Human Corneal Epithelial Cells Research Articles

Evaluation of the Possible Influence of Povidone Iodine (PVI) Solution and Polyhexanide (PHMB) on Wound Healing in Corneal Epithelial Regeneration.

This study investigates the possible toxic effects of the preoperative antiseptic substances povidone iodine (PVI) and polyhexanide (PHMB; Serasept® 2) on wound healing in ophthalmology. To assess this impact, human telomerase-immortalized corneal epithelial (hTCEpi) cells and human telomerase-immortalized conjunctival epithelial (hCjE) cells were exposed to 1% and 5% PVI or 0.04% PHMB for different periods to evaluate the cytotoxicity of these two antiseptics. Furthermore, the toxicity of these antiseptics was investigated in a human tissue-specific corneal epithelial construct and porcine eye culture model. The results reveal the high cytotoxicity of PVI and PHMB in the hTCEpi and hCjE in monolayer cell culture models, independent of the incubation time and concentration of these substances. However, after hTCEpi cell differentiation into a tissue-specific corneal epithelial construct, contact with these antiseptics for the relevant preoperative time did not alter cPARP1 or Ki67 expression. Furthermore, the wound-healing process in the porcine cornea was not significantly influenced after incubation with these antiseptics. In summary, corneal and conjunctival epithelial cell lines are very sensitive to PVI and PHMB, whereas no significant alterations were found in intact tissue-specific corneal epithelial constructs or porcine corneas. Therefore, we could not identify PVI and PHMB as reasons for postoperative eye irritation.

KCNK5 Regulating Potassium Efflux and Inducing Pyroptosis in Corneal Epithelial Cells Through TNFSF10-Mediated Autophagy in Dry Eye.

The purpose of this study was to elucidate the involvement of potassium two pore domain channel subfamily K member 5 (KCNK5)-mediated potassium efflux in the pathogenesis of dry eye and to unravel the underlying molecular mechanisms. To induce experimental dry eye in adult wild-type C57BL/6 mice, scopolamine was administered via subcutaneous injection, and the mice were subjected to desiccating stress. To create an in vitro model of dry eye, desiccation stress was applied to the human corneal epithelial cell line (HCE-T). Intracellular potassium concentration was quantified using inductively coupled plasma mass spectrometry. Cellular death was assessed through lactate dehydrogenase assays. Gene expression profiling was conducted through both RNA sequencing and quantitative real-time PCR. Protein analysis was carried out through Western blotting and immunofluorescence staining. Assessment of the corneal epithelial defect area was conducted through fluorescein sodium staining. Tear secretion was quantified using the phenol red cotton thread method. Potassium efflux was observed to further facilitate corneal epithelial pyroptosis. KCNK5 exhibited upregulation in both in vivo and in vitro models of dry eye. The overexpression of KCNK5 was observed to induce potassium efflux and activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis in vitro. Silencing KCNK5 effectively mitigated pyroptosis in dry eye. Additionally, the overexpression of KCNK5 results in the downregulation of TNF superfamily member 10 (TNFSF10) and subsequent impairment of autophagy. TNFSF10 supplementation could promote autophagy and mitigate pyroptosis in dry eye. The upregulation of KCNK5 mediates TNFSF10 to impair autophagy and induce pyroptosis in dry eye. Consequently, targeting KCNK5 may represent a novel and promising approach to therapeutic intervention in the management of dry eye.

The evaluation of cytoxicity of ocular hypotensive therapy to cultured human corneal epithelial cells

