Corneal Epithelial Model Research Articles

Evaluation of Eye Irritation Potential of Solid Substance with New 3D Reconstructed Human Cornea Model, MCTT HCE(TM).

The eye irritation potential of drug candidates or pharmaceutical ingredients should be evaluated if there is a possibility of ocular exposure. Traditionally, the ocular irritation has been evaluated by the rabbit Draize test. However, rabbit eyes are more sensitive to irritants than human eyes, therefore substantial level of false positives are unavoidable. To resolve this species difference, several three-dimensional human corneal epithelial (HCE) models have been developed as alternative eye irritation test methods. Recently, we introduced a new HCE model, MCTT HCETM which is reconstructed with non-transformed human corneal cells from limbal tissues. Here, we examined if MCTT HCETM can be employed to evaluate eye irritation potential of solid substances. Through optimization of washing method and exposure time, treatment time was established as 10 min and washing procedure was set up as 4 times of washing with 10 mL of PBS and shaking in 30 mL of PBS in a beaker. With the established eye irritation test protocol, 11 solid substances (5 non-irritants, 6 irritants) were evaluated which demonstrated an excellent predictive capacity (100% accuracy, 100% specificity and 100% sensitivity). We also compared the performance of our test method with rabbit Draize test results and in vitro cytotoxicity test with 2D human corneal epithelial cell lines.

Evaluation of cationic nanosystems with melatonin using an eye-related bioavailability prediction model

In this study, two types of nanosystems, namely lecithin/chitosan nanoparticles and Pluronic® F127/chitosan micelles, have been prepared and evaluated for their potential for the ocular delivery of melatonin, which is known to exert an ocular hypotensive effect. The melatonin content, particle size, zeta potential and in vitro drug release properties were studied as a function of the presence of chitosan in the nanosystem. Lecithin/chitosan nanoparticles were evaluated in terms of the mucoadhesive properties by a newly established method based on HCE-T cells, also used in in vitro biocompatibility and permeability studies.Lecithin/chitosan nanoparticles were significantly larger than the corresponding F127/chitosan micelles (mean diameter of 241.8 vs. 20.7nm, respectively) and characterised by a higher surface charge (22.7 vs. 4.3mV, respectively). The HCE-T cell viability assay did not show significant toxic effects of nanosystems investigated at the (relevant) chitosan concentration tested. The permeability study results confirmed the permeation enhancing effect of F127, which was hindered in the presence of chitosan. Lecithin/chitosan nanoparticles were characterised by prominent mucoadhesive properties and prolonged melatonin release, which was shown to control melatonin permeation across an in vitro corneal epithelial model. Such properties demonstrate the potential for nanoparticles to provide an extended pre-corneal residence time of melatonin, ensuring higher eye-related bioavailability and extended intraocular pressure reduction compared to melatonin in both aqueous and micelle solutions.

Identification of cornifelin and early growth response-1 gene as novel biomarkers for in vitro eye irritation using a 3D reconstructed human cornea model MCTT HCE™.

Evaluation of the eye irritation is essential in the development of new cosmetic products. Draize rabbit eye irritation test has been widely used in which chemicals are directly applied to rabbit eye, and the symptoms and signs of eyes are scored. However, due to the invasive procedure, it causes substantial pain and discomfort to animals. Recently, we reported in vitro eye irritation test method using a 3D human corneal epithelial model (MCTT HCE™) which is reconstructed from remaining human tissues after a corneal transplantation. This model exhibited an excellent predictive capacity for 25 reference chemicals (sensitivity 100%, specificity 77% and accuracy 88% vs. GHS). To improve the test performance, we explored new biomarkers for the eye irritation through transcriptomic approach. Three surfactants were selected as model eye irritants that include sodium lauryl sulfate, benzalkonium chloride and triton X-100. After test chemicals were treated, we investigated differentially expressed genes through a whole-gene microarray (Affymetrix GeneChip(®) Human Gene 2.0 ST Array, 48,000 probes). As a result, we identified that mRNAs of cornifelin (CNFN), a constituent of the insoluble cornified cell envelope of stratified squamous epithelia, and early growth response-1 (EGR1), a nuclear transcriptional regulator, were significantly up-regulated by all three irritants. Up-regulation of CNFN and EGR1 was further confirmed by Q-RT-PCR, and immunohistochemistry revealed increased level of CNFN in irritant-treated tissues, supporting the relevance of CNFN and EGR1 as new biomarkers for eye irritation.

