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1 From the Moorfields Eye Hospital and the 2 Institute of Ophthalmology, London, United Kingdom.
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Abstract |
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METHODS. Primary human corneal epithelial cells were cultured with defined keratinocyte serum-free medium. The cells were incubated with hypromellose (hydroxypropylmethylcellulose 0.3%) with and without benzalkonium chloride 0.01%, saliva with differing osmolalities, 100% serum, and 50% serum (1:1 vol/vol with chloramphenicol 0.5%) for varying times and concentrations. Toxicity was examined in four ways. Microvillous density was assessed with scanning electron microscopy. Cell membrane permeability and intracellular esterase activity were analyzed after staining with fluorescent calcein-AM/ethidium homodimer and cellular adenosine triphosphate (ATP) was quantified using a luciferin-luciferase–based assay.
RESULTS. The toxicity ranking of the tear substitutes correlated in all assays. The ATP assay was the most sensitive, followed by ethidium cell permeability, and finally the esterase activity. Preserved hypromellose was more toxic than the unpreserved preparation. Among natural tear substitutes, natural saliva was most toxic. Isotonic saliva and 50% serum were of similar toxicity, and 100% serum was least toxic. Natural tear substitutes were—except for natural saliva—less toxic than unpreserved hypromellose. Hypotonicity, but not amylase, was the major toxic effect associated with saliva. The dilution of serum with chloramphenicol induced toxicity.
CONCLUSIONS. This is the first toxicity study using human primary corneal epithelial cells cultured under fully defined conditions as an in vitro model. Cellular ATP is a sensitive parameter for quantifying toxicity. Isotonic saliva and serum offer greater therapeutic potential for severely aqueous-deficient dry eyes than do pharmaceutical tear substitutes.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The biomechanical properties of pharmaceutical tear substitutes have been optimized.5 It is also well established that the addition of preservatives should be avoided in tear substitutes, because they break down the functional barrier and induce morphologic changes in corneal epithelial cell membranes.6 7 8 The most widely used preservative, benzalkonium chloride (BAC), is retained within epithelial cell membranes for several days. High concentrations of this cationic detergent lead to cell necrosis, whereas concentrations as low as 0.0001% induce growth arrest and apoptotic cell death. Unpreserved artificial tears with slightly hypotonic or physiologic electrolyte composition and biologic buffers improve corneal epithelial barrier function and patient comfort.9 10
However, few attempts have been made to copy the complex nutritional properties of natural tears in pharmaceutical tear substitutes. Fibronectin11 and growth factors such as epidermal growth factor (EGF)12 13 or basic fibroblast growth factor (bFGF)14 are known to accelerate corneal epithelial wound healing through stimulation of proliferation and migration and antiapoptotic effects.15 16 They have been evaluated as single agents in vitro and in vivo, but only vitamin A has been established in routine clinical treatment of dry eyes on a wider basis,17 because of limitations of stability as well as efficacy of single compound preparations.10 18 19 20 21 Recently, the use of natural tear substitutes such as autologous serum or submandibular gland saliva have been advocated for the treatment of intractable aqueous-deficient dry eyes.22 23 They are unpreserved, nonallergenic, and provide nutritional factors found in natural tears.24 25 Disadvantages are potential instability and compositional differences compared with natural tears, including higher concentrations of transforming growth factor (TGF)-ß in serum and high amylase activity and low osmolality in saliva.
We performed a laboratory study on cultured human corneal epithelial cells to compare the toxicity of natural with pharmaceutical tear substitutes. We also tried to determine the role of factors such as amylase, the hypotonicity and variations in preparation, such as serum dilution and antibiotic additives.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Serum drops were prepared as recently described either as undiluted serum (100%) or as serum (50%) diluted 1:1 vol/vol with unpreserved 0.5% chloramphenicol eye drops (produced by the pharmacy of Moorfields Eye Hospital), which also contained boric acid 1.5%, borax 0.3%, and purified water.26 It was stored at -20o for a maximum of 3 months. Submandibular gland saliva was collected from healthy volunteers from the sublingual caruncle with a cup fixed by mild vacuum to the surrounding mucosa.27 The saliva was filter sterilized (0.2-µm pore size; Whatman, Maidstone, UK) and used immediately in the experiments.
