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From the Department of Pharmaceutics, University of Kuopio, Finland.
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Abstract |
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METHODS. Immortalized human corneal epithelial (HCE) cells were grown on filters, with various filter materials and coating procedures. In the optimal case, HCE cells were grown on polyester filters coated with rat tail collagen gel containing fibroblast cells. Transepithelial electrical resistance (TER) was measured during the growth of the cells to evaluate the epithelial differentiation and tightness of the epithelial cell layers. Transmission electron microscopy (TEM) was used to show the formation of tight junctions, desmosomes, and microvilli. Cellular morphology was characterized by light microscopy. Permeabilities of 3H-mannitol and 6-carboxyfluorescein were determined, to evaluate the intercellular spaces of the epithelium. Rhodamine B was used as a lipophilic marker of transcellular permeability. Permeabilities of the excised rabbit corneas were determined in side-by-side diffusion chambers.
RESULTS. The TER values of the corneal epithelial cultures were 200 to 800
· cm2, depending on the culture conditions. In
optimal conditions, cultured corneal epithelium consisted of five to
eight cell layers, TER was at least 400 · cm2, and
the most apical cells were flat, with tight junctions, microvilli, and
desmosomes. The permeability coefficients
(Pcell, 10-6 cm/sec) for
3H-mannitol, 6-carboxyfluorescein, and rhodamine B were
1.42 ± 0.36, 0.77 ± 0.40, and 16.3 ± 4.0,
respectively. Corresponding values (at 10-6 cm/sec) for
the isolated rabbit corneas were 0.38 ± 0.16, 0.46 ± 0.27,
and 18.1 ± 4.0, respectively.
CONCLUSIONS. The TER, morphology, and permeability of the cultured corneal epithelial cells resemble those of the intact cornea. This cell culture model may be useful in evaluation of corneal drug permeation and its mechanisms.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo studies of ocular drug absorption usually require killing at least five animals at each time point in the drug concentration profile. Because full pharmacokinetic analysis includes many time points, the number of rabbits used per study is often more than 20.7 Although rabbits are the only widely used animal model in ocular pharmacokinetics, this model has several problems, among which is the rabbit’s low blinking frequency compared with the human’s.8
In general, cell culture models offer many potential advantages in the analysis of drug transport and metabolism.9 Of particular importance, they open possibilities to decrease the number of animal experiments in this type of research. In addition, these systems offer potential for manipulation of the environment or cellular properties as a means of addressing the mechanistic questions of drug permeation in living cells. From the standpoint of drug discovery and drug formulation, cell culture models can be used to expedite identification of compounds or formulations with favorable pharmacokinetic properties and to evaluate structure–absorption and structure–metabolism relationships on a large scale. Development of new test methods of in vitro absorption is of utmost importance in current drug discovery, because the modern combinatorial and automated methods of drug synthesis produce numerous compounds, but only in minute quantities. Therefore, maximal predictive information should be gained from the in vitro use of small quantities of the test substances.
A corneal cell culture model could be useful in testing the permeability of ocular drugs and formulations. The model should be based on corneal epithelial cells, because the epithelium is the barrier that limits permeation.10 The published models are based on primary cells,11 12 13 but these cells usually stop growing after one or two passages and after storage in liquid nitrogen, they revive weakly or not at all. New rabbit cells must be isolated frequently; therefore, these methods may not be optimal for larger scale screening of new compounds and formulations.
Immortalized cell lines can be grown continuously, and they should be more practical for testing of permeability. SIRC cells are a corneal cell line from rabbits that was recently described for permeability testing.14 15 Unfortunately, these cells exhibit a fibroblast phenotype, which decreases their value as a model.16 The human corneal epithelial (HCE)-T in vitro model of human corneal epithelium17 18 19 represents a three-dimensional culture for HCE-T cells grown on a collagen membrane to provide a species- and tissue-specific equivalent of the human corneal surface in vivo. Araki-Sasaki et al.20 established an immortalized HCE cell line that has properties of normal corneal epithelial cells. This cell line continues to grow for more than 400 generations through 100 passages, and the cells can be frozen and revived. Recently, Saarinen-Savolainen et al.21 demonstrated the usefulness of this cell line in testing the toxicity of ophthalmic drugs and pharmaceutical excipients, but so far no permeability model based on immortalized HCE cells has been presented.
