Corneal Epithelial Cell Monolayers Research Articles

Cannabidiol-Loaded Mixed Polymeric Micelles of Chitosan/Poly(Vinyl Alcohol) and Poly(Methyl Methacrylate) for Trans-Corneal Delivery.

Ocular drug delivery is challenging due to the very short drug residence time and low permeability. In this work, we produce and characterize mucoadhesive mixed polymeric micelles (PMs) made of chitosan (CS) and poly(vinyl alcohol) backbones graft-hydrophobized with short poly(methyl methacrylate) blocks and use them to encapsulate cannabidiol (CBD), an anti-inflammatory cannabinoid. CBD-loaded mixed PMs are physically stabilized by ionotropic crosslinking of the CS domains with sodium tripolyphoshate and spray-drying. These mixed PMs display CBD loading capacity of 20% w/w and sizes of 100–200 nm, and spherical morphology (cryogenic-transmission electron microscopy). The good compatibility of the unloaded and CBD-loaded PMs is assessed in a human corneal epithelial cell line. Then, we confirm the permeability of CBD-free PMs and nanoencapsulated CBD in human corneal epithelial cell monolayers under liquid–liquid and air–liquid conditions. Overall, our results highlight the potential of these polymeric nanocarriers for ocular drug delivery.

Tuning corneal epithelial cell adhesive strength with varying crosslinker content in silicone hydrogel materials.

PurposeTo quantify the effect of silicone hydrogel crosslink density on the adhesion at corneal epithelial cells/silicone hydrogel contact lens interface.MethodsA custom-built rheometer, referred to as the live cell monolayer rheometer, was used to measure the adhesive strengths between corneal epithelial cell monolayers and silicone hydrogel lens surfaces. The resulting stress relaxations of senofilcon A–derived silicone hydrogel materials with varying crosslinking densities and delefilcon A were tested. Senofilcon A–like materials labeled L1, L2, L3, L4, and L5 contained crosslinker concentrations of 1.2, 1.35, 1.5, 1.65, and 1.8 wt%, respectively. The residual modulus measured from the live cell monolayer rheometer provided a direct indication of adhesive attachment.ResultsWithin the senofilcon-derived series, the adhesive strength shows a surprising minimum with respect to crosslink density. Specifically, L1 (1.20%) has the highest adhesive strength of 39.5 ± 11.2 Pa. The adhesive strength diminishes to a minimum of 11.2 ± 2.1 Pa for L3, whereafter it increases to 14.5 ± 2.5 Pa and 18.1 ± 5.1 Pa for L4 and L5, respectively. The delefilcon A lens exhibits a comparable adhesive strength of 27.8 ± 6.3 Pa to L1.ConclusionsThese results demonstrated that increasing the crosslink density has a nonmonotonic influence on the adherence of lenses to mucin-expressing corneal epithelial cells, which suggests a competition mechanism at the cell/lens interface.Translational RelevanceBecause the adhesiveness of contact lenses to ocular tissues may impact the comfort level for lens wearers and affect ease of removal, this study suggests that lens adhesion can be optimized through the control of crosslink density.

Biocompatibility of Nanocellulose-Reinforced PVA Hydrogel with Human Corneal Epithelial Cells for Ophthalmic Applications.

Transparent composite hydrogel in the form of a contact lens made from poly(vinyl alcohol) (PVA) and cellulose nanocrystals (CNCs) was subjected to in vitro biocompatibility evaluation with human corneal epithelial cells (HCE-2 cells). The cell response to direct contact with the hydrogels was investigated by placing the samples on top of confluent cell layers and evaluating cell viability, morphology, and cell layer integrity subsequent to 24 h culture and removal of the hydrogels. To further characterize the lens–cell interactions, HCE-2 cells were seeded on the hydrogels, with and without simulated tear fluid (STF) pre-conditioning, and cell viability and morphology were evaluated. Furthermore, protein adsorption on the hydrogel surface was investigated by incubating the materials with STF, followed by protein elution and quantification. The hydrogel material was found to have affinity towards protein adsorption, most probably due to the interactions between the positively charged lysozyme and the negatively charged CNCs embedded in the PVA matrix. The direct contact experiment demonstrated that the physical presence of the lenses did not affect corneal epithelial cell monolayers in terms of integrity nor cell metabolic activity. Moreover, it was found that viable corneal cells adhered to the hydrogel, showing the typical morphology of epithelial cells and that such response was not influenced by the STF pre-conditioning of the hydrogel surface. The results of the study confirm that PVA-CNC hydrogel is a promising ophthalmic biomaterial, motivating future in vitro and in vivo biocompatibility studies.