Are coffee silverskin extracts safe for topical use? An in vitro and in vivo approach

Recent changes in regulatory requirements and social views on animal testing have incremented the development of reliable alternative tests for predicting skin and ocular irritation potential of products based on new raw materials. In this regard, botanical ingredients used in cosmetic products are among those materials, and should be carefully reviewed concerning the potential presence of irritant constituents. In particular, cosmetic products used on the face, in vicinity of the eyes or that may come in contact with mucous membranes, should avoid botanical ingredients that contain, or are suspected to contain, such ingredients. In this study, we aimed to evaluate the effect of a new cosmetic ingredient, namely, coffee silverskin (CS), with an in vitro skin and ocular irritation assay using reconstructed human epidermis, EpiSkin™, and human corneal epithelial model, SkinEthics™ HCE, and an in vivo assay. Three different extracts of CS were evaluated. The histology of the models after extracts applications was analysed. The in vitro results demonstrated that extracts were not classified as irritant and the histological analyses proved that extracts did not affect both models structure. The content of caffeine, 5-hydroxymethyl furfural and chlorogenic acid was quantified after the epidermal assay. The in vivo test carried out with the most promising extract (hydroalcoholic) showed that, with respect to irritant effects, these extracts can be regarded as safe for topical application.

Morphological Characterization of a Reconstructed Human Corneal Epithelial Model (LabCyte CORNEA-MODEL) as an Alternative to the Draize Eye Test for the Assessment of Eye Irritation

Morphological Characterization of a Reconstructed Human Corneal Epithelial Model (LabCyte CORNEA-MODEL) as an Alternative to the Draize Eye Test for the Assessment of Eye Irritation

Charakterisierung der Tränenfilmdynamik am Beispiel einer Trehaloseapplikation zur Behandlung des Trockenen Auges

The implementation of additional modalities for tear film break-up time characterisation expands the application range of the Oculus® Keratograph 5M. The aim of the present study was to evaluate the possibilities for non-invasive break-up time analysis using this device. Furthermore we applied the Oculus® Keratograph 5M to characterise possible modifications of tear break-up time after application of Thealoz® eye drops (Théa Pharma). The device allowed for a precise and solid topographical analysis of tear film dynamics. We could show that at four weeks after treatment, trehalose solution was a better treatment for dry eye compared with saline. These results are in agreement with our previous in vitro findings concerning the protective role of trehalose on desiccation-caused cell death in a corneal epithelial model.

Merkel-like cells in the murine conjunctival stroma

Merkel cells, discovered by Friedrich Sigmund Merkel in 1875, are found in multiple regions of the skin and some mucosa and make contact with specialized nerve fibers, participating in the perception of touch. However, Merkel cells have thus far not been described on the ocular surface. The purpose of this study was to investigate the distribution and ultrastructure of Merkel cells on the ocular surface and study changes in their number and ultrastructure after corneal epithelial wounding. Entire mouse ocular surfaces were obtained and stained by antibodies and tracking dye on fixed whole-mount specimens. The distribution and ultrastructure of specific fluorescence-positive cells were analyzed using deconvolution microscopy and transmission electron microscopy (TEM), respectively. The corneal epithelial wound-healing model was employed to observe the ultrastructural changes of these CK8-positive cells. We found that CK8-positive cells and FM1-43-positive cells were mainly located in the stromal layer, but not in the epithelial basal layer, of the forniceal conjunctiva. Our TEM results indicate that these cells possess the unique characteristic structures of Merkel cells, including electron-dense membrane-surrounded granules and spine-like protrusions of variable lengths, and demonstrate the formation of Merkel cell-neurite complexes. After corneal epithelial wounding, these cells exhibited rapid cell shrinkage and nuclear lobulation. Thus, Merkel-like cells were found in the conjunctival stroma of the ocular surface and may play an important role in maintaining the normal physiological function of the ocular surface.