The purpose of this study was to establish an in vitro corneal permeability model using an immortalized HCE cell line. We suggest that this model may be useful as a predictive kinetic model in vitro, because the cultured corneal epithelium exhibits morphologic characteristics and permeability similar to those observed in the intact cornea.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transepithelial Electrical Resistance
Transepithelial electrical resistance (TER) was metered (Endohm;
World Precision Instruments, Sarasota, FL) at different phases of cell
growth. TER was used as an indicator of epithelial differentiation and
epithelial tightness. TER data were corrected for low-background TER by
using a blank filter containing the possible coating materials and
culture medium. The TERs of the filter without HCE cells were 47
· cm2 on polyester filters and 28
· cm2 on polycarbonate filters. TERs of
blank filters were not dependent on filter coating. At the end of each
permeability experiment, TER was measured to detect the condition of
the cells. The TER data were not changed during the permeability
studies.
Permeation Studies with Cultured Cells
The permeation studies were performed by using
3H-mannitol (molecular weight [MW], 182;
specific activity, 19.7 Ci/mmol; NEN Life Science Products, Inc.
Boston, MA) and 6-carboxyfluorescein (MW 376; Sigma) as hydrophilic
markers for characterizing the paracellular permeation between the
epithelial cells. The experiments were performed after cell growth in
different conditions. The penetration study with
3H-mannitol was initiated by adding 2.6 ml
serum-free medium to the basolateral side (receiver side) and 1.5 ml
serum-free feeding medium containing 3H-mannitol
(0.5 µCi/ml) to the apical side (donor side). At 30, 60, 90, 120,
150, 180, 240, and 300 minutes, aliquots of 100 µl were withdrawn
from the receiver chamber and replaced with an equal volume of blank
medium. Then 400 µl scintillation liquid (Optiphase Supermix; Milton
Keynes, Wallac, UK) was added to every sample, and the radioactivities
were measured using a liquid scintillation and luminescence counter
(1450 MicroBeta Trilux; Wallac, Turku, Finland).
The transport was studied also with 6-carboxyfluorescein (Sigma) as a probe, in a manner similar to the 3H-mannitol experiments, except that balanced salt solution (BSS; BSS Plus; Alcon, Fort Worth, TX) was used as a buffer solution and the concentration of 6-carboxyfluorescein on the apical side was 10 µM. The salt solution was used because the red color of the medium could disturb the fluorescence measurements. The sample volume was 200 µl. Concentrations of 6-carboxyfluorescein were determined using a fluorescence plate reader (model FL 500; Bio-Tek Instruments, Inc., Burlington, VT) with 485-nm excitation and 530-nm emission filters.
The lipophilic marker was rhodamine B (MW 479; Sigma) in BSS. The donor concentration of rhodamine B was 10 µM, and samples were taken at 10, 20, 30, 45, 60, 75, 90, 105, and 120 minutes. Concentrations of rhodamine B were determined by the fluorescence plate reader with 530-nm excitation and 590-nm emission filters. To increase the fluorescence intensity, methanol was added into the samples (the final concentration of methanol was 40% vol/vol). Otherwise, the procedure was identical with the 6-carboxyfluorescein experiments. To avoid buildup of the limiting hydrodynamic diffusion layers all permeation studies were performed at 37°C using a horizontal plate mixer (Heidolph Inkubator 1000 and Titramax 1000; Heidolph Electro GmbH & Co., Kelheim, Germany).
Light Microscopy
Confluent corneal epithelial cells were fixed in 2%
paraformaldehyde-0.5% glutaraldehyde phosphate buffer, dehydrated in a
graded ethanol series (80%, 94%, 99%) and embedded in paraffin.
Sections of 5 µm were cut and stained in hematoxylin and eosin
solutions. The specimens were examined with a light microscope
(Microphot-Fxa; Nikon, Tokyo, Japan).
Male albino New Zealand strain rabbits, weighing approximately 3 kg, were used to study the morphology of normal rabbit cornea. The corneas were fixed in 4% buffered formalin solution, dehydrated in a graded ethanol series, embedded in paraffin, cut in 5-µm sections, and stained with hematoxylin-eosin for light microscopy.
Transmission Electron Microscopy
The cultured cells were fixed in 2.5% glutaraldehyde, postfixed
with 1% osmium tetroxide, dehydrated in graded alcohols, embedded in
resin (LX-112; Ladd Research Industries, Inc., Burlington, VT) and cut
in 60-nm sections. The specimens were viewed and photographed on a
transmission electron microscope (JEM 1200 EX; JEOL, Tokyo, Japan).