The osmolality of saliva was adjusted with 1 to 6 µl 20% sodium
chloride solution added per 100 µl undiluted saliva. The osmolality
and pH of 20-µl samples were determined before and after adjustment
using standard freeze-point osmometry (Advanced Microosmometre-3;
Vitech Scientific, Partridge Green, UK) and a miniature pH meter
(pH-boy; Camlab, Cambridge, UK). Human 
-amylase type XIII-A
(Sigma, Poole, UK) was dissolved in defined keratinocyte serum-free
medium (defined K-SFM; Gibco, Paisley, UK) to give activities of 0.8,
4, 20, 50, 100, and 200 IU/ml. In humans, this ranges between 44 IU/ml
for unstimulated and 170 IU/ml for stimulated submandibular gland
saliva.28
Amylase activity in a 50-µl test
preparation was assayed according to Bernfeld,29
using
modified starch as substrate (1% wt/vol in PBS). The addition of
amylase was found not to alter pH and osmolality of the medium.
Epithelial Cell Culture
Primary human corneal epithelial cells were obtained from
corneoscleral rims after trephination of corneal grafts at Moorfields
eye bank. After incubation at 37°C for 2 hours with 1.2 IU/ml neutral
protease (Dispase II; Boehringer–Mannheim, Mannheim,Germany) the
epithelium was stripped off with gentle scraping from the limbus to the
center into PBS. This was centrifuged at 100g for 5 minutes,
and the cells suspended in K-SFM supplemented with 100 IU/ml
penicillin, 100 µg/ml streptomycin, 5 ng/ml EGF, 2.5 mg/ml bovine
pituitary extract (Gibco), and 0.03 mM calcium chloride.30
The cells were cultured at 37°C with 5% CO2 in
95% humidified air until 80% confluent and were expanded by using
routine cell culture techniques. In addition immortalized human corneal
epithelial cells (ATCC11515) from the American Type Culture Collection
(ATTC; Rockville, MD) were cultured.
Evaluation of Test Model
Immunohistochemistry with mouse monoclonal antibodies (ICN-flow;
ICN, Basingstoke, UK), using the alkaline phosphatase
method,31
stained the cells strongly positive for human
cytokeratin-3. The effect of varying volumes (60, 100, and 200 µl) of
fluid in the well over 4, 6, and 10 days on adenosine triphosphate
(ATP) counts was measured. The pH and osmolality of test drugs were
measured before and 2 hours after exposure to exclude toxicity due to a
potential absence of buffer capacity in natural tear substitutes.
Toxicity Studies
All experiments were performed on cells at passages 2 to 4
plated at a density of 15,000 cells/well. The cells were plated either
for morphologic evaluation on 16-well glass slides (Nunc, Napierville,
IL) or for the ATP assay in 96-well flat bottom plates (Falcon,
Plymouth, UK). The cells were incubated for at least 24 hours with
K-SFM supplemented with bovine pituitary extract. When approximately
60% confluence was reached, the medium was withdrawn, and the wells
were washed twice with PBS. The cells were then incubated for a further
18 hours with defined K-SFM supplemented only with
penicillin-streptomycin.
The wells were washed twice with 200 µl PBS and incubated with 200 µl prewarmed single test drug. Each microplate was used to test six to seven single agents as well as a maximum inhibitor (BAC: 1%) and a minimal inhibitor (defined K-SFM) in triplicate. The following seven exposure times were used with neat test drug: 10 and 30 minutes, 1, 2, 4, 6, and 24 hours. In analogy to a drop of 38 µl applied to a normal tear volume of 7 µl which is then washed out by the subsequent tear turnover of 1.2 µl/min, the concentration of an initially neat fluid volume after 25 minutes and 1, 2, and 3 hours was calculated. These were 50%, 20%, 4%, and 0.8%. The test drug was diluted in defined K-SFM to these concentrations and applied for 2 hours. These calculations may overestimate the retention time for normal eyes5 but are likely to be more accurate for absolute aqueous-deficient dry eyes.
Scanning electron microscopy (SEM) was used to evaluate time response. The viability staining and the ATP assay were used to assess time and dose response. The ATP assay was the most sensitive of the three assays used. Therefore, the effect of 10 minutes’ application of test drugs with subsequent PBS wash and 48-hour incubation with defined K-SFM, as well as 2-hour incubation with saliva of varying osmolality or defined K-SFM supplemented with increasing amylase activity, was assessed with the ATP assay only.
Endpoint Assays
For all endpoint assays the test drug was removed and the wells
washed twice. For SEM, specimens were fixed in Karnovsky’s fixative,
dehydrated through ascending alcohol concentrations, critical point
dried, mounted, and sputter coated with 7 to 10 nm gold before
examination with a scanning electron microscope (model 6100; JEOL,
Tokyo, Japan). The surface morphology of the cells was evaluated at 10
minutes and 6 hours of exposure.