BACKGROUND: Corneal epithelial defect is a corneal epithelial cells’ (CEpC) disruption of various origin. In patients with corneal epithelial defect, glaucoma is a frequent concomitant disease demanding prescription of hypotensive medications, most of which containing benzalkonium chloride.
 AIM: The aim of this study is to evaluate cytotoxicity of antiglaucoma drugs, as well as benzalkonium chloride (BAK), to human CEpC in vitro.
 MATERIALS AND METHODS: The study was carried out on primary cultures of human CEpC. Cytotoxicity of dorzolamide, brimonidine, timolol (dilutions 1/100, 1/50, 1/20, 1/10, a 24-hour exposure) and of BAK was estimated on the model of intact epithelium (monolayer). Benzalkonium chloride was evaluated in concentrations equals to its concentration in tested ophthalmic solutions. Cytotoxicity of dorzolamide, brimonidine, timolol (dilutions 1/100, 1/20, a 48-hour exposure) was evaluated on the model of corneal epithelial defect (damage of the monolayer). The medication’s cytotoxicity was estimated by cellular changes (phase-contrast microscopy) and by the MTS-test’s results.
 RESULTS: Among BAK-free medications: dorzolamide (1/50, 1/20, 1/10 dilutions), brimonidine (1/10 dilution) — induce CEpCs’ pathological changes, whereas timolol (all tested dilutions) is non-toxic. BAK-preserved drugs: dorzolamide, brimonidine, timolol (1/100, 1/50, 1/20, 1/10 dilutions) — induce CEpCs’ damage, their viability reduction, and corneal epithelial defect closure inhibition. BAK shows similar effect in tested concentrations.
 CONCLUSIONS: Cytotoxicity of antiglaucoma drugs is attributed to their component — benzalkonium chloride. Administration of preserved drugs is not reasonable in eyes with corneal epithelial defect of various origin.

U-Net Convolutional Neural Network for Real-Time Prediction of the Number of Cultured Corneal Endothelial Cells for Cellular Therapy.

Corneal endothelial decompensation is treated by the corneal transplantation of donor corneas, but donor shortages and other problems associated with corneal transplantation have prompted investigations into tissue engineering therapies. For clinical use, cells used in tissue engineering must undergo strict quality control to ensure their safety and efficacy. In addition, efficient cell manufacturing processes are needed to make cell therapy a sustainable standard procedure with an acceptable economic burden. In this study, we obtained 3098 phase contrast images of cultured human corneal endothelial cells (HCECs). We labeled the images using semi-supervised learning and then trained a model that predicted the cell centers with a precision of 95.1%, a recall of 92.3%, and an F-value of 93.4%. The cell density calculated by the model showed a very strong correlation with the ground truth (Pearson's correlation coefficient = 0.97, p value = 8.10 × 10-52). The total cell numbers calculated by our model based on phase contrast images were close to the numbers calculated using a hemocytometer through passages 1 to 4. Our findings confirm the feasibility of using artificial intelligence-assisted quality control assessments in the field of regenerative medicine.

Research progress of corneal endothelial cell regeneration and replacement

AbstractCorneal endothelial cells (CECs) are crucial for the maintenance of corneal transparency and normal visual function. Corneal endothelial dysfunction can lead to corneal edema, opacity, and even blindness. Due to the limited proliferative capacity of human CECs and the global shortage of donor cornea, corneal endothelial regeneration and replacement always represent the most challenge in the basic research and clinical treatment of corneal diseases. Although there is a potential existence of corneal endothelial progenitor cells, the efficiency of Descemet stripping without endothelial keratoplasty remains controversial. In recent years, significant advancements have been made in the field of cultured endothelial cell regeneration and artificial material replacement. Here, we reviewed the current research and clinical progress of corneal endothelial cell regeneration and replacement, including the in vitro cultivation of primary human CECs, in vitro differentiation of stem cell‐derived CECs, tissue‐engineered corneal endothelium, and fabrication of artificial corneal endothelium. We also discussed the remaining questions regarding innovating clinical preventive and therapeutic strategies for corneal endothelial dysfunction.

Effects of particulate matter exposure on the expression of the SARS-CoV-2 ACE2 receptor in ocular surface tissues and cells.

