Corneal Tissue Model Research Articles

304 (PB292): Effects of Nectin-4 targeted Bicycle Toxin Conjugate, zelenectide pevedotin (BT8009), and enfortumab vedotin on a human corneal tissue model

304 (PB292): Effects of Nectin-4 targeted Bicycle Toxin Conjugate, zelenectide pevedotin (BT8009), and enfortumab vedotin on a human corneal tissue model

Construction of Dome-Shaped 3D Corneal Epithelial Tissue Models Based on Eyeball-Shaped Gel Microspheres.

By overcoming interspecies differences and mimicking the in vivo microenvironment, three-dimensional (3D) in vitro corneal models have become a significant novel tool in contemporary ophthalmic disease research. However, existing 3D corneal models struggle to replicate the actual human corneal environment, especially the dome-shaped physiological structure with adjustable curvature. Addressing these challenges, this study introduces a straightforward method for fabricating collagen/chitosan-alginate eyeball-shaped gel microspheres with a Janus structure via a two-phase aqueous system, used subsequently to construct in vitro 3D corneal epithelial tissue models. By adjusting the diameter ratio of collagen/chitosan to alginate droplets, we can create eyeball-shaped gel microspheres with varying curvatures. Human corneal epithelial cells were seeded on the surfaces of these microspheres, leading to the formation of in vitro 3D corneal epithelial tissues characterized by dome-like multilayers and tight junctions. Additionally, the model demonstrated responsiveness to UVB exposure through the secretion of reactive oxygen species (ROS) and proinflammatory factors. Therefore, we believe that in vitro 3D corneal epithelial tissue models with dome-shaped structures hold significant potential for advancing ophthalmic research.

Enhancing Corneal Drug Penetration Using Penetratin for Ophthalmic Suspensions.

Eye drops, including solutions and suspensions, are essential dosage forms to treat ophthalmic diseases, with poorly water-soluble drugs typically formulated as ophthalmic suspensions. In addition to low bioavailability, suspensions exhibit limited efficacy, safety, and usability due to the presence of drug particles. Improving bioavailability can reduce the drug concentrations and the risk of problems associated with suspended drug particles. However, practical penetration enhancers capable of improving bioavailability remain elusive. Herein, we focused on penetratin (PNT), a cell-penetrating peptide (CPP) that promotes active cellular transport related to macromolecule uptake, such as micropinocytosis. According to the in vitro corneal uptake study using a reconstructed human corneal epithelial tissue model, LabCyte CORNEA-MODEL24, PNT enhanced the uptake of Fluoresbrite® YG carboxylate polystyrene microspheres without covalent binding. In an ex vivo porcine eye model, the addition of 10 µM PNT to rebamipide ophthalmic suspension markedly improved the corneal uptake of rebamipide; however, the addition of 100 µM PNT was ineffective due to potentially increased particle size by aggregation. This article provides basic information on the application of PNT as a penetration enhancer in ophthalmic suspensions, including the in vitro and ex vivo studies mentioned above, as well as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay and storage stability at different pH values.

Exploring Cell-Penetrating Peptides as Penetration Enhancers in Eye Drop Formulations Using a Reconstructed Human Corneal Epithelial Model.

Ocular tissues function as biological barriers that hinder drug delivery, depending on the target tissue and route of administration, and must be overcome to achieve the desired therapeutic effect. Penetration enhancers have long been investigated to improve corneal drug penetration via eye drop instillation; however, further development is warranted owing to potential safety concerns. In the present study, we focused on cell-penetrating peptides (CPPs) as a penetration enhancer to address the requirements and explored CPP candidates suitable for corneal drug delivery. Using a reconstructed human corneal epithelial tissue model, LabCyte CORNEA-MODEL24 as an alternative to animal testing that is expected to have higher reproducibility than extracted eyeballs and octa-arginine (R8) as a representative model CPP with simple structure, we investigated the enhancement of 6-carboxyfluorescein (6-FAM) uptake by fluorescence imaging and the potential of eye irritation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Also, surface plasmon resonance (SPR) evaluated the interaction between R8 and model compounds, suggesting that the stronger interaction could facilitate the corneal uptake of compounds. A comparative screening study of corneal uptake using various CPPs showed that the CPPs other than R8 also have the potential to enhance the corneal uptake of 6-FAM. In particular, penetratin (PNT) showed stronger fluorescence intensity. Through these findings, this manuscript provides beneficial information for the development of a novel corneal penetration enhancer with CPPs. In the future, it is expected that the basic findings with R8 will be verified to be applicable to other CPPs for development as penetration enhancers for eye drop formulation.