Permeability Experiment with Excised Cornea
All animal experiments conformed to the ARVO Statement for the
Use of Animals in Ophthalmic and Vision Research. Male and female New
Zealand albino rabbits (age 6–12 months, weight 3.0–4.6 kg) were
killed by injecting a lethal dose of fentanyl and fluanisone (Hypnorm;
Janssen Pharmaceutica, Beerse, Belgium) into the marginal ear vein. The
cornea was dissected with a scleral ring (5 mm laterally from the
limbus). The cornea was attached to a corneal holder with a rubber band
(made in our laboratory) using a tissue mounting tool (Physiologic
Instruments; San Diego, CA), and the lens and iris were carefully
removed. The corneal holder with cornea was mounted between two
side-by-side diffusion chambers (Snapwell; Navicyte, Sparks, NV). The
exposed corneal surface area was 0.6 cm2. Blank
BSS buffer (6.5 ml) was added to the endothelial side and the donor
solution (6-carboxyfluorescein and rhodamine B, both at a concentration
of 100 µM, dissolved in 5.5 ml BSS buffer) was added to the
epithelial side. The solutions in each chamber were mixed by bubbling
an O2-CO2 (95:5) mixture at
the rate of three to four bubbles per second and maintaining the
temperature at 35 ± 1°C in a circulating water bath. At
intervals of 30 minutes, until 240 minutes, samples of 500 µl were
taken from the endothelial side and replaced by an equal volume of
blank BSS buffer. The 6-carboxyfluorescein and rhodamine B were
analyzed, as described earlier. Permeability of
3H-mannitol in isolated corneas was studied, as
described by Suhonen et al.22
Data and Statistical Analysis
Apparent permeability coefficients
(Ptot, in centimeters per second) of
the cultured HCE cells and filter together were calculated
as
 | (1) |
The cultured corneal epithelium and the support (filter, extracellular
matrix, feeder fibroblasts) caused serial resistance against drug
permeation. The resistance for drug permeability was provided both by
the barrier of the support (Rsup) and
cell layer (Rcell). Total resistance
of the system (Rtot) was defined as
 | (2) |
 | (3) |
 | (4) |
The Mann-Whitney test was used to study the statistical significance of the differences between the permeabilities of model compounds in the isolated rabbit cornea and cultured HCE cells. P < 0.05 was considered to show a statistically significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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· cm2 (Fig. 1)
, and the Pcell of
3H-mannitol was 10 to 20 x
10-6 cm/sec, regardless of the filter or coating
material (data not shown). Under the air–liquid interface the
transepithelial TER of the cultured human corneal epithelium increased
for 2 to 3 weeks to approximately 200 to 800
· cm2, depending on the culture
conditions (Fig. 1)
. Permeability was determined when the TER was at
least 85% of peak level. The time when the resistance reached a
constant level varied from 3.5 to 5 weeks after seeding, depending on
the coating material of the filters (Fig. 1)
. Figure 2
shows the relationship between TER and
Pcell, for
3H-mannitol in different cases. With decreasing
TER, the permeability coefficient was increased. At a TER of 400 to 800
· cm2,
Pcell was 1 to 2 x
10-6 cm/sec. TER was similar before the
permeability experiment and after the experiment.
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· cm2 (Fig. 2)
.
Therefore, the permeabilities of 6-carboxyfluorescein (paracellular
permeant) and rhodamine B (transcellular permeant) were studied using
polyester filters coated with collagen-containing fibroblasts. In
cultured corneal epithelium (with collagen and fibroblasts on
polyester), rhodamine B penetrated the cell layers 21 and 11 times
faster than hydrophilic 6-carboxyfluorescein or
3H-mannitol, respectively. In excised rabbit
cornea, the differences were 39- and 48-fold, respectively. The HCE
cell culture model was more permeable to
3H-mannitol than isolated rabbit cornea
(P < 0.05), whereas in the case of rhodamine B and
6-carboxyfluorescein, no significant differences were seen (Table 1)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, a model of drug permeability was developed using immortalized epithelial cells from human cornea. Three earlier studies have described cell culture models that use primary epithelial cells from rabbit cornea.11 12 13 However, immortalized cells have practical advantages such as revival after freezing, no need for frequent and tedious cell isolation, and use of fewer rabbits. Such a model should resemble an in vivo cornea as closely as possible. We used immortalized epithelial cells from human cornea, because the epithelium is the main barrier to drug absorption.10 Furthermore, use of human cells is advantageous in a cell culture model, because humans and rabbits may differ significantly in metabolic enzymes and active transporters. Such cell culture models are, however, dependent on optimal cell culture conditions.
