WO1999051763A1 - Systeme de culture d'organes de la cornee destine a des essais de toxicite chimique de produits de consommation - Google Patents

Systeme de culture d'organes de la cornee destine a des essais de toxicite chimique de produits de consommation Download PDF

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Publication number
WO1999051763A1
WO1999051763A1 PCT/US1999/007277 US9907277W WO9951763A1 WO 1999051763 A1 WO1999051763 A1 WO 1999051763A1 US 9907277 W US9907277 W US 9907277W WO 9951763 A1 WO9951763 A1 WO 9951763A1
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tested
compound
test parameter
corneal
cultured
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PCT/US1999/007277
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English (en)
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Fu-Shin X. Yu
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The Schepens Eye Research Institute, Inc.
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Priority to AU35484/99A priority Critical patent/AU3548499A/en
Publication of WO1999051763A1 publication Critical patent/WO1999051763A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics

Definitions

  • a mammalian whole eye or a cornea-containing portion thereof, or, preferably, a cornea or portion thereof is cultured under defined conditions as an organ culture system for chemical toxicity tests, e.g., of consumer products.
  • Corneas or corneal tissue may be advantageously cultured together with a limbal-conjunctival ring.
  • bovine or porcine corneas are used (or corneas from any other mammal wherein sufficient test material is available) .
  • the ex vivo organ culture system of the invention compared to cell culture, far more closely resembles an in vivo testing system in terms of maintaining the corneal architecture and natural interaction between the epithelial cells and other cells residing in the ocular surface, including eratocytes in the stroma and limbal epithelial cells.
  • Another key advantage of this invention is that end-points or reference standards of specific tests can be precisely characterized. Examples include such tests as determining the activation of well-known, stress- responsive transcription factors in response to chemical stimuli, or assessing corneal structural and functional changes caused by chemical exposure. Other parameters that can be measured include changes in, e.g., corneal transparency or opacity, transepithelial permeability and -3-
  • kits for using the system include, e.g., quantitation controls such as bovine or porcine eyes in culture and ready for testing.
  • Fig. 1 is a diagram of the corneal organ culture model system for chemical toxicity testing according to the method of the invention
  • Fig. 2 depicts the corneal organ culture system at different stages of the method (2A-2E) ;
  • Fig. 3 depicts an electrophoresis mobility shift assay (EMSA) for NF-KB activation in organ-cultured, corneal epithelial cells exposed for 1 hour to different chemical compounds; and
  • ESA electrophoresis mobility shift assay
  • Fig. 4 depicts an EMSA for AP-1 activation in organ- cultured, corneal epithelial cells exposed for 1 hour to different chemical compounds. -4 -
  • the ex vivo organ culture system of the invention for the sensitive and specific assay of chemically induced activation of stress-responsive transcription factors or other changes in a mammalian eye or cornea, may be used to replace the Draize eye test, a standard procedure using live rabbits for identifying potential human eye irritants. Specific details of the ex vivo system are illustrated in Figs. 1 and 2.
  • This new system is based on organ culture of, e.g., bovine or porcine corneas, whole eyes, or a corneal tissue-containing portion thereof, and allows the effects of tested substances to be evaluated.
  • Exemplary substances include chemical compounds or mixtures of compounds (hereinafter commonly referred to as "chemicals").
  • the system of the invention serves as an effective tool for the prediction of ocular irritations caused by diverse classes of chemicals.
  • Eye irritation that results from chemical exposure affects the anterior ocular surface, primarily the cornea. Approximately 75% of the historically relevant, Draize rabbit eye irritation test scores were derived directly from corneal effects. Thus, a logical component to the ex vivo assessment of chemically induced damage to the ocular surface is the evaluation of corneal effects. There are three cell types in the cornea: the stratified epithelium covering the anterior surface, the keratocytes interspersed in the stroma, and the single cell layer of the endothelium. -5-
  • the first line of cellular defense of the cornea against eye irritants is the impermeable barrier of the epithelium, which is vulnerable to chemical insults.