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test.

Vitrigel-EIT (eye irritancy test) methods provide a brief assay system for estimating the changes of barrier function and architecture after exposing test chemicals to a human corneal epithelium model

Vitrigel-EIT (eye irritancy test) methods provide a brief assay system for estimating the changes of barrier function and architecture after exposing test chemicals to a human corneal epithelium model

Vitrigel-Eye Irritancy Test Method Using HCE-T Cells

We previously reported that the time-dependent relative changes of transepithelial electrical resistance (TEER) after exposing four different chemicals to a human corneal epithelium (HCE) model were well correlated to the potential of ocular irritancy. Meanwhile, we recently developed a collagen vitrigel membrane (CVM) chamber possessing a scaffold composed of high-density collagen fibrils equivalent to connective tissues in vivo as a three-dimensional culture tool. The CVM chamber is useful for biomedical assays and immunohistology using cryosections that are inappropriate to be performed using the conventional Millicell chamber with a polyethylene terephthalate membrane. In this study, we aimed to develop a new eye irritancy test (EIT) method called "Vitrigel-EIT method" that can facilitate to briefly and accurately estimate the widespread irritancy of test chemicals by applying the TEER assay system to a HCE model fabricated in the CVM chamber. HCE-T cells (a HCE-derived cell strain) were cultured in the CVM chamber for 6 days, and consequently, the Vitrigel-HCE model possessing the following characteristics of HCE in vivo was formed: six cell layers with specific protein expressions and their barrier function. Time-dependent profiles of TEER values after exposing 30 test chemicals to the HCE model were converted into the scores of three indexes (time lag, intensity, and plateau level), and each chemical was successfully classified into irritant or nonirritant category by utilizing the criteria for the indexes, resulting in the excellent correlation with Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification (sensitivity: 100%, specificity: 75%, accuracy: 90%). These data suggest that the widespread eye irritancy of chemicals can be predicted without false negatives by the Vitrigel-EIT method. Interestingly, the disruption of tight junctions was immunohistologically observed after exposing not only irritants but also three compounds classified as nonirritant by GHS but found positive in our Vitrigel-EIT method confirming a possible mild irritant property.

Integrin alpha v beta 6 contributes to maintaining corneal epithelial barrier function

The corneal epithelial barrier dysfunction is associated with a number of disorders of the cornea. This study aims to investigate the role of integrin alpha v beta 6 (avb6) in maintaining the corneal epithelial barrier function. In this study, the association of avb6 and endosome/lysosome in the human corneal epithelial cell line, HCE cells, was observed with immunocytochemistry. The corneal epithelial barrier function was assessed with HCE monolayers in Transwells. The results showed that avb6 was observed attaching to endosomes in HCE cells. Knockdown of avb6 resulted in disturbing the fusion of endosome/lysosome. Tumor necrosis factor (TNF)-α could bind avb6 to form a complex that interfered with the fusion of endosome/lysosome in HCE cells. With the HCE monolayer as a corneal epithelial barrier model, the knockdown of avb6 or exposure to TNF-α markedly increased the epithelial barrier permeability to a macomolecular protein, ovalbumin, in Transwell system. After passing through the deficient epithelial barrier, the ovalbumin molecules still conserved the antigenicity. We conclude that the integrin avb6 plays an important role in the fusion of endosome/lysosome in corneal epithelial cells; inhibition of avb6 results in corneal epithelial barrier dysfunction.