In our study, the feeding medium of the cells included 15% FBS. Sometimes, however, high concentrations of FBS may disturb cell proliferation and differentiation,28 and some groups have reported improved differentiation of the corneal epithelium in serum-free medium.19 29 We also cultivated the cells in lower FBS concentrations (2%, 5%, and 10%), but in these conditions the cells did not survive (data not shown). Thus, the serum concentration was maintained at 15%, and the other medium components were similar to those described by Araki-Sasaki et al.20 As described for the primary corneal epithelial cells,30 the air–liquid interface was critical for the differentiation of HCE cells in culture. No flat apical cells or proper barrier was obtained without air lifting.
Other important factors in development of the culture model are the filter material, its pore size, and the coating components on the filter (i.e., the extracellular matrix). Polycarbonate, with or without collagen, is used in most cultures of corneal cells.12 13 31 For barrier formation with HCE cells, however, polyester filters were better than polycarbonate filters (Fig. 2) , and the clear polyester membrane also provided better cell visibility for light or phase–contrast microscopy.
Collagen facilitates the growth and differentiation of corneal epithelial cells in culture.12 30 31 32 33 34 Collagen coating helps the cells to attach to the cultivating bed and stimulates their proliferation and differentiation. Human corneal epithelium cultured on collagen gels can synthesize and deposit basement membrane components such as laminin and type IV collagen.34 35 The mixture of collagen and laminin further improved the culture model and in our studies represented the basal lamina (Fig. 2) .
3T3 fibroblast cells from mice are used as a feeder layer for differentiation of the epithelial cells. The interactions between the fibroblasts and corneal epithelial cells stimulate the differentiation of the corneal epithelial primary cells.31 36 For culturing immortalized HCE cells, we coated the filters with a mixture of collagen and fibroblasts to provide a substrate resembling a corneal stroma. The fibroblasts did not divide in the collagen matrix, but they functioned as feeder cells for HCE cells. Based on morphology and on the barrier properties, a polyester filter with collagen and fibroblasts appeared to be the best conditions for HCE cell culture.
The apical surface of the corneal epithelium contributes more than half of the total electrical resistance of the cornea,37 and the top two layers are the most important part of the cornea in limiting the permeability of hydrophilic drugs.2 10 Therefore, the most apical cell layers are the most important part of the HCE permeability model. Of importance, the HCE cell culture model shows tight junctions and desmosomes in the flattened apical cell layer. Although the apical part of the cultured corneal epithelium appears to include the major structural features, it is also noted that the wing cells and basal cells in the cultured epithelium are not organized as well as they are in the intact cornea (Fig. 3) . Obviously, the HCE permeability model does not include stroma or endothelium, but these layers are not critical barriers to corneal drug absorption.
In optimal conditions, the TERs of the HCE cell cultures were 400 to
800 · cm2. This is approximately 10 times
lower than the TER of 3 to 10 k · cm2 in
the excised rabbit cornea.38
39
40
In addition, the
permeability of the HCE cell cultures for hydrophilic markers mannitol
and 6-carboxyfluorescein was two to four times higher than in the
isolated rabbit cornea (Table 1)
, although the difference for
6-carboxyfluorescein was not statistically significant. These markers
have a very low lipophilicity, and they permeate biological membranes
only through the intercellular space. The higher permeability and lower
TER of the HCE cell cultures suggest that the cell culture exhibits a
slightly larger intercellular space (i.e., the pore size or pore
density is larger) compared with rabbit cornea. Pore size and pore
density will be evaluated in a quantitative manner in future studies.