Particulate matter (PM) has been reported to be one of the risk factor for COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, although the ocular surface is deeply affected by both PM exposure and SARS-COV-2 infection, no studies have investigated the effects of PM exposure on the ocular route of SARS-COV-2 infection. To this end, we explored the effects of PM on the expression of SARS-COV-2-associated receptors and proteins in ocular surface. Herein, short- and long-term PM-exposed rat models were established by topically administering PM for 3 and 10days, respectively. Immortalized human corneal epithelial cells (HCECs) and human conjunctival epithelial cells (HCjECs) were exposed to PM. ACE2, TMPRSS2, CD147, and ADAM17 expression levels were measured by western blot analysis. Our results show that short-term PM exposure had little effect on the expressions of ACE2, TMPRSS2, and CD147 in ocular surface tissues. However, long-term PM exposure decreased the ACE2 expression in conjunctival tissues and increased the CD147 expression in corneal or conjunctival tissues. PM exposure reduced the ACE2 expression by increasing the ADAM17 expression and ACE2 shedding level in HCECs and HCjECs. Our findings suggest that long-term PM exposure down-regulate the expression of the SARS-CoV-2 receptor ACE2 in conjunctival tissues through ADAM17-dependent ACE2 shedding. However, long-term PM exposure up-regulates the expression of another SARS-CoV-2 receptor CD147 in ocular surface tissues, accompanied by ocular surface damage and cytotoxicity. This study provides a new insight into uncovering potential risk factors for infection with SARS-CoV-2 via the ocular route.

The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation

Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintenance of ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.

Novel anti‐evaporative lipid‐based formulation for dry eye disease treatment

Purpose: The majority of dry eye disease (DED) patients suffer from the tear film lipid layer (TFLL) instability issues associated with increased evaporation of water from the ocular surface. Therefore, we developed a liposomal formulation based on selected TFLL lipid classes that would restore the tear film stability and retard the evaporation of the aqueous tear fluid.Methods: The liposomal formulation was prepared by the thin film hydration method and subsequently characterized intermittently by standard techniques for at least 6 months. The mitochondrial metabolic activity (MTT) assay with human corneal epithelial (HCE) cells was utilized to assess the formulation toxicity in vitro. The evaporation resistance properties of the formulation were determined in vitro by the Langmuir trough methods, while a DED cellular model engendered by exposing the HCE cells to benzalkonium chloride (BAC) enabled additional assessment of the in vitro efficacy. In vivo safety was evaluated in rats and rabbits in one‐week studies. The conditions of the animal eyes were assessed by a non‐invasive biomicroscope, an optical coherence tomography, and histological staining of the dissected eyes.Results: The lipid‐based formulation with favourable pharmaceutical and functional properties (e.g., the evaporation resistance, and the promotion of HCE cell recovery after damage) was developed successfully, while also the physical stability of the formulation was found promising. No evidence of toxicity was observed during in vitro and in vivo experiments.Conclusions: The results suggest that the developed formulation is well‐tolerated and possesses properties and a safety profile compatible with ocular administration. Utilized in vitro efficacy models imply that the developed formulation is a promising candidate towards more efficient DED treatment. In vivo efficacy remains to be clarified in future studies.

Mechanotransduction and corneal endothelium homeostasis: Application in tissue engineering

Aim: Globally, there is a significant shortage of corneas available in the eye banks. This high demand has triggered a new platform for addressing the issue by using biomaterials. Mechanical cues play a key role in regulating fundamental cellular functions, as demonstrated by numerous studies conducted on many types of cells. This project's goal is to engineer an effective Descemet's membrane (DM) by changing the stiffness of compressed collagen scaffolds so that it maximizes the functional properties of Descemet's membrane to fully support endothelial cells.Methods: This study entails creation of collagen substrates of varying stiffness which enabled to scrutinize the effect of different biomechanical signals on human corneal endothelial cells (HCECs) behaviour. Thereafter, the viability, function, and responses of HCECs were compared to determine the best scaffold to support corneal endothelial cells. Collagen scaffolds were characterized: light transmittance, scaffold stiffness values, and surface topography by Atomic Force Microscope. Studying HCECs' responses and behaviour on scaffolds of different stiffness has been done by assessing the cell viability and immunofluorescence labelling for Yes‐associated protein (YAP), ZO‐1, and Na/K ATPase.Results: The study found higher growth rate of stiff scaffolds and a confluent monolayer of HCECs in comparison to soft scaffolds. This was accompanied by an increase in nuclear YAP. Typical functional markers were expressed by HCECs on a stiff collagen scaffold compared to a softer scaffold. Collagen scaffolds with a different stiffness do affect nuclear YAP, therefore demonstrating mechanosensitivity of corneal endothelial cells.Conclusion: In conclusion, these results convey that tissue engineering of the human corneal endothelium can determine the scaffold which mimics the best DM biomechanical properties. Hence, very soon scaffold improvements is viable if the role of mechanotransduction is explained for endothelial cell homeostasis.