In Vitro and In Vivo Comparative Toxicological Study of a New Preservative-Free Latanoprost Formulation

To compare in vitro, on the human reconstituted corneal epithelial SkinEthics model, and in vivo, using an acute rabbit toxicological model, the effects of a benzalkonium chloride (BAK)-preserved solution of latanoprost and a preservative-free (PF) latanoprost solution. In vitro, the three-dimensional (3D) reconstituted human corneal epithelia (HCE) were treated with PBS, BAK-latanoprost, PF-latanoprost, or 0.02% BAK for 24 hours followed or not followed by a 24 hour post incubation recovery period. Cellular viability was evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test at 24 hours and the apoptotic cells were counted using TUNEL labeling on frozen sections at 24 hours and 24 hours plus 24 hours. In vivo, rabbits received 50 μL of the same solutions, which were applied at 5 minute intervals a total of 15 times. Ocular surface toxicity was investigated using slit lamp biomicroscopy examination, conjunctival impression cytology (CIC), and corneal in vivo confocal microscopy (IVCM). Standard immunohistology also assessed inflammatory CD45-positive cells. In vitro, BAK-latanoprost and 0.02% BAK induced significant apoptosis in the apical layers that correlated with the significant decrease of cell viability as assessed by the MTT test. PF-latanoprost slightly decreased cell viability and few apoptotic cells were found in the superficial layers, without reaching statistical significance compared with PBS. In vivo, clinical observation and IVCM images showed the lowest ocular surface toxicity with PBS and PF-latanoprost, while BAK-latanoprost and BAK induced abnormal corneoconjunctival aspects. PF-latanoprost showed the lowest CIC score, close to the PBS score and induced fewer CD45-positive cells in both the limbus and the conjunctiva compared with BAK and latanoprost, as assessed by immunohistology. We confirm that rabbit corneoconjunctival surfaces presented better tolerance when treated with PF-latanoprost compared with the standard BAK-latanoprost preparation or the BAK solution.

Plastic Compressed Collagen Constructs for Ocular Cell Culture and Transplantation: A New and Improved Technique of Confined Fluid Loss

Purpose: Cultured limbal epithelial cell transplantation is a commonly used clinical treatment for ocular surface repair. We have previously shown that plastic compressed (PC) type I collagen constructs are a suitable substrate for human limbal epithelial cell (HLEC) culture for transplantation. For this process to achieve compliance with Good Manufacturing Practice, and therefore be suitable for therapeutic cell therapy manufacture, the original method required substantial modification.Methods: The compression method was changed from unconfined (highly variable reproducibility) to confined compression (CC) (highly reproducible manufacture) and we assessed whether this altered the physical characteristics of the substrate. We have measured transparency, assessed scanning electron microscope images of the surface and performed live/dead cell viability assays of cells within the constructs. HLECs were then cultured on the surface of both types of construct and the resulting cell phenotype characterized.Results: We have determined that the change in process does not alter the physical characteristics of the substrate. Furthermore, there is no change to the substrate’s ability to support HLEC culture and maintenance of a mixed population of stem and differentiated cells. Additionally, cells were able to form a confluent sheet and multilayer to produce an intact epithelium.Conclusions: This modification allows scaling up of the process in a well-plate format, which is essential for creation of multiple corneal epithelial models for in vitro testing. This improvement to the original plastic compression method also allows the process to be employed in custom-made cassettes, the design of which takes into consideration the manufacturing and regulatory requirements for delivery of a cell therapy.

Differential contribution of hypertonic electrolytes to corneal epithelial dysfunction

The surface of the eye is covered by a protective tear film consisting of an aqueous-mucus layer and a superficial lipid layer, which are vital for light refraction and protection of vision. The aqueous component of the tear film contains electrolytes and a large variety of proteins, peptides and glycoproteins secreted by the lacrimal and meibomian glands, and apical cells of the corneal and conjunctival epithelia (Ohashi, Y. et al., 2006; Ruiz-Ederra, J. et al., 2009). Maintenance of a precise osmotic gradient of electrolytes between the tear film and the ocular surface epithelia is of paramount importance in regulating cell function and homeostasis. An imbalance of electrolytes is a hallmark of many pathologies, including dry eye, a disease affecting between 6 and 43 million people in the United States alone (Gilbard, J.P., 1994; Pflugfelder, S.C., 2011).