  • measurement of the cellular response of the epithelium to irritants provides a reliable parameter for testing chemical toxicity. Severe irritation causing complete epithelial cell loss will result in anterior stromal damage, including increased thickening of the stroma and apoptosis of keratocytes (Jester, Petroll et al . , 1998; Jester, Li et al., 1998).
  • Organ culture serves as an appropriate model for chemical safety tests, the corneal architecture being well maintained via the use of air interface organ culture techniques and the development of, e.g., agar, agarose, agar-collagen or agarose-collagen gel for filling the endothelial corneal concavity.
  • agar, agarose, agar-collagen or agarose-collagen gel for filling the endothelial corneal concavity.
  • Several parameters have been determined as indicating corneal integrity. These parameters can be separately or simultaneously measured in the method of the invention to determine the stress-related responses of the ex vivo corneal model system to diverse classes of chemicals.
  • the protooncogens c-fos and c-j un are members of the immediate early response class, whose expression is rapidly and transiently induced following a wide variety of stimuli.
  • the transcription factor AP-1 activation protein-1
  • the transcription factor NF- ⁇ B acts as a central, coordinating regulator.
  • NF- ⁇ B binding activity within minutes.
  • the activation of these two transcription factors in the corneal organ culture system of the invention, in response to toxicants can be measured with a standard electrophoresis mobility shift assay (EMSA) .
  • ESA electrophoresis mobility shift assay
  • a dosage response curve for each chemical tested can be obtained from the measurement.
  • Complementary assays determining NF- ⁇ B activation (by nuclear staining of activated forms) and AP-1 expression (by PCR determination of c-jun and c-fos mRNA levels) can also be assessed.
  • the activation of other kinds of genes may also be the subject of chemical safety testing: heat shock proteins such as Heat shock protein 70, cytokines such as interleukin 1 - alpha and interleukin 8, and enzymes such as cyclooxygenase 2, a key enzyme with prostaglandin synthase activity.
  • the activation of these genes can be determined by measuring their promoter activity by a reporter gene, (e.g. luciferase, ⁇ -galactosidase, green fluorescence protein) , or by measuring their enzymatic activity. Since each distinct group of genes may be involved in a unique response process, the measurement of gene expression and/or activation from different groups should be considered as multiple, but intrinsically related, end-points.
  • Organ culture also permits assessing other parameters, e.g., the effects of substances such as chemicals on the ocular surface.
  • Some parameters may include changes in corneal morphology, transepithelial permeability and transepithelial electrical resistance, and corneal transparency, as well as epithelial wound healing and leakage of cellular enzymes (e.g., transketolase) into the -7 -
  • anatomic abnormality can be induced by chemicals into the corneal culture system of the invention and detected by histological examination of cryostat section of the sampled cornea.
  • chemicals can cause programmed cell death (apoptosis) of keratocytes in organ cultures. Individual apoptotic cells in corneal cryostat sections can be detected by the TUNEL assay (Moore et al . , 1998). The percentage of apoptotic keratocytes would directly correlate to the toxicity of a chemical.
  • toxic chemicals may cause cloudiness or opacity in the cornea, which is normally transparent. (See Fig.
  • Transepithelial permeability and transepithelial electrical resistance are two physiological relevant parameters for evaluating the barrier function of the corneal epithelium, i.e., corneal health. These properties are likely to be affected by damage caused by chemicals added to the organ culture system during testing. Transepithelial permeability can be assessed by measuring the retention of fluorescein (which usually is not permeable through corneal epithelium) (Tchao 1988), and -8-
  • transepithelial electrical resistance can be measured using an Epithelial Voltohmmeter (Kruszewski et al., 1997; Ward et al., 1997; Gautheron et al., 1992).
  • bovine or porcine eyes or cornea in an organ culture test system offers several advantages for ex vivo chemical tests.
  • bovine and porcine eyes are the by-products of the meat industry; no live animal would be sacrificed for the tests. These eyes are readily available resources worldwide; therefore, large-scale tests are possible.
  • both bovine and porcine eyes are very similar to those of humans, anatomically and biochemically. Thus, the data obtained by using these eyes are relevant to the human response to chemicals.
  • human eyes from eye banks might be used in a similar fashion for some key tests.