P12-52 Evaluating ocular side effects of systemic medications utilizing the in vitro reconstructed human corneal epithelial tissue model

P12-52 Evaluating ocular side effects of systemic medications utilizing the in vitro reconstructed human corneal epithelial tissue model

Pharmacological evaluation of innovative eye drop formulations containing TS-polysaccaride, hyaluronic acid and glycyrrhizin for irritative ocular diseases using in vitro reconstituted human corneal epithelium model

In vitro reconstructed human corneal tissue models are closer to in vivo human corneal tissue in term of morphology, biochemical and physiological properties, and represent a valid alternative to animal use for evaluating the pharmacological effects ophthalmic topically applied medical devices. In this experimental work the in vitro reconstructed human corneal tissues have been used for assessing the potential beneficial effects of an innovative ophthalmic formulation containing hyaluronic acid, glycyrrhizin and TS-polysaccharide for the treatment of symptomatic states on the eye surface including dry eye, itching, foreign body sensation and redness due allergic reaction. Corneal tissues have been treated with benzalkonium chloride for 24 h to induce cell damage and then treated with the tested items for 16 h. After the incubation period, tissue viability, TNF-α, IL-6 and MMP-9 have been assessed. Diclofenac has been used as reference anti-inflammatory drug. The novel formulation protected the tissues against benzalkonium chloride damage, while exerted a mild but not significant reduction of the anti-inflammatory mediator TNF-α.

In vitro reconstructed 3D corneal tissue models for ocular toxicology and ophthalmic drug development.

Testing of all manufactured products and their ingredients for eye irritation is a regulatory requirement. In the last two decades, the development of alternatives to the in vivo Draize eye irritation test method has substantially advanced due to the improvements in primary cell isolation, cell culture techniques, and media, which have led to improved in vitro corneal tissue models and test methods. Most in vitro models for ocular toxicology attempt to reproduce the corneal epithelial tissue which consists of 4-5 layers of non-keratinized corneal epithelial cells that form tight junctions, thereby limiting the penetration of chemicals, xenobiotics, and pharmaceuticals. Also, significant efforts have been directed toward the development of more complex three-dimensional (3D) equivalents to study wound healing, drug permeation, and bioavailability. This review focuses on in vitro reconstructed 3D corneal tissue models and their utilization in ocular toxicology as well as their application to pharmacology and ophthalmic research. Current human 3D corneal epithelial cell culture models have replaced in vivo animal eye irritation tests for many applications, and substantial validation efforts are in progress to verify and approve alternative eye irritation tests for widespread use. The validation of drug absorption models and further development of models and test methods for many ophthalmic and ocular disease applications is required.

Induction of Irritation and Inflammation in a 3D Innervated Tissue Model of the Human Cornea.

Detection of slight changes in the chemical, thermal, and physical environments of the ocular surface is necessary to protect eyesight. The cornea, as the most densely innervated peripheral tissue in the body, can be damaged as a result of caustic chemical exposure. Such damage can be painful and debilitating, thus underscoring the need to understand mechanisms of ocular irritation. Both ethical and translational limitations regarding the use of animal subjects in part drive the need to develop relevant in vitro cell and tissue models that emulate the physiology of the human cornea. In this study, we utilized our 3D in vitro cornea-like tissue model to study the effects of irritation mediated by transient receptor potential (TRP) channels vanilloid 1 and ankyrin 1 (TRPV1; TRPA1) in response to allyl isothiocyanate (AITC) stimulation. Changes in gene expression were analyzed to characterize wound healing responses of the epithelial, stromal, and neuronal cell populations in the corneal tissue models. Key findings of the study include indications of wound healing, such as stromal myofibroblast differentiation and epithelial barrier re-establishment, amplification of pro-inflammatory cytokines, and downstream ECM protein remodeling due to irritation with the addition of sensory innervation. This study further establishes this in vitro tissue model as a useful tool for studying corneal irritation in vitro in a holistic manner with promise as a novel and sensitive tool for studying chemical exposures and subsequent responses.

A core-skirt designed artificial cornea with orthogonal microfiber grid scaffold

Artificial cornea is an effective treatment option for cases of severe corneal loss. In this study, we prepared a core-skirt designed artificial cornea with orthogonal microfiber grid scaffold. We fabricated PCL orthogonal microfiber grid scaffolds by a direct writing technique, and then combined them with compressed collagen (CC) to obtain a sandwich-like CC/P (where P is used to represent the PCL microfiber grid scaffold). PHEMA hydrogel and the CC/P served as the core and the skirt, respectively, with the P also serving as an intermediate between the two. The physical properties of the artificial cornea, including the morphology, the mechanical properties and the light transmittance, were evaluated. SEM images showed an effective connection and a lack of phase separation at the interface between the core and the skirt, and the skirt formed a highly porous scaffold that promoted tissue biointegration. In addition, we used the skirt structure to construct a corneal tissue model containing two cells types: corneal stromal stem cells (CSSCs) and mouse hippocampal neurons. The results showed that the cells could grow and differentiate well, and the orthogonal microfiber grid scaffold fibers were good guides for the structural growth of CSSCs and neuronal axons.

Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model

The purpose of the current work was to utilize a three dimensional (3D) corneal epithelial tissue model to study dry eye disease and oxidative stress-related corneal epithelial injuries for the advancement of ocular therapeutics. Air-liquid interface cultures of normal human corneal epithelial cells were used to produce 3D corneal epithelial tissues appropriate for physiologically relevant exposure to environmental factors. Oxidative stress was generated by exposing the tissues to non-toxic doses of ultraviolet radiation (UV), hydrogen peroxide, vesicating agent nitrogen mustard, or desiccating conditions that stimulated morphological, cellular, and molecular changes relevant to dry eye disease. Corneal specific responses, including barrier function, tissue viability, reactive oxygen species (ROS) accumulation, lipid peroxidation, cytokine release, histology, and gene expression were evaluated. 3D corneal epithelial tissue model structurally and functionally reproduced key features of molecular responses of various types of oxidative stress-induced ocular damage. The most pronounced effects for different treatments were: UV irradiation - intracellular ROS accumulation; hydrogen peroxide exposure - barrier impairment and IL-8 release; nitrogen mustard exposure - lipid peroxidation and IL-8 release; desiccating conditions - tissue thinning, a decline in mucin expression, increased lipid peroxidation and IL-8 release. Utilizing a PCR gene array, we compared the effects of corneal epithelial damage on the expression of 84 oxidative stress-responsive genes and found specific molecular responses for each type of damage. The topical application of lubricant eye drops improved tissue morphology while decreasing lipid peroxidation and IL-8 release from tissues incubated at desiccating conditions. This model is anticipated to be a valuable tool to study molecular mechanisms of corneal epithelial damage and aid in the development of therapies against dry eye disease, oxidative stress- and vesicant-induced ocular injuries.

Assembly and Application of a Three-Dimensional Human Corneal Tissue Model.

The cornea provides a functional barrier separating the outside environment from the intraocular environment, thereby protecting posterior segments of the eye from infection and damage. Pathological changes that compromise the structure or integrity of the cornea may occur as a result of injury or disease and can lead to debilitating effects on visual acuity. Over 10 million people worldwide are visually impaired or blind due to corneal opacity. Thus, physiologically relevant in vitro approaches to predict corneal toxicity of chemicals or effective treatments for disease prior to ocular exposure, as well as to study the corneal effects of systemic, chronic conditions, such as diabetes, are needed to reduce use of animal testing and accelerate therapeutic development. We have previously bioengineered an innervated corneal tissue model using silk protein scaffolds to recapitulate the structural and mechanical elements of the anterior cornea and to model the functional aspects of corneal sensation with the inclusion of epithelial, stromal, and neural components. The purpose of this unit is to provide a step-by-step guide for preparation, assembly, and application of this three-dimensional corneal tissue system to enable the study of corneal tissue biology. © 2019 by John Wiley & Sons, Inc.

Characterization of drug metabolizing enzymes, transporters and permeation in a human organotypic corneal tissue model

Characterization of drug metabolizing enzymes, transporters and permeation in a human organotypic corneal tissue model

Human Corneal Tissue Model for Nociceptive Assessments.

New in vitro tissue models to mimic in vivo conditions are needed to provide insight into mechanisms involved in peripheral pain responses, potential therapeutic strategies to address these responses, and to replace animal models for such indications. For example, the rabbit cornea Draize test has become the standard method used for decades to screen ophthalmic drug and consumer product toxicity. In vitro tissue models with functional innervation have the potential to replace in vivo animal testing and provide sophisticated bench tools to study ocular nociception and its amelioration. Herein, full thickness, innervated, 3D human corneal tissues are grown under physiologically relevant culture conditions to study nociceptive-related responses, by mimicking ocular environmental cues, including intraocular pressure (IOP) and tear flow (TF). Capsaicin, a chili pepper-derived irritant known to cause a burning sensation in mammalian tissues is utilized as a nociceptive stimulant to induce pain, while subsequent serum treatment is used to mimic healing. Pain mediators released upon capsaicin stimulation and cell regrowth after serum treatment are characterized to assess ocular responses in this new, innervated, human corneal tissue system for comparison of outcomes to established animal and related responses.