Infection and Replication of Influenza Virus at the Ocular Surface.

Although influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subjected to aerosol inoculation. The temperature at which cells were cultured postinoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to that at 37°C, and this effect was slightly greater for the conjunctivitis isolates than for the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multilayer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited the hemagglutination of both ocular and nonocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of nonocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells but that this is more likely for ocular-tropic viruses, as they are better able to maintain their infectivity during passage through the tear film.IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza virus tropism have yet to be fully elucidated. Here, we sought to identify factors that limit the ability of most influenza viruses to cause ocular infection. Although ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors that influence infection and replication in this noncanonical site may point toward novel determinants of tropism in the respiratory tract.

Corneal cell adhesion to contact lens hydrogel materials enhanced via tear film protein deposition.

Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS), borate buffered saline (BBS), or Sensitive Eyes Plus Saline Solution (Sensitive Eyes), either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes) exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.

Corneal Wound Healing, a Newly Identified Function of CAP37, Is Mediated by Protein Kinase C Delta (PKCδ)

The neutrophil-derived granular protein, CAP37, an innate immune system molecule, has antibiotic and immunomodulatory effects on host cells, including corneal epithelial cells. We previously showed that CAP37 modulates corneal epithelial cell migration, adhesion, and proliferation, and that protein kinase C delta (PKCδ) mediates CAP37-induced chemotaxis of these cells. The objective of this study was to investigate the hypothesis that CAP37 facilitates corneal wound healing through the PKC signaling pathway. The standard "scratch" assay performed on monolayers of corneal epithelial cells was used to measure the in vitro effect of CAP37 on wound closure. In vivo wound healing in response to CAP37 was measured using a mouse corneal epithelium abrasion model. In vitro and in vivo wound closure were monitored over 48 hours. The PKCδ was visualized during wound closure in cell monolayers and corneal epithelium by immunohistochemistry. The importance of PKCδ in CAP37-induced corneal wound healing was assessed by siRNA. We found that CAP37 accelerated wound closure in vitro and in vivo. Maximal closure occurred with concentrations of CAP37 between 250 and 500 ng/mL. Topical applications on mouse cornea led to re-epithelialization of the cornea by 24 hours. Immunohistochemistry of in vitro and in vivo wounds revealed a local increase of PKCδ along the wound edge in CAP37-treated cell monolayers and corneas, compared to untreated controls. CAP37-induced corneal wound healing was significantly reduced in vivo upon treatment with PKCδ siRNA. These findings support the hypothesis that CAP37 facilitates corneal wound healing through the activation of PKCδ.

<em>Ex Vivo</em> Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1. Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

<em>Ex Vivo</em> Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity. Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Effect of a novel multipurpose contact lens solution on human corneal epithelial barrier function