  • bovine and porcine eyes both provide ' enough corneal epithelial cells for easy use in the required tests outlined in this application.
  • Bovine or porcine eyes are obtained from the abattoir within several hours (e.g. five) of slaughter.
  • the eyes are placed individually onto a container similar to egg cartons with the epithelial-side up, and they are transported to the laboratory on ice in a moisture chamber (or cooler) .
  • the ocular surface should be disinfected using 0.1% povidine-iodine solution. It is optional when the eye or cornea is tested shortly after preparation.
  • Corneal-scleral rims with approximately 5 mm of the limbal conjunctiva present, are excised and rinsed in sterilized phosphate buffered saline as described in Foreman et al., 1996. (See Fig. 2A. )
  • the excised corneas are placed epithelial-side down into a sterile "cup" containing minimal essential medium (MEM) to prevent drying of the epithelium.
  • the cups made with silicone rubber, are designed to allow the scleral rims to rest on the cup edges without damage to the corneal epithelium.
  • MEM minimal essential medium
  • the endothelial corneal concavity is then filled with MEM containing 0.5-1% agar or, preferably, agarose, with or without 1 mg/ml of rat tail tendon collagen maintained at 37 °C. This mixture is allowed to gel. Corneas are then inverted and then transferred to a 60 mm dish, as illustrated in Fig. 1 and Fig. 2D.
  • Corneas are cultured in the test medium for typically 0.5-6 hours, preferably about 1 hour.
  • a portion of the cornea immersed in a medium containing a test substance e.g., a chemical, for approximately 0.5-6 hours, preferably about 1 hour, is another way the corneal culture system of the invention may be used to test toxicity of a substance.
  • a test substance e.g., a chemical
  • the whole eye may also be used for corneal organ culture.
  • the posterior part of the eye is immersed in a culture medium that covers the eye up to the limbal conjunctiva region, leaving the corneal epithelium exposed to the air.
  • the test chemicals are added to the medium and applied to the surface of the epithelium and incubated for a time within 0.5-6 hours, pref. about 1 hour.
  • the corneal surface is washed with PBS, and the epithelial cells are removed from the corneas and transferred to a 1.5-ml tube using a scalpel blade.
  • Cells are resuspended in a buffer containing 20 mM Hepes-KOH (pH 7.0), 1 mM DTT, 1 mM EDTA, 200 mM KC1, 20% glycerol, 0.1 mM phenylmethyl-sulfonyl fluoride (PMSF) and 0.1% NP40 by pipetting the cells up and down.
  • the cell suspensions are - 11-
  • the supernatants are transferred to new tubes and can be used, e.g., for protein determination and for the electrophoresis mobility shift assay. They can also be stored in liquid N 2 for future use.
  • a 20 ⁇ l volume containing 3-4 ⁇ g of epithelial extract is mixed with 4 ⁇ l of 5 x binding buffer (20 mM HEPES, pH 7.5, 50 mM KC1, 1 mM DDT, 2.5 mM MgCl 2 , 20% Ficoll) , 2 ⁇ g of poly(dl-dC) as nonspecific competitor DNA, 2 ⁇ g of bovine serum albumin, and 10,000-15,000 cpm Cerenkov radiation of the labeled oligonucleotide.
  • samples are loaded on a 4% non- denaturing polyacrylamide gel and run in 0.5 x TBE buffer (89 mM Tris, 89 mM boric acid, 1 mM EDTA), pH 8.3.
  • sequences of the oligonucleotides used to detect the DNA binding activity are: for NF-KB : 5 ' -AGCTTCAGAGGGGATTTCCGAGAGG-3 ' ; and for AP-1 : 5 ' -CGCTTGATGAGTGAGCCGGAA-3 ' .
  • the oligonucleotides are annealed with their complementary strands and labeled using T4 polynucleotide kinase and [ ⁇ - 32 P] ATP.
  • the labeled probes are separated from free nucleotides by ethanol precipitation. This is a standard technique used in research and industrial laboratories investigating DNA-binding activity. -12-
  • the amount of oligonucleotides with shifted mobility is dependent on the concentration of activated transcription factors.
  • the activation of NF- ⁇ B or AP-1 in each test sample can be quantitated by Phospholmager analysis and presented as activity relative to the control, untreated corneal epithelium.
  • the data obtained by the assays described above should quantitatively reflect the changes in the corneas caused the chemical exposure. Although a detected alteration may not be considered as a direct cause (s) of "pain” or "irritation,” any identified quantitative correlation could be used as an ex vivo biochemical endpoint/marker for predicting ocular irritation.
  • Fig. 3 shows an EMSA for NF- ⁇ B activation in organ- cultured epithelial cells in response to various chemicals, in accordance with the invention.
  • the excised corneas in organ culture were treated with benzalkonium chloride (BAK) , isopropanol (ISO) , or H 2 0 2 , or left untreated (in medium as control C) and incubated for about 1 hour at 37 °C and 5% C0 2 .
  • the corneal epithelial cells were then collectd by scraping.