Characterization of the proteome changes in an in vitro hyperosmotic dry eye model employing human corneal epithelial cells

Aims/Purpose: The detrimental effects of tear hyperosmolarity on human corneal epithelial (HCE) cells is a primary cause of dry eye syndrome (DES). However, the molecular mechanisms underlying inflammatory processes and cellular dysfunction in HCE are largely unknown. This study characterized the proteome changes in an optimized hyperosmolarity‐induced DES model in vitro.Methods: An in vitro hyperosmolar (400–550 mOsm) DES model was established in a simian virus 40 (SV40)‐immortalized HCE cell line. Cells incubated in medium with physiological osmolarity (312 mOsm) were used as control. Cell viability, cell death and intracellular reactive oxygen species (ROS) levels were analysed with MTS, Annexin‐V‐FLUOS, crystal violet and DCFDA assays. Mass spectrometry‐based proteomics analyses characterized the proteome and elucidated the underlying molecular changes.Results: The HCE cell viability was significantly decreased in a concentration‐dependent manner with increasing hyperosmolarity compared to control at 48 hr (450 mOsm, −47%, p = 2.7 × 10−8; 500 mOsm, −71%, p = 1.1 × 10−4; 550 mOsm, −84%, p = 2.6 × 10−5). Notably, cell adherence was significantly decreased (p = 5.8 × 10−9), while levels of ROS (p = 3.6 × 10−2) and apoptosis (p = 3.6 × 10−2) were significantly increased at 450 mOsm compared to control. Proteomics analysis identified ~2000 proteins (FDR <1%) and, as many as 215 proteins were found to be significantly (p < 0.05) differentially abundant in the 450 vs. 312 mOsm. The effects of high hyperosmolarity (450 mOsm)‐induced damage resembling DES on the HCE cell were further demonstrated by the expression of proteins involved in the activation of inflammatory response (p = 5.5 × 10−3), apoptosis (p = 5.3 × 10−4) and generation of ROS (e.g., glutathione S‐transferase P) (p = 7.1 × 10−3). Moreover, many differentially expressed proteins were also implicated in the regulation of cell viability (p = 1.1 × 10−6) and organization of cytoplasm (e.g., cyclase associated actin cytoskeleton regulatory protein 1) (p = 2.4 × 10−2).Conclusions: In summary, an optimized hyperosmolarity‐induced DES cell culture model for proteomics analysis was established, and specific alterations in the proteome were characterized in this model. This in vitro model would be instrumental for testing therapeutic targets for DES in the future.