Purpose To explore the effect of a novel multipurpose contact lens solution (MPS) on the junction protein distribution and barrier function of cultured human corneal epithelial cell monolayers. Methods Cultured human corneal epithelial cells (HCEpiC) were exposed to a novel MPS (MPS A; Biotrue™, Bausch & Lomb) at 50%, 75% and 100% for 10 or 30 min. Four commercially available MPS products, MPS B (AQuify, Ciba Vision), MPS C (COMPLETE MPS Easy Rub, AMO), MPS D (OPTI-FREE Express, Alcon) and MPS E (OPTI-FREE RepleniSH, Alcon) were tested in parallel. Tight junction structure and integrity were evaluated by confocal microscopy using ZO-1 antibody and scanning microscopy (SEM). Quantitative evaluation of MPSs on epithelial barrier function was determined by measuring transepithelial electrical resistance (TEER) across HCEpiC grown on Transwell Clear permeable supports and on electric cell-substrate impedance sensing (ECIS) electrode arrays. Results Overall after exposure to the three concentrations (50%, 75%, and 100%) of MPS A, ZO-1 distribution and fluorescent intensity on the cell surface appeared similar to the media control with continuous tight junctions and clear intercellular junctions. At all measured time points after exposure to MPS A (50% or 75%) there was also no effect on the TEER using both resistance methodologies, and SEM showed that MPS A appeared similar to the Hank's balanced salt solution (HBSS) control. In cells exposed to MPS D there was a dose-dependent change in the distribution of ZO-1, some cell detachment, and a decrease in monolayer resistance at all time points measured. Ultrastructurally, MPS D caused gross changes, including damage to cell junctions and plasma membranes. To a lesser extent, the remaining three commercial MPS products demonstrated some effects on tight junction ZO-1 distribution and/or TEER. Conclusions Based on the in vitro measurements of tight junction protein expression, monolayer integrity, and transepithelial electrical resistance, the novel multipurpose contact lens solution (MPS A) did not alter corneal barrier function as compared to media, PBS or HBSS control. Clinical significance of the observed differences in epithelial barrier function among the MPSs tested needs further investigation.

NC-1059: A Channel-Forming Peptide That Modulates Drug Delivery across In Vitro Corneal Epithelium

The goal of this study was to determine whether a synthetic peptide, NC-1059, can modulate the corneal epithelium to increase the permeation of therapeutic agents across this barrier. An in vitro system employing transformed human corneal epithelial (THCE) cells was optimized for this study. Culture conditions were identified to promote formation of a confluent monolayer that rapidly develops a substantial transepithelial electrical resistance. Electrical parameters were measured with a modified Ussing flux chamber, and solute flux was quantified with fluorescently labeled compounds. NC-1059 causes a concentration-dependent increase in short-circuit current and an increase in transepithelial electrical conductance when assessed in modified Ussing chambers. The effect of NC-1059 on transepithelial electrical resistance was reversible. To test for paracellular permeability and size exclusion, FITC-labeled dextran ranging in size from 10 to 70 kDa was used. Dextran permeated the corneal cell monolayer in the presence, but not the absence, of NC-1059. Fluorescein sodium and carboxyfluorescein were then used as low molecular weight markers with similar NC-1059-modulated kinetics being observed. Maximum permeation for the fluorescein derivatives occurred 30 to 90 minutes after a 5-minute NC-1059 exposure. A prototypical drug, methotrexate, also exhibited increased permeation in the presence of NC-1059. NC-1059 enhances drug permeation across cultured corneal epithelial cell monolayers by transiently affecting the paracellular pathway. Thus, NC-1059 is a lead compound for development of cotherapeutic agents to enhance access and effectiveness of ophthalmic compounds.

Incorporation of cell-adhesion peptides into collagen scaffolds promotes corneal epithelial stratification

The objective of this study was to examine the effects of cell-adhesion peptides incorporated into collagen scaffolds on corneal epithelial cell stratification. Peptides (YIGSR, YIGSRIKVAV, IKVAVYIGSR and negative control YISGR) were first chemically attached to dendrimers. The peptide-modified dendrimers were then used as collagen cross-linkers. This permitted the incorporation of the peptides into the bulk structure of the collagen gels. The amount of peptide incorporated into the collagen gels was determined by 125I radiolabelling to be between 0.064 and 6.4 μg/mg collagen for YIGSR, and between 0.1187 to 11.87 μg/mg collagen for YIGSRIKVAV and IKVAVYIGSR. Corneal epithelial cell monolayers were grown on the surface of the collagen scaffolds and then exposed to conditions that promoted stratification as a stratified epithelial layer is desired in a tissue-engineered cornea. It was found that all of the incorporated peptides promoted stratification of the cells with the exception of the negative control YISGR. A synergistic effect of the combined sequences from laminin was observed, with the orientation of the peptide sequences having a great impact on the ability of the materials to promote cell stratification.