For live-dead viability staining, the cells were incubated with 100 µl of 4 µM ethidium homodimer-1/2 µM calcein-AM (Molecular Probes, Leiden, The Netherlands) for 30 minutes at room temperature. The cells were then immediately viewed with a fluorescence microscope at 485 nm excitation and 515 nm emission wavelength. The nonfluorescent calcein-AM is converted into green fluorescent polyanionic calcein by intracellular esterase, indicating active cell metabolism.32 Ethidium homodimer is excluded by viable cells but permeates damaged cell membranes, binds to nucleic acids, and results in red fluorescence. The number of green, red, and bicolored cells was counted in three wells per 10 fields of 315 x 210 µm each at 400-fold magnification. The percentage of cells with exclusive green fluorescence (interpreted as no cell membrane damage) and of green and bicolored fluorescence (interpreted as detectable esterase activity) was calculated.
For the ATP assay all reagents were from DCS Innovative Diagnostik Systeme (Hamburg, Germany). Cellular ATP was extracted by adding 200 µl PBS and 50 µl cell extraction reagent to each well, using a multichannel pipette. The cells were left at least 20 minutes at room temperature before 20 µl of culture extract was transferred and mixed with 50 µl luciferin-luciferase counting reagent, previously equilibrated for 20 minutes to room temperature, into the wells of a white 96-well assay plate (Dynex, Chantilly, VA). The resultant luminescence was read immediately using a luminometer (Dynatech ML1000; Dynex). An ATP standard curve was performed for all studies using 0.05-ml aliquots of a 250 ng/ml ATP standard serially diluted 1:3 in dilution buffer.33 34 All experiments were at least performed twice.
Data Evaluation and Statistical Methods
The percentage of cell growth inhibition (CGI) for each drug and
test situation was calculated
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The index area under the curve was calculated using the trapezoidal
rule. The IC50 value is the test drug
concentration in culture medium that induces a 50% reduction of ATP
levels and was determined by interpolation. Percentage of coefficient
of variation was calculated by SD/means. The Wilcoxon signed-rank test
for nonparametric data was used to determine the significance of
differences. Probabilities were corrected for multiple tests using
Bonferroni’s method and were considered to be significant at
P 
0.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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20% (P < 0.00001). Cell membrane permeability,
however, was more sensitive and increased with 4% preserved
hypromellose (P < 0.001), 50% natural saliva
(P < 0.01), and 100% unpreserved hypromellose
(P < 0.001) or isotonic saliva (P <
0.001). The 50% (P < 0.4) or 100% serum
(P < 0.6) showed no significant effect at 2 hours of
incubation.
ATP Assay
The loss of cellular ATP levels as time and dose responses are
shown in Figure 4
. At the 2-hour time point, 100% serum resulted in significantly less
inhibition than 50% serum or natural saliva. Isotonic saliva was less
toxic than natural saliva (P < 0.01). Serum in the
50% and 100% preparation (P < 1 x
10-5), as well as isotonic
saliva preserved cellular ATP significantly better than unpreserved
hypromellose (P < 0.01). Natural saliva was not
significantly different from unpreserved hypromellose
(P = 0.09). The 50% serum without chloramphenicol
reduced ATP concentrations significantly less than 50% serum with
chloramphenicol (P = 0.049). Short time exposures of 10
minutes with subsequent incubation in culture medium for 2 days showed
no delayed effect of higher dilution of natural tear substitutes. Table 2
gives the figures calculated for the IC50 and the
area under the dose–response curve (AUC) of all drugs tested in the
ATP assay. Table 3
summarizes, categorizes, and ranks the tested tear substitutes.
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Discussion |
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Validity of Cell Culture Models for Cytotoxicity Tests
To reduce the number of animals used in the preclinical evaluation
of ocular toxicity of substances the Draize test has been substituted
by in vitro tests.39
40
Cultured epithelial cells of
animal41
42
and human corneal43
and
conjunctival8
44
origins have been used as substrate for
these. Monolayered cultures of corneal epithelial cells have been shown
to be equally sensitive as three-dimensional corneal constructs for
evaluation of acute toxicity.45
These tests should be
performed under standardized conditions, and undefined supplements
should be avoided in the culture medium. To allow extrapolation
for the clinical situation, human rather than animal and primary rather
than immortalized cells should be used.46
To the best of
our knowledge, our study is the first to meet these three conditions
simultaneously.