Cold plasma treatment for fungal keratitis

Aims/Purpose: Fungal keratitis is serious ocular infection caused by any of the fungi species. It is treated with antifungal medications, in worst scenario with cornea transplantation. The therapy is still challenging due to poor drug penetration into the cornea. Cold plasma, produced by ionization of argon gas, contains especially reactive species but also UV radiation which damage microorganisms. The aim of this study is to determine the effect of cold plasma on human corneal epithelial cells (HCE), corneal tissue and multiple pathogen microorganisms to assess the opportunity of cold plasma treatment for fungal keratitis.Methods: The effect of cold plasma on HCE was analysed by the light microscopy, Live/Dead assay, metabolic activity assay, and immunohistochemistry. Disinfecting potential was analysed by plating prevalent yeasts (Candida albicans, C. krusei, C. parapsilosis, C. glabrata) on agar plates and treated with cold plasma. Plasma treatment of infected human donor corneas with C. albicans was determined by measurement of O.D. of liquid culture.Results: The treatment of HCE by cold plasma led to the change of the morphology, significantly lower viability, metabolic activity and proliferation of the cells. Yeast strains were significantly eradicated completely after 5 min of cold plasma treatment (p ≤ 0.001). Cold plasma therapy of the infected human donor corneas showed the decrease of the infection in all tested time intervals with statistical significance (p ≤ 0.01, p < 0.05 and p ≤ 0.001 respectively for 1, 2 and 5 min treatment).Conclusions: Cold plasma treatment showed negative impact on the culture of HCE. The analysis of its effect on the whole corneal tissue is in progress. On the contrary, a significant reduction of CFU was observed in all examined Candida spp. and significant decrease of infection was determined in infected corneas. Therefore, the cold plasma technology showed a potential to be an effective treatment for fungal keratitis.

In‐depth study of the mucoadhesive and lubricating properties of Hyalistil SYNFO and other sodium hyaluronate‐based eye drops

Purpose: Several sodium hyaluronate (SH) eye drops formulations are widely used for the management of dry eye disease (DED). Hyalistil SYNFO is an eye drops based on SH and xanthan gum and osmoprotectants (glycine and betaine). The aim of our study was to evaluate viscosity and mucoadhesion profiles of Hyalistil SYNFO with respect to other SH‐based eye drops, and to investigate their ability to protect the corneal surface from dehydration.Methods: The physicochemical properties, in term of viscoelastic and mucoadhesive behaviour, were evaluated by means of a rheometer, TA‐XT2 texture analyzer and zeta potential determination. An in vitro dehydration model was used to demonstrate the lubricant effect of Hyalistil SYNFO compared to other commercially available SH‐based eye drops on human corneal epithelial cells (HCE).Results: The rheological profiles indicated that Hyalistil SYNFO exhibits a viscoelastic behaviour under shear strain showing a characteristic curve owing to its specific molecular properties. Evaluations of detachment force of tested formulations resulting in a strengthening of the mucin‐formulation interface showed that Hyalistil SYNFO exhibited a stronger and significant mucoadhesion calculated as percentage compared to mucin and other SH‐based products. The occurring interaction between formulation and mucin was also demonstrated by the variations in zeta potential measured by the mucin‐eye drops mixture compared to mucin or product alone. The in vitro study on HCE cells demonstrated that Hyalistil SYNFO, compared to others formulation, was able to protect corneal epithelial cells from dehydration.Conclusions: The pseudoplastic profile and increased mucoadhesive strength, due to the presence of both SH and xanthan gum, may lead to an improved residence time of Hyalistil SYNFO on the corneal epithelium ensuring relief during the treatment of DED. Moreover, the effect against dehydration improves eye lubrication and corneal cell protection.

Comparative Analysis of the Osmoprotective Effects of Daily Disposable Contact Lens Packaging Solutions on Human Corneal Epithelial Cells.