Mutation of retS, encoding a putative hybrid two-component regulatory protein in Pseudomonas aeruginosa, attenuates multiple virulence mechanisms

Two-component regulatory systems play an important role in bacterial virulence. We report that mutation of a Pseudomonas aeruginosa gene designated retS (previously designated fimK; accession number PA4856) encoding a putative hybrid two-component regulator, attenuates multiple virulence mechanisms. The retS mutant was selected from a Tn 5 transposon library of the cytotoxic P. aeruginosa strain PA103 based upon expression of a small-colony phenotype suggestive of reduced surface-associated “twitching” motility, a property dependent upon type IV pili. Subsequent analysis revealed that the mutant expressed pilin, albeit at lower levels than wild-type PA103. In a murine model of corneal infection, retS mutation was associated with delayed disease development and altered pathology. In vitro, retS mutants demonstrated loss of acute cytotoxic activity towards corneal epithelia as determined by trypan blue exclusion and by LDH release assays ( P < 0.0001). This coincided with loss of ExsA-regulated type III secretion. Mutation of retS also impaired ExsA-independent pathogenic mechanisms. When compared to the exsA mutant of PA103, retS mutants exhibited reduced epithelial adherence and invasion and reduced intracellular survival within the cells after invasion. Time-lapse video microscopy revealed that retS mutants, compared to exsA mutants, had a reduced capacity to access, and move along, the basal cell surfaces of corneal epithelial cell monolayers. Taken together, these data suggest that the protein encoded by retS regulates various properties of P. aeruginosa including both ExsA-dependent and ExsA-independent virulence mechanisms.

Ultraviolet Transmittance of Human Limbal Epithelial Cells Cultured on Human Amniotic Membranes

Purpose: To evaluate ultraviolet (UV) A and B transmittance by human limbal epithelial cells cultured on human amniotic membranes. Methods: Human limbal epithelial cells were taken from the limbus of donor corneas and were cultured on human amniotic membranes with inactivated 3T3 fibroblasts for 2 to 4 weeks. Then, the cultured cells were examined histologically. Next, cells from different culture periods were irradiated with UV-A (365 nm) or UV-B (302 nm) at energy levels ranging from 50 to 800 μ W/cm2, and UV transmittance was measured with a UV light meter. Results: Histological examination revealed a monolayer of corneal epithelial cells on the amniotic membrane after 2 weeks of culture, and a layer of 3–4 cells was formed after 4 weeks. Transmittance of UV-A and UV-B was highest by the amniotic membrane alone, followed in decreasing order by limbalepithelial cells cultured on amniotic membranes for 2 weeks, 3 weeks, and 4 weeks. Conclusions: These results indicate that UV absorbance increases in proportion to the number of limbal epithelial cell layers in cultures on amniotic membranes. Limbal epithelial cells may need to be cultured until 3–4 layers are formed in order to prevent ocular damage by UV light after transplantation.

Insulin, not leptin, promotes in vitro cell migration to heal monolayer wounds in human corneal epithelium.

To investigate the effects of insulin and leptin on in vitro wound healing of transformed human corneal epithelial cell monolayers and to identify cellular (migration versus proliferation) and intracellular signaling mechanisms. Scratch wounds were created in monolayers of an immortalized human corneal epithelial cell (HCEC) line. The wounded monolayers were exposed to insulin and leptin. Wound areas were measured every hour after wounding for up to 8 hours. Phosphoinositide 3-kinase (PI3-kinase) and mitogen-activated protein (MAP)-kinase signaling was analyzed with Western blot. The actions of insulin were also examined after incubation with inhibitors to extracellular signal regulated kinase (ERK 1/2) and PI3-kinase. The presence of insulin, but not leptin facilitated closure of wounds created in corneal epithelial cell monolayers. Phosphorylation of ERK 1/2 and Akt was stimulated after exposure of the monolayers to insulin. Inhibitors of PI3-kinase and ERK 1/2 prevented or reduced insulin-induced corneal wound healing, respectively. Exposure of corneal epithelium to insulin facilitated closure of in vitro small wounds through enhanced cell migration instead of proliferation, which depended on ERK 1/2 and PI3-kinase signaling. These data suggest a mechanism by which insulin may influence corneal wound healing in vitro. In vivo, disruptions to the insulin signaling pathway observed in diseases such as diabetes might account for the delayed wound healing and corneal defects.