  • NF- ⁇ B activation was determined using radiolabeled oligonucleotides encompassing the consensus motifs of NF-KB.
  • the gel binds in Fig. 3 indicate NF- ⁇ B binding.
  • Fig. 4 depicts an EMSA for AP-1 activation in organ- cultured corneal epithelial cells in response to various chemicals, in accordance with the invention.
  • the excised corneas in organ culture were treated with benzalkonium chloride (BAK), isopropanol (ISP), or H 2 0 2 , or left untreated (in medium as control C) , and incubated for approximately 1 hour at 37 °C and 5% C0 2 .
  • the epithelial cells were collected by scraping.
  • AP-1 activation was determined by electrophoresis mobility shift assay using radiolabeled oligonucleotides encompassing the consensus motifs of AP-1.
  • the gel bands in Fig. 4 indicate AP-1 binding.
  • AP-1 DNA-binding activity was significantly increased in response to H 2 0 2 or decreased in response to BAK or isopropanol, and the observed changes were concentration-dependent.
  • TKT leakage is measured as follows. Cultured bovine corneas are exposed to test substances for 1 hr and the culture media collected. TKT levels in the collected media are assayed by colorimetric determination of 7-sedoheptulose phosphate production (Sax et al, 1997; Takevchi, et al, 1984) . A dose- dependent curve of TKT leakage for each test chemical can be derived, allowing the evaluation of both eye irritation and recovery of the epithelium.
  • corneal epithelium heals a wound rapidly. Damage caused by irritants could inhibit or even prevent wound closure.
  • exposure to test chemicals prolonged healing times of a scraping wound (Jung et al, 1993) .
  • the cytotoxicity of test chemicals is evaluated by assessing their effects on the healing of an epithelial debridement wound.
  • the epithelial debridement wound can be made with a 5-mm trephine as described in Guo et al, 1998 or Yu et al, 1998. In normal bovine organ culture, this wound is re-covered by epithelium within 36 hours (Yu et al, unpublished results) .
  • test chemicals may be assessed under two different sets of conditions: (i) corneas in culture are pretreated with a substance of chemical in MEM, followed by epithelial wounding and wound healing in the absence of the test chemical; or (ii) a wound of normal epithelium is allowed to heal in the presence of the test chemical.
  • the wounded corneas are allowed to heal in culture with no chemical treatment (37°C in a humidified 5% C0 2 incubator) , as the control. Any remaining corneal defect after a suitable incubation time (e.g. 32 hr) are stained with Richardson's stain and the corneas photographed. The diameter of the remaining defect can be measured, and the size of the uncovered area can then be calculated, yielding the rate of wound healing.
  • a suitable incubation time e.g. 32 hr
  • test substance that permit wound closure (permissive, or practically non-irritating chemical) , inhibit wound closure (inhibitory, or minimal or mild irritant) , or prevent wound closure (cytotoxic, or severe to extreme irritant) .
  • This approach allows chemicals and concentrations to be ranked according to their toxicity, and these rankings can be compared to those published in the literature for both in vitro and in vivo tests, and statistical correlations can be established.
  • This sensitive, functional test might become an important endpoint for our ex vivo system in the evaluation of the irritancy potential of consumer products.
  • the corneal organ culture system of the invention can permit the reduction or elimination of the use of animals for safety tests and allow for easy manipulation of test parameters under well-defined conditions, in a similar manner to cell culture.
  • the ex vivo organ culture and testing system of the invention is much more physiologically relevant to the human condition than conventional cell cultures, and the data obtained are much more reliable in terms of predictability for human in vivo response.
  • the end-points permitted by ex vivo tests, as described here, range from cellular response to ultrastructure alternations. Combination of these parameters should give realistic assessments of the toxicity of a chemical to the eye (eye irritation) .
  • the assays described are commonly used and easily standardized. A set of internal controls can be established, allowing quantitation and elimination of differences from different experiments. Many different products, such as consumer products, chemicals, formulations, solvents, powder and dusts, can be tested for eye irritation with the ex vivo system of the invention. Therefore, this system has applications in cosmetic, pharmaceutical, chemical, biotechnology, food and environmental industries, as well as other industries.
  • Bovine corneal opacity and permeability test an in vitro assay of ocular irritancy. Fundam Appl Toxicol. 18:442-449.