Limited human corneal donor material for experimental purposes is one reason that in vitro studies are often performed on cultured animal cells. We found corneoscleral rims of short-term, preserved donor material used for keratoplasty a suitable source of primary human corneal epithelial cells. Also, human corneal epithelial cells tend to become senescent after passage 5,43 which can significantly increase variability and introduce bias. We therefore used a low-calcium medium30 and performed experiments only on cells up to passage 4. Thus, the coefficient of variation in our model was only 12%, which is representative of the genetic and phenotypic heterogeneity of normal tissue.34
The relevance of in vitro obtained toxicity data is sometimes challenged, because culture conditions cannot substitute for the complex physical and molecular interactions of tear film and ocular surface in vivo. However, in vitro evaluation may allow more appropriate conclusions for severely aqueous-deficient dry eyes, because these are more susceptible to toxicity36 37 47 and have a reduced tear turnover. In these evaluations, frequent application of tear substitutes or salivary gland transplantation result in an eye-bath–like exposure. Lastly, test conditions such as drug volume and incubation time were standardized and pH and osmolality controlled and found to be stable within physiological limits. Although cell culture models cannot provide neural pathways that are important in epithelial integrity in the long term, we believe that the model used here is fully defined and allows extrapolation to the acute toxicity of pharmaceutical and natural tear substitutes on human corneal epithelium in vivo.
Relevance of End Point Assays
We assessed and defined toxicity as a loss of cell membrane
microvilli and barrier function, as well as intracellular esterase
activity and ATP. SEM is an accepted way to visualize drug-induced
alterations of the corneal epithelial cell morphology, such as loss of
cell coherence and microvilli.41
48
However, it is
expensive and technically demanding. Fluorescent viability staining
provides information beyond morphology, because it visualizes
functional alterations of the cell membrane integrity and cell
metabolism. It has previously been used to evaluate the viability of
corneal donor tissue.32
In our study, cell membrane
permeability was a more sensitive parameter than intracellular esterase
to determine toxicity in these assays. Tetrazolium salt assays such as
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay have been used extensively to quantify cytotoxicity. However, the
luminescence-based ATP assay offers even better sensitivity and
reliability for 96-well plate assays.49
50
51
All three
endpoint tests generally agreed in the ranking of the drug toxicity,
but the ATP assay was able to quantify a significant cell inhibition
after 2 hours of incubation with both serum preparations that was not
detected with the viability stain. It also was easier and faster to
perform.
Comparison of Cytotoxicity of Pharmaceutical and Natural Tear
Substitutes
Our results show that natural tear substitutes are—with the
exception of natural saliva—less toxic than pharmaceutical tear
substitutes. Autologous serum is used in early clinical studies for
aqueous-deficient dry eyes at concentrations of 10% to
100%.24
26
Our data show that undiluted serum maintains
ATP levels better than 50% serum. This supports the clinical
observation that undiluted serum is required for a sufficient effect in
some patients.26
Although reported to be relatively
nontoxic, chloramphenicol 0.5% eye drops used to dilute serum to 50%
reduced ATP levels more than the addition of culture medium
supplemented with penicillin-streptomycin.52
Recent clinical studies suggest that saliva can be beneficial in severely aqueous-deficient dry eyes. Data on the toxicity of saliva on the ocular surface is limited to the report that the incubation of corneal tissue in parotid saliva does not result in histologically detectable enzymatic digestion.53 Our study confirms that amylase, due to its specificity for complex carbohydrates, is not harmful to cultured epithelial cells. The low osmolality of natural saliva was the major factor contributing to its toxicity.
Although moderately hypotonic solutions of 160 mOsm/l reduce the signs and symptoms10 54 caused by the hypertonicity of aqueous-deficient dry eye tears, substitutes of 75 mOsm/l were found to be irritating.55 In rabbits, hypotonic pharmaceutical tear substitutes delayed the recovery of corneal epithelium.10 Our data show that, when incubated for 2 hours with saliva, ATP is best maintained at 180 to 300mOsm/l. Lower or higher osmolalities inhibit cellular metabolism. This can be reproduced in immortalized cells, which confirms the importance of this basic mechanism of toxicity. The tonicity of salivary tears provided by gland transplantation is due to surgical denervation and contact with the ocular surface higher (165 ± 74 mOsm; range, 88–310) than in natural saliva.25 It may therefore be less toxic in vivo than unpreserved hypromellose. However, at the low end of the range, toxicity is likely and may, with continuous lubrication, result in the microcystic epithelial edema observed in selected cases.56
Factors Responsible for the Beneficial Effect of Natural Tear
Substitutes
Natural tear substitutes, unlike palliative pharmaceutical tear
substitutes, may address parts of the underlying multifactorial
pathogenesis of dry eye. Saliva and tears both lubricate mucous
membranes. They have similar biochemical and physiological features.