New Human Organotypic Corneal Tissue Model for Ophthalmic Drug Delivery Studies.

The purpose of the current work was to develop a physiologically relevant, in vitro human three-dimensional (3D) corneal epithelial tissue model for use in ophthalmic drug development. Normal human corneal epithelial cells were cultured at the air-liquid interface to produce the 3D corneal tissue model. Corneal barrier was determined by measuring transepithelial electrical resistance (TEER). Quantitative PCR arrays were utilized to investigate expression of 84 phase I/II metabolizing enzymes and 84 drug transporter genes. Permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients. Finally, different formulations of latanoprost and bimatoprost were administered and drug absorption and tissue viability and integrity were investigated. Histologic assessment and TEER of the corneal tissue model revealed tissue structure, thickness, and barrier formation (1000 ± 146 Ω·cm2) comparable to native human corneal epithelium. The 3D corneal tissue expressed tight junctions, mucins, and key corneal epithelial detoxification enzymes. Drug-metabolizing enzyme and transporter gene expression in 3D corneal tissue and excised human corneal epithelium were highly correlated (r2 = 0.87). Coefficients of permeation for model drugs in the tissue model and excised rabbit corneas also showed a high correlation (r2 = 0.94). As expected, latanoprost and bimatoprost free acids had much lower permeability (Papp = 1.2 × 10-6 and 1.9 × 10-6) than the corresponding prodrugs (Papp = 2.5 × 10-5 and 5.6 × 10-5), respectively. The presence of 0.02% benzalkonium chloride in ophthalmic formulations significantly affected tissue barrier and viability. The newly developed 3D corneal tissue model appears to be very useful for evaluation of corneal drug permeability and safety during ophthalmic drug development.

Function-Related Protein Expression in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Models

Fuchs endothelial corneal dystrophy (FECD) is a corneal pathology that affects the endothelial cell's ability to maintain deturgescence, resulting in a progressive loss of corneal transparency. In this study, we investigated the expression of function-related proteins in corneal endothelial cells using FECD or healthy corneal endothelial cells, either in a cell culture two-dimensional model or in an engineered corneal endothelium three-dimensional tissue model. No statistically significant difference in gene regulation was observed for the function-related families ATP1, SLC4, SLC16, AQP, TJP, and CDH between the FECD and the healthy cell models. Similarly, no difference in barrier integrity (transendothelial electrical resistance measurements and permeability assays) was observed invitro between FECD and healthy cultured cells. Protein expression of the key function-related families was decreased for Na+/K+-ATPase α1 subunit, monocarboxylate transporters 1 and 4 in native exvivo end-stage FECD specimens, whereas it returned to levels comparable to that of healthy tissues in the engineered FECD model. These results indicate that cell expansion and tissue engineering culture conditions can generate a corneal endothelium from pathologic FECD cells, with levels of function-related proteins similar to that of healthy tissues. Overall, these results explain why it is possible to reform a functional endothelium using corneal endothelial cells isolated from nonfunctional FECD pathologic specimens.

Extracellular Matrix and Integrin Expression Profiles in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Model

Primary corneal endothelial cell (CEC) cultures and 3D-engineered tissue models were used to study the aberrant deposition of extracellular matrix (ECM) in a vision impairing pathology known as Fuchs endothelial corneal dystrophy (FECD). CECs were isolated from excised Descemet membranes of patients with end-stage FECD. CECs isolated from healthy corneas served as controls. Microarray gene profiling was performed on postconfluent cultures of healthy and FECD cells. Protein expression analyses were conducted on tissue models that were engineered by seeding an endothelium on previously devitalized human stromal carriers. The engineered endothelia were kept in culture for 1–3 weeks to reform the endothelial monolayer. Protein expression of integrin subunits α4, α6, αv, and β1, as well as laminin, type IV collagen, fibronectin, clusterin, and transforming growth factor β-induced protein (TGFβIp) was then assessed by immunofluorescence. Microarray analysis showed nonstatistical twofold downregulation of collagen-coding genes (COL4A4, COL8A2, and COL21A1) and a twofold upregulation of the COL6A1, laminin α3 gene LAMA3, and integrin subunit α10 gene ITGA10 in FECD cells. Fibronectin type III domain containing 4 (FNDC4) and integrin β5 (ITGB5) genes was significantly upregulated in FECD cells. Immunostainings demonstrated that the protein expression of the integrin subunits α4, α6, αv, and β1, type IV collagen, as well as laminin remained similar between native and engineered endothelia. TGFβIp expression was found on the stromal side of both FECD and healthy Descemet's membrane, and only one out of three FECD specimens was positive for the clusterin protein. Interestingly, the ECM protein fibronectin was also found to have a stronger presence on engineered FECD tissues, a result consistent with the native FECD specimens. To conclude, this study allowed to identify fibronectin deposition as one of the first steps in the pathogenesis of FECD, as defined by our engineered tissue model. This opens the way to an entirely new perspective for in vitro pharmacological testing of new therapies for FECD, the leading indication for corneal transplantation in North America.