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Abstract

L'invention concerne la culture d'yeux, de cornées ou de parties contenant une cornée de mammaliens dans des conditions définies comme système de culture d'organes. Comparé à la culture cellulaire, le système selon l'invention ressemble beaucoup plus à un système d'essai in vivo pour ce qui est du maintien de l'architecture de la cornée et de l'interaction naturelle entre cellules épithéliales et autres cellules résidant à la surface oculaire. Le système de culture d'organes selon l'invention est précis, efficace, économique et fiable pour l'évaluation du potentiel d'un produit chimique ou d'une formulation à causer une irritation ou une lésion des yeux; ce système peut également s'utiliser dans des tests d'innocuité de produits de consommation.
PCT/US1999/007277 1998-04-02 1999-04-01 Systeme de culture d'organes de la cornee destine a des essais de toxicite chimique de produits de consommation WO1999051763A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011067384A1 (fr) * 2009-12-03 2011-06-09 Novagali Pharma Sa Procede in vitro d'essai de toxicite et d'efficacite d'un materiau d'essai sur une surface oculaire
US9220694B2 (en) 2006-07-28 2015-12-29 Santen Sas Emulsion compositions containing cetalkonium chloride
CN110070922A (zh) * 2017-08-22 2019-07-30 苏州市药品检验检测研究中心（苏州市药品不良反应监测中心） 一种化学品眼刺激性的评价方法

Citations (1)

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US4959319A (en) * 1985-08-01 1990-09-25 Skelnik Debra L Process of corneal enhancement

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US4959319A (en) * 1985-08-01 1990-09-25 Skelnik Debra L Process of corneal enhancement

Non-Patent Citations (2)

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Title
DOUGHTY M J: "EVALUATION OF SHORT-TERM BOVINE EYE STORAGE PROTOCOL FOR THE ENUCLEATED EYE TOXICITY TEST", TOXICOLOGY IN VITRO., ELSEVIER SCIENCE., GB, vol. 11, 1 January 1997 (1997-01-01), GB, pages 229 - 240, XP002919275, ISSN: 0887-2333, DOI: 10.1016/S0887-2333(97)00011-8 *
FOREMAN D M, ET AL.: "A SIMPLE ORGAN CULTURE MODEL FOR ASSESSING THE EFFECTS OF GROWTH FACTORS ON CORNEAL RE-EPITHELIALIZATION", EXPERIMENTAL EYE RESEARCH., ACADEMIC PRESS LTD., LONDON., vol. 62, 1 January 1996 (1996-01-01), LONDON., pages 555 - 564, XP002919276, ISSN: 0014-4835, DOI: 10.1006/exer.1996.0065 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9220694B2 (en) 2006-07-28 2015-12-29 Santen Sas Emulsion compositions containing cetalkonium chloride
US9956289B2 (en) 2006-07-28 2018-05-01 Santen Sas Emulsion compositions containing quaternary ammonium compounds
US10842873B2 (en) 2006-07-28 2020-11-24 Santen Sas Methods for preparing oil-in-water emulsions comprising cetalkonium chloride
US11612658B2 (en) 2006-07-28 2023-03-28 Santen Sas Oil-in-water emulsions comprising cetalkonium chloride and methods of making and using the same
WO2011067384A1 (fr) * 2009-12-03 2011-06-09 Novagali Pharma Sa Procede in vitro d'essai de toxicite et d'efficacite d'un materiau d'essai sur une surface oculaire
CN110070922A (zh) * 2017-08-22 2019-07-30 苏州市药品检验检测研究中心（苏州市药品不良反应监测中心） 一种化学品眼刺激性的评价方法
CN110070922B (zh) * 2017-08-22 2022-10-04 苏州市药品检验检测研究中心（苏州市药品不良反应监测中心） 一种化学品眼刺激性的评价方法

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DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase