µM 8-BrcAMP or 100 µM
forskolin on apparent permeability coefficients for fluorescein passage
across BeWo monolayers at 37°C
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DISCUSSION |
The BeWo cells form a monolayer of cells with regular microvilli on the
apical surface, consistent with the general characteristics of the
human trophoblast (19, 27, 28). The cell line is readily passaged, and
the stability of the BeWo cell line has been previously noted (20). A
few reports also exist on the growth of BeWo monolayers on a
semipermeable membrane (4, 5). However, variations in cell culture
density, passaging methods, splitting ratios, and extracellular matrix
will significantly influence the cellular composition of a cell line
(2). In particular, the growth and composition of the BeWo cell line
have been shown to be sensitive to the make up of the extracellular
matrix (12). In this study, we have described the experimental
conditions for the growth of BeWo cells on a porous substrate and
placement in a side-by-side diffusion apparatus (2) for the purpose of
developing a model to study transplacental transport and metabolism in
vitro.
Passive diffusion is the primary mechanism by which xenobiotics cross
the placental barrier (7, 22, 25). An appropriate in vitro system would
be expected to provide at least a qualitative representation of the
trophoblast sieving of molecules observed in the placenta. BeWo
monolayers form a molecular weight-selective barrier after ~5 days in
culture. The passive permeation of selected hydrophilic markers across
the BeWo monolayers was found to be linearly related to molecular size.
The calculated Pe
was also proportional to the water diffusion coefficients for the
markers (not shown), consistent with the trans-trophoblast passage of small hydrophilic solutes in the placenta through aqueous paracellular routes (7, 22, 25).
Although the TEER of a cell monolayer is poorly correlated with
permeability to larger hydrophilic markers (1), very reproducible and
maximal TEER values were also observed for BeWo 5 days postseeding at a
density of 100,000 cells/cm2. The
TEER values of this system were low compared with reports of other
laboratories (4); however, differences in growth substrates, temperature, buffers, instrumentation, and other lab-to-lab variables can account for the differences. As expected, EDTA treatment of the
cells, likely chelating extracellular free calcium and disrupting intercellular junctions, substantially decreased the TEER value. In
contrast, the inhibition of sodium channels by amiloride did not seem
to play an important role in TEER of BeWo monolayers. Generally, these
results appeared consistent with placental ion-transfer characteristics
described in previous reports (24).
A few studies (4, 5) have examined the morphological and functional
polarity of BeWo monolayers grown on permeable supports. In those
studies, polarized transport of transferrin by BeWo monolayers has been
reported (4, 5). Functional polarized transport of the fatty acid
linoleic acid has also been demonstrated in plasma membrane vesicles
prepared from human syncytiotrophoblasts by Lafond et al. (15) but not
in BeWo monolayers. We were able to demonstrate a polarized transport
of linoleic acid across the BeWo monolayers favoring the
apical-to-basolateral direction. The transport was saturable, enhanced
in the presence of albumin, and could be inhibited by excess unlabeled
fatty acid and low temperature, all features of a carrier-mediated
process and consistent with the work of Lafond et al. (15). In fact,
the estimated Km
of ~7.4 µM for unbound linoleic acid transport by BeWo monolayers here was in close agreement with the 7.9 µM reported by Lafond et al.
(15) with human brush border vesicles. In other studies, we were also
able to demonstrate apparent preferential binding of
[3H]dihydroalprenolol
to the basolateral surface of the BeWo monolayers, an observation that
was in agreement with the ~10-fold greater binding of
dihydroalprenolol on basolateral membranes of the human placental
barrier (3). Collectively, the studies of earlier researchers (12, 14)
and our results here affirmed that BeWo monolayers exhibit
morphological and functional polarity when grown on permeable supports.
Moreover, these studies indicated that the BeWo monolayers may have a
number of applications in characterizing the nature and role of
polarized carrier mechanisms at the placental barrier.
BeWo cells are not capable of spontaneously differentiating to the
syncytiotrophoblast in culture (23). This is in contrast to primary
cultures of cytotrophoblasts, which aggregate and form a syncytia
typical of the in vivo placental barrier (14). Forskolin and 8-BrcAMP,
stimulants of differentiation and inhibitors of proliferation, were
added to the BeWo cultures to assess the role of differentiation on
monolayer permeability. The treatment of BeWo monolayer cultures with
forskolin and 8-BrcAMP did stimulate BeWo cell differentiation, and
morphologically the BeWo cells did take on an appearance similar to the
syncytialized primary cultures. These morphological changes were
consistent with earlier reports (28). The forskolin or 8-BrcAMP-induced
alteration of the differentiation of the BeWo cells may not only alter
morphology but could also regulate transport systems. Our studies
suggested that induction of the differentiation of BeWo cells did not
substantially alter passive transport of fluorescein. By contrast, the
forskolin pretreatment enhanced the transport of linoleic acid and
developed more polarity in the transport process. Longer treatments
with forskolin resulted in cell sloughing and an increase in monolayer permeability resulting from syncytia formation. There have been no
reports for comparison on the effects of forskolin on the passive permeability of trophoblasts in culture. With respect to nutrient transporters, in BeWo cellular or monolayer uptake studies, forskolin has been reported to increase transferrin uptake transport (26); however, there was no effect on
L-alanine transport (17).
Forskolin treatment of BeWo cells does stimulate the development of
asymmetry in the neutral amino acid transporter, suggesting that
trophoblast fusion and differentiation is accompanied by significant
membrane specialization (8). The altered passage of linoleic acid
across the BeWo monolayers in this study provides further indication that, in addition to neutral amino acid carriers, other transporters may be similarly regulated during trophoblast fusion and
differentiation. In future studies, the effects of forskolin on BeWo
transporters might be considered a routine part of the process of
characterizing carrier mechanisms.
In conclusion, the human choriocarcinoma cell line BeWo (b30) was
cultured on semipermeable membranes, formed confluent monolayers in
~5 days, and was morphologically similar to primary cultures of human
trophoblasts, except in the extent of differentiation. The passive
seiving properties of the BeWo monolayers were defined by establishing
the permeability with respect to fluorescein and FITCDs of varying
molecular weight. The BeWo monolayers developed a reproducible TEER
indicative of ion transport, and the monolayers were functionally
polarized with respect to linoleic acid transport. BeWo cells grown on
permeable supports could be induced to undergo differentiation in the
presence of forskolin and 8-BrcAMP. Under our growth and experimental
conditions, we have extended some of the observations of other
laboratories (5, 6, 17, 21) and support the notion that BeWo monolayers
might be further exploited as a polarized trophoblast layer to examine
transplacental transport and metabolism. In current studies, we have
established that BeWo monolayers also retain inducible cytochrome
P-450 enzymes, peptidases, and surface
lectin binding typical of the human placenta (F. Liu, X. Zhang, M. Soares, and K. L. Audus, unpublished observations). Ongoing research in
our laboratory also includes application of this in vitro system to the
study of transport and metabolism of drugs and drugs of abuse. On the
basis of these results, the BeWo cell monolayers appear to be an
appropriate in vitro system to investigate the transplacental transport
of certain nutrients and xenobiotics.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Dr. Jerome F. Strauss III for valuable
comments and assistance in establishing the primary human trophoblast
cultures.
 |
FOOTNOTES |
This study was supported by the National Institute of Drug Abuse
(N01DA-4-7405). Support for the Cellular and Molecular
Biopharmaceutics Handling Laboratory was provided by Corning Costar.
Address for reprint requests: K. L. Audus, Dept. of Pharmaceutical
Chemistry, Univ. of Kansas, Lawrence, KS 66045.
Received 31 March 1997; accepted in final form 21 July 1997.
 |
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