Semifluorinated alkane based systems for enhanced corneal penetration of poorly soluble drugs

Semifluorinated alkanes (SFAs) are amphiphilic liquids that can dissolve hydrophobic drugs to form clear solutions. This study evaluated the potential of two SFAs to act as vehicle for topical ocular drug delivery. After confirming ocular safety, an ex vivo corneal penetration model was developed to determine drug distribution and corneal bioavailability. Hydrophobic dye distribution in the different corneal layers was visualised under a confocal microscope. Corneal bioavailability of cyclosporine A (CsA) dissolved in perfluorobutylpentane (F4H5) or perfluorohexyloctane (F6H8) was compared to commercially available CsA ophthalmic emulsions, Restasis® and Ikervis®. Precorneal residence of the four test vehicles containing the hydrophobic dye was also compared using an ex vivo corneal tissue model. Preferential accumulation of the hydrophobic dye in the corneal epithelium was observed with higher amounts detectable when delivered via the SFAs compared to Restasis or Ikervis. A significant improvement in corneal CsA penetration was observed after application of a single dose of 0.05% CsA in F4H5 and F6H8 when compared to Restasis with the area under curve over 4 h (AUC(0–4h)) being at least 8-fold greater for both SFAs (p < .0001). Moreover, the AUC(0–4h) of 0.1% CsA in F4H5 was almost 5-fold greater than Ikervis (p < .0001). Finally, the precorneal residence time of both SFA solutions was significantly longer than that of the commercial emulsions with the AUC(0–60min) being 2- to 11-fold greater. This study demonstrated that SFAs can significantly improve the local bioavailability of hydrophobic drugs by increasing corneal penetration as well as prolonging precorneal residence. They therefore offer a promising new platform for topical drug delivery to the eye.

In vitro reconstructed human corneal tissue model: Applications to corneal oxidative stress, dry eye, and ophthalmic drug delivery

In vitro reconstructed human corneal tissue model: Applications to corneal oxidative stress, dry eye, and ophthalmic drug delivery

Diabetic complications in the cornea

Diabetic corneal alterations, such as delayed epithelial wound healing, edema, recurrent erosions, neuropathy/loss of sensitivity, and tear film changes are frequent but underdiagnosed complications of both type 1 (insulin-dependent) and type 2 (non-insulin-dependent) diabetes mellitus. The disease affects corneal epithelium, corneal nerves, tear film, and to a lesser extent, endothelium, and also conjunctiva. These abnormalities may appear or become exacerbated following trauma, as well as various surgeries including retinal, cataract or refractive. The focus of the review is on mechanisms of diabetic corneal abnormalities, available animal, tissue and organ culture models, and emerging treatments. Changes of basement membrane structure and wound healing rates, the role of various proteinases, advanced glycation end products (AGEs), abnormal growth and motility factors (including opioid, epidermal, and hepatocyte growth factors) are analyzed. Experimental therapeutics under development, including topical naltrexone, insulin, inhibitors of aldose reductase, and AGEs, as well as emerging gene and cell therapies are discussed in detail.

Invitro 3D corneal tissue model with epithelium, stroma, and innervation.

The interactions between corneal nerve, epithelium, and stroma are essential for maintaining a healthy cornea. Thus, corneal tissue models that more fully mimic the anatomy, mechanical properties and cellular components of corneal tissue would provide useful systems to study cellular interactions, corneal diseases and provide options for improved drug screening. Here a corneal tissue model was constructed to include the stroma, epithelium, and innervation. Thin silk protein film stacks served as the scaffolding to support the corneal epithelial and stromal layers, while a surrounding silk porous sponge supported neuronal growth. The neurons innervated the stromal and epithelial layers and improved function and viability of the tissues. An air-liquid interface environment of the corneal tissue was also mimicked invitro, resulting in a positive impact on epithelial maturity. The inclusion of three cell types in co-culture at an air-liquid interface provides an important advance for the field of invitro corneal tissue engineering, to permit improvements in the study of innervation and corneal tissue development, corneal disease, and tissue responses to environmental factors.