Contact lens (CL) wear challenges the balance of the ocular surface environment by increasing water evaporation and tear osmolarity. Maintaining ocular surface homeostasis during CL wear remains a goal of lens manufacturers and an important consideration for eye care professionals. The purpose of this study was to measure the metabolic activity and inflammatory responses of a transformed human corneal epithelial cell (THCEpiC) line under hyperosmotic conditions in the presence of CL packaging solutions. CL packaging solutions sampled from seven daily disposable silicone hydrogel CL blister packages were prepared at 25% and made hyperosmolar (400 mOsm/kg) with NaCl. THCEpiCs were incubated with each solution for 24 hr, after which cell culture supernatants were collected. THCEpiC metabolic activity was determined by an alamarBlue assay. Concentrations in cell culture supernatants of inflammatory cytokine (interleukin [IL]-6) and chemokine (IL-8), as well as monocyte chemoattractant protein-1 (MCP-1), were quantitated by specific enzyme-linked immunosorbent assays. THCEpiC metabolic activity under hyperosmolar conditions decreased in the presence of somofilcon A and senofilcon A solutions (p=0.04 and 0.004, respectively), but no other solution (all p≥0.09). Concentrations of IL-6 increased in the presence of delefilcon A, somofilcon A, narafilcon A, and senofilcon A solutions (all p≤0.001), but no other solution (all p≥0.08), while those of IL-8 increased in the presence of all solutions (all p≤0.03) but kalifilcon A (p>0.99), and those of MCP-1 increased in the presence of delefilcon A, verofilcon A, somofilcon A, and stenfilcon A solutions (all p<0.0001), but no other solution (all p>0.99). CL packaging solutions differ in their capacity to inhibit epithelial inflammation. THCEpiC inflammatory response was less in the presence of a CL packaging solution containing osmoprotectants than in solutions lacking osmoprotectants under moderately hyperosmolar conditions in vitro. Clinical studies are warranted to further substantiate the benefit of osmoprotectants.

Tetracycline: structural characterization and antimicrobial properties of its water-soluble di-anionic bi-sodium salt.

The new water-soluble di-anionic bi-sodium salt of tetracycline (TC), an antibiotic in clinical use, with the formula {[TC]2-[Na+(MeOH)(H2O)] [Na+]·(H2O)}n (TCNa) was synthesized. The compound was characterized by m.p., attenuated total reflectance-Fourier transform infra-red (ATR-FTIR) spectroscopy, and ultraviolet (UV) and proton nuclear magnetic resonance (1H NMR) spectroscopy in the solid state and in solution. The molecular weight (MW) was determined by cryoscopy. The crystal structure of TCNa was also determined by X-ray crystallography. The antibacterial activity of TCNa was evaluated against the bacterial species Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) by means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and inhibition zones (IZs). Moreover, the ability of the compound to eradicate biofilm formation was also evaluated. The results are compared with those obtained for the commercially available drug TCH2. The in vitro and in vivo toxicities of TCNa were tested against human corneal epithelial cells (HCECs) and Artemia salina.

In vitro Assay to Examine the Function of Tears on Corneal Epithelial Cells.

Tears contain numerous secreted factors, enzymes, and proteins that help in maintaining the homeostatic condition of the eye and also protect it from the external environment. However, alterations to these enzymes and/or proteins during pathologies such as mechanical injury and viral or fungal infections can disrupt the normal ocular homeostasis, further contributing to disease development. Several tear film components have a significant role in curbing disease progression and promoting corneal regeneration. Additionally, several factors related to disease progression are secreted into the tear film, thereby serving as a valuable reservoir of biomarkers. Tears are readily available and can be collected via non-invasive techniques or simply from contact lenses. Tears can thus serve as a valuable and easy source for studying disease-specific biomarkers. Significant advancements have been made in recent years in the field of tear film proteomics, lipidomics, and transcriptomics to allow a better understanding of how tears can be utilized to gain insight into the etiology of diseases. These advancements have enabled us to study the pathophysiology of various disease states using tear samples. However, the mechanisms by which tears help to maintain corneal homeostasis and how they are able to form the first line of defense against pathogens remain poorly understood and warrant detailed in vitro studies. Herein, we have developed an in vitro assay to characterize the functional importance of patient isolated tears and their components on corneal epithelial cells. This novel approach closely mimics real physiological conditions and could help the researchers gain insight into the underlying mechanisms of ocular pathologies and develop new treatments. Key features • This method provides a new technique for analyzing the effect of tear components on human corneal epithelial cells. • The components of the tears that are altered in response to diseases can be used as a biomarker for detecting ocular complications. • This procedure can be further employed as an in vitro model for assessing the efficacy of drugs and discover potential therapeutic interventions.

Protective effect of acteoside on hydrogen peroxide-induced oxidative damage in human corneal epithelial cells

Protective effect of acteoside on hydrogen peroxide-induced oxidative damage in human corneal epithelial cells

Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells.