Pathogenesis of corneal infection: binding of Pseudomonas aeruginosa to specific phospholipids.

Clinical isolates of Pseudomonas aeruginosa were examined for binding interactions with phospholipids of corneal epithelium. Thin-layer chromatography (TLC) of lipids extracted from corneal epithelia followed by staining with an ammonium molybdate spray reagent revealed three phospholipid components, PL1, PL2, and PL3. The chromatographic mobility of PL1 was similar to that of the phospholipid standards phosphatidylinositol (PI) and phosphatidylserine (PS), which were not well resolved from one other; PL2 and PL3 comigrated with the standards phosphatidylcholine and phosphatidylethanolamine, respectively. By use of a TLC-bacterial overlay procedure, 35S-labeled P. aeruginosa organisms were shown to bind to PL1 but not to PL2 or PL3. P. aeruginosa binding to PL1 was concentration dependent. Alkaline methanolysis abolished the binding. PL1 was separated into two components, PL1-I and PL1-S, by chromatography on borate-treated TLC plates. Both PL1-I and PL1-S contained binding sites for P. aeruginosa. Mass spectral analysis identified PL1-I and PL1-S as PI and PS, respectively. Radiolabeled P. aeruginosa organisms were subsequently shown to bind to commercially available bovine PI and PS and synthetic dipalmitoyl-PS but not to other phospholipid standards, including bovine SM and PC or synthetic dioleoyl- and distearoyl-PC. A control Escherichia coli strain did not bind to either PS or PI. Tetramethylurea, a disrupter of hydrophobic associations, did not influence the binding of P. aeruginosa to PS or PI. P. aeruginosa bound to the monolayers of corneal epithelial cells. P. aeruginosa binding to the monolayer cultures as well as to rabbit corneas pretreated with exogenous PS and PI was significantly higher than that to those preincubated with PC or medium alone. The data suggest that phospholipids PS and PI present in mucus or on the cell surface may function as P. aeruginosa receptors and contribute to selective bacterium-host interactions responsible for initial colonization.

An in vitro study of ophthalmic antiviral agent toxicity on rabbit corneal epithelium

Using an in vitro system we measured the corneal epithelial cytotoxicity and the antiviral activity of the antiviral agents idoxuridine (IDU), trifluridine (TFT), ethyldeoxyuridine (EDU), and ( E)-5-(2-Bromovinyl)-2′-deoxyuridne (BVDU). Confluent rabbit corneal epithelial cell cultures were established, and the antiviral agents were added for 5, 30, or 60 min at a range of concentrations including that used clinically (IDU 0.1%, TFT 1.0%, BVDU 0.1%, EDU 2.0%). Twelve hour [ 3H]thymidine incorporation then was measured and expressed as % inhibition of control cultures. In separate experiments confluent corneal epithelial cell monolayers were inoculated with 10 4 plaque forming units (PFU) of HSV type 1 (McKrae strain) for 1 h, and IDU 0.1%, TFT 1.0%, and BVDU 0.1% were added to the culture for determination of PFU inhibition. Significant dose-, but not time-dependent, toxicity was observed at the clinical concentrations of IDU, TFT, and EDU. Toxicity was absent for BVDU. TFT and IDU were the most toxic, and EDU was of intermediate toxicity. IDU, TFT, and BVDU showed significant antiviral activity in this corneal epithelial cell culture system (TFT > BVDU >IDU). The results of this in vitro study paralleled the findings of previous in vivo corneal epithelial toxicity studies of IDU, TFT, and BVDU. Our data, however, suggest that EDU has a potential for clinical toxicity and further studies are recommended. Our model may be useful in the future toxicologic study of new antiviral agents.