For lubrication, both contain mucin and are relatively viscous. For
antimicrobial protection, they have similar concentrations of lysozyme,
lactoferrin, IgA, and 2-macroglobulin.57
58
59
To
maintain cellular health, they are rich in EGF and vitamin A. These
factors can also be found in serum. EGF and fibronectin concentrations
in saliva and serum are higher than in normal tears.60
61
62
However, the concentration of vitamin A in serum is higher (46 µg/ml)
than in saliva (51 ng/ml) and this may explain why isotonic saliva is
not as effective in maintaining cellular ATP levels as 100%
serum.24
63
In summary, our data show that natural saliva has a cytotoxic effect on corneal epithelial cells that is comparable to unpreserved pharmaceutical tear substitutes. Iso-osmolar saliva promises better clinical potential for treatment of severe aqueous-deficient dry eyes than do pharmaceutical tear substitutes. Undiluted serum maintains cellular viability best. The addition of antibiotics reduces this effect. Although the use of natural tear substitutes has been anecdotal so far, this approach seems promising for the therapy of aqueous-deficient dry eyes. Determining the beneficial factors responsible for the therapeutic effect of natural tear substitutes on epithelial viability, as well as their required concentration and stability, should be subject of future studies. Human primary corneal epithelial cells cultured under fully defined conditions offer an in vitro model for this, and cellular ATP is a very sensitive parameter to quantify toxicity. Such studies should ultimately allow the formulation of pharmaceutical tear substitutes with equally supportive properties.
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Acknowledgements |
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Footnotes |
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Submitted for publication April 20, 2000; revised September 13, 2000; accepted October 6, 2000.
Commercial relationships policy: N.
Corresponding author: Gerd Geerling, Augenklinik der Medizinischen Universität Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. ggeerling{at}ophtha.mu-luebeck.de
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) The first time and concentration that were
significantly different from the minimal inhibitor. (A,
B) Esterase extinction as time response on incubation with
neat test drug: Significant esterase extinction was induced by
preserved hypromellose (P < 0.0001),
unpreserved hypromellose, and natural and isotonic saliva
(P < 0.01). The 50% or 100% serum had no effect
(P > 0.99). (C, D) Esterase
extinction as a concentration response with 2 hours of
incubation. A loss of esterase activity was induced by
preserved (P < 0.00001) and unpreserved hypromellose
(P < 1 x
10-16), as well as natural
(P < 0.01) and isotonic saliva (P <
1 x 10-12). No
significant effect was observed with 50% (P = 0.14)
and 100% serum (P = 0.92). (E,
F) Cell membrane permeability as time response on incubation
with neat test drug: Cell membrane permeability was induced by
preserved and unpreserved hypromellose, isotonic saliva
(P < 0.0001), natural saliva, and 50% serum
(P < 0.01). No significant effect on cell membrane
permeability was observed for 100% serum at 24 hours
(P = 0.06). (G, H)
Cell membrane permeability as a concentration response with 2 hours of
incubation: Preserved and unpreserved hypromellose or isotonic saliva
(P < 0.001) and natural saliva (P <
0.01) permeabilized cell membranes. The 50% (P = 0.4)
or 100% serum (P = 0.6) had no such effect.
) The first time and concentration at which the ATP level
was significantly different from the minimal inhibitor. (A,
B) Time response on incubation with neat test drug: For
BAC-preserved hypromellose, P < 1 x
10-6; for all other test
drugs, P < 0.01). (C, D)
Ten-minute exposure in varying concentrations with subsequent 48 hours
of incubation in culture medium: No delayed effect of higher dilution
of natural tear substitutes or unpreserved hypromellose on ATP levels
was observed. (E, F) Dose response on 2-hour
incubation: For BAC-preserved hypromellose, P < 0.001;
for all other tear substitutes, P< 0.01. ATP response to
osmolality or amylase 10: (G) Cellular ATP measurement after
2 hours’ exposure to amylase in defined K-SFM adjusted to varying
activities; (H) ATP levels measured in primary and
immortalized human corneal epithelial cells after 2 hours’ exposure to
saliva with the osmolality adjusted to varying levels.