Corneal Epithelium in Ocular Pharmacology

AbstractPurpose Corneal epithelium is the main barrier of ocular drug absorption form the tear fluid to the anterior chamber. The aim was to generate corneal epithelial cell culture model for drug permeability testing.Methods Human corneal epithelial cell line was used and cultured on filters to build the model. The model was characterised in terms of permeability, electrical resistance, intercellular space, and gene expression of drug transporters and 11,000 other genes. Human corneal epithelium and rabbit cornea were used as positive controls.Results The results indicate that the corneal epithelial model has similar morphology and permeability characteristics and intercellular space as the normal corneal epithelium. However, significant differences were seen in the gene expression patterns.Conclusion Even though tight corneal epithelial organotypic culture model can achieve some features of the corneal epithelium, it deviates from the in vivo counterpart substantially. Thus the model has only limited utility.

Corneal Complications in Streptozocin-Induced Type I Diabetic Rats

PURPOSE. This study seeks to characterize corneal functions and complications in a streptozocin (STZ)-induced rat model of type I diabetes mellitus (DM) and to understand the pathogenesis of diabetic keratopathy. METHODS. DM was induced via STZ injection in Sprague-Dawley rats. Body weight, length, and corneal size were measured and compared with the age-matched normal controls. Corneal morphology and histology were evaluated with slit lamp, digital confocal microscopy and hematoxylin and eosin staining. Tear secretion was measured with cotton threads, and corneal sensitivity was determined with an esthesiometer. Protein expression and distribution were assessed with Western blotting and immunohistochemistry. Wound healing was determined using an in vivo corneal epithelial debridement model. RESULTS. Compared with the normal control rats, STZ rats had reduced body weight, and body length, but minimally affected corneal size. No significant changes in ocular surface regularity, corneal thickness, and morphology were noted in diabetic corneas. STZ rats showed stronger Rose Bengal staining, decreased tear secretion, slightly attenuated sensitivity, less innervation, delayed epithelial wound healing, and impaired epidermal growth factor receptor signaling in their corneas. While the expression of adherens junction protein β-catenin, and tight junction proteins occludin and ZO-1 was unchanged, the formation of these junctions after wound closure was delayed. CONCLUSIONS. Pathogenesis of diabetic keratopathy involves multiple tissues and/or cell types and several events including reduced tear secretion, impaired innervation, weakened cell junction, and altered wound responses. These insights may prove useful for the clinical translation of evolving strategies for the management and treatment of diabetic corneal complications.

Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman’s membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5–6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air–liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test.

Biliverdin Rescues the HO-2 Null Mouse Phenotype of Unresolved Chronic Inflammation Following Corneal Epithelial Injury

PURPOSE. The heme oxygenase system (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory pathway based on its ability to modulate leukocyte migration and to inhibit the expression of inflammatory cytokines and proteins by its products biliverdin/bilirubin and carbon monoxide. Corneal injury in HO-2 null mice leads to impaired healing and chronic inflammatory complications, including ulceration and neovascularization. The authors examined whether topically administered biliverdin can counteract the effects of HO deficiency in a corneal epithelial injury model. METHODS. HO-2 null mice were treated with biliverdin 1 hour before epithelial injury and twice a day thereafter. Reepithelialization and neovascularization were assessed by fluorescein staining and vital microscopy, respectively, and were quantified by image analysis. Inflammation was quantified by histology and Gr-1-specific immunofluorescence, and oxidative stress was assessed by DHE fluorescence. RESULTS. Treatment with biliverdin accelerated wound closure, inhibited neovascularization and reduced epithelial defects. It also reduced inflammation, as evidenced by a reduction in the appearance of inflammatory cells and the expression levels of inflammatory and oxidant proteins, including KC and NOXs. CONCLUSIONS. The results clearly show that biliverdin, directly or through its metabolism to bilirubin by biliverdin reductase-the expression of which is increased after injury-rescues the aberrant inflammatory phenotype, further underscoring the importance of the HO system in the cornea for the execution of an ordered inflammatory and reparative response.

Analysis of BAK-preserved and unpreserved antiallergic eye drops on a 3D-reconstituted corneal epithelial model

Analysis of BAK-preserved and unpreserved antiallergic eye drops on a 3D-reconstituted corneal epithelial model

The role of electrical signals in murine corneal wound re‐epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.