Nevertheless, permeability of the HCE cell cultures for hydrophilic markers (0.8–1.4 x 10-6 cm/sec; Table 1 ) is low enough to allow the screening of drug candidates. Ophthalmic drugs are usually administered as eye drops, and their sites of action are often in the inner eye structures. These drugs are small and lipophilic enough for transcellular permeation. They exhibit corneal permeability between 5 and 30 x 10-6 cm/sec (e.g., betaxolol, dexamethasone, timolol),41 which is 4 to 20 times higher than the paracellular permeability of mannitol and carboxyfluorescein in the HCE cell culture model. To verify that the HCE cell culture is useful in screening, the permeability of a lipophilic transcellular marker rhodamine B was determined. As expected, rhodamine B exhibited high permeability of the HCE cell culture (16 x 10-6 cm/sec), practically the same as in the isolated rabbit cornea (Table 1) . Furthermore, the permeabilities of 6-carboxyfluorescein and rhodamine B are in the same range with the previously reported values for these compounds in the excised rabbit cornea.42
The difference in permeability of rhodamine B and 6-carboxyfluorescein was 21-fold, although not quite as large as in the isolated rabbit cornea (39-fold). In contrast to our model, Kawazu et al.12 reached only a 4.6-fold difference between hydrophilic atenolol (Pcell 2.6 x 10-5 cm/sec) and lipophilic alprenolol (10.8 x 10-5 cm/sec) using primary corneal epithelial cell cultures. Our results indicate that the HCE cell culture model can discriminate between the permeabilities of lipophilic and hydrophilic drugs. In addition, comparison of the permeabilities in the HCE cell culture model (Table 1) with a database of more than 100 compounds41 shows that the values for both hydrophilic drug (6-carboxyfluorescein) and lipophilic drug (rhodamine B) match the average corneal permeability for the drugs with similar hydrophilicity-lipophilicity very well.
Only sparse information is available about the permeability of the human cornea for drugs. The rabbit corneas and rabbits in vivo are the current standard methods in ocular drug delivery studies. Therefore, we compare the cell culture model to the rabbit cornea instead of the human cornea. The morphology of the human and rabbit cornea are similar: The main permeability barrier in both cases is in the apical tight layer of the corneal epithelium. Human cornea and its epithelium (0.52 mm and 50–70 µm) are thicker than the rabbit cornea (0.35–0.42 mm) and its epithelium (40 µm). Permeabilities of cyclophosphamide and four carbonic anhydrase inhibitors have been compared in human and rabbit cornea.8 43 44 The permeabilities are similar, but the paracellular permeability in rabbit cornea may be less than in human cornea.8 43 44 This type of difference was also seen in this study: The paracellular space of the HCE culture model appeared wider than in the rabbit cornea. The existing morphologic and permeability data suggest that the rabbit cornea is a fairly good model. Studies with cultured human corneal epithelial cells will provide further information about the similarities and, importantly, also about the differences between the human and rabbit corneal epithelial transport processes relevant to drug absorption.
It appears that the permeability barrier of the HCE culture is comparable to that of rabbit cornea. Therefore, this model can be used to predict ocular absorption of the drugs that permeate through the cornea by passive diffusion. The extent of applicability of this model also depends on the expression of the active transporters, efflux proteins, metabolic enzymes, surface proteins, and mucins in the cultured cells. These aspects of the cultured model should be elucidated in future studies, because it is possible that the expression profiles of the immortalized cell line, primary cells, and animal model in vivo are different. An in vitro cell culture model does not take into account the issue of contact time on the corneal surface. Before an experimental setup is developed for such cell culture studies, the impact of pharmaceutical formulations with prolonged precorneal contact should be investigated in vivo in animal studies.
In conclusion, we have developed an in vitro model of the corneal epithelium using immortalized human cells. This model resembles intact cornea with morphologically identifiable desmosomes, tight junctions, microvilli, and cell layers with apical flat cells. The discrimination capacity of the hydrophilic and lipophilic markers and their overall permeabilities in the cell culture model suggest that this model can be used to evaluate corneal drug permeabilities and the mechanisms of ocular drug absorption.
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Acknowledgements |
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Footnotes |
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Submitted for publication April 11, 2001; revised July 2, 2001; accepted July 30, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Arto Urtti, Professor of Biopharmaceutics, Department of Pharmaceutics, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland. arto.urtti{at}uku.fi
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), with type 1 collagen gel coating (
),
with type 1 collagen gel coating containing fibroblast cells (•),
with type 1 collagen gel coating containing laminin (
), and with
collagen coating without air–liquid interface (
). The cells were
cultured also on polycarbonate filters coated with collagen (
) or
collagen and fibroblasts (
). The data in the figure are of
representative individual experiments.