The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs. Ca++ sources for TPMD-induced Ca++ waves were explored using Ca++ channel inhibitors and Ca++-free media. TPMDs and TPMD Ca++ Wvs were observed in all cornea epithelial models examined, often times showing oscillating Ca++ levels. The sarcoplasmic reticulum Ca++ ATPase inhibitors thapsigargin and CPA reduced TPMD Ca++ Wvs. TRP V1 antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs. Ca++-free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) did not affect TPMD Ca++ Wvs. These results provide a direct demonstration of corneal epithelial cell TPMDs and TPMDs in in vivo cells from a live animal. TPMDs were observed following gentle eye rubbing, a routine corneal epithelial cell mechanical stress, indicating TPMDs and TPMD Ca++ Wvs are common features in corneal epithelial cells that likely play a role in corneal homeostasis and possibly pathophysiological conditions. Intracellular Ca++ stores are the primary Ca++ source for corneal epithelial cell TPMD Ca++ Wvs, with TRPV1 Ca++ channels providing Ca++ in MCECs but not HCECs. Corneal epithelial cell TPMD Ca++ Wv propagation is not influenced by gap junctions or ATP.

Copolymerized Polymers Based on Cyclodextrins and Cationic Groups Enhance Therapeutic Effect of Rebamipide in the N-Acetylcysteine-Treated Dry Eye Model.

We aimed to prepare a β-cyclodextrin (β-CD) polymer using radical polymerization with co-monomers, 6-deoxy-6-(2-methacryloyloxyethylsuccinamide)-β-cyclodextrin (CD-MSAm) and N,N,N-trimethyl-N-(2-hydroxy-3-metacryloyloxopropyl)-ammonium chloride (QA) to design cyclodextrins suitable for use in ophthalmology. In addition, we evaluated their solubility and inclusion properties with rebamipide (REB), a poorly soluble drug, and investigated the usefulness of the β-CD polymer and REB (REB@CDQA) combination in treating dry eye. The β-CD polymer (CD-MSAm-co-QA, CDQA) based on CD-MSAm/QA was prepared via radical polymerization, and the usefulness of REB@CDQA in treating dry eye was evaluated using a rabbit treated with N-acetylcysteine (dry eye model). The solubility of the CDQA powder was higher than that of the β-CD powder, and 80 nm colloids were observed in the CDQA solution. No corneal toxicity was observed in human corneal epithelial cells or rat corneas treated with 0.2% CDQA solution. The levels of REB dissolved in the CDQA solution were higher than those of the β-CD solution. Moreover, the application of the CDQA solution enhanced REB retention in the cornea and attenuated the transcorneal penetration of REB. In addition, instillation of REB@CDQA enhanced the volume of the lacrimal fluid and normalized the reduced mucin levels in the dry eye model. The extent of tear film breakup was attenuated by REB@CDQA instillation. The CDQA solution enhanced the solubility of REB, and the combination of CDQA and REB enhanced the drug content in the corneal tissue. Moreover, the therapeutic effect on dry eye was higher than that of REB suspensions without CDQA.

The protective effect of silver nanoparticles' on epithelial cornea cells against ultraviolet is accompanied by changes in calcium homeostasis and a decrease of the P2X7 and P2Y2 receptors

PurposeThe aim of the study was to evaluate the effect of silver nanoparticles hydrocolloids (AgNPs) on human corneal epithelial cells. Epithelial cells form the outermost and the most vulnerable to environmental stimuli layer of the cornea in the eye. Mechanical stress, UV radiation, and pathogens such as bacteria, viruses, and parasites challenge the fragile homeostasis of the eye. To help combat stress, infection, and inflammation wide portfolio of interventions is available. One of the oldest treatments is colloidal silver. Silver nanoparticle suspension in water is known for its anti-bacterial anti-viral and antiprotozoal action. However, AgNPs interact also with host cells, and the character of the interplay between corneal cells and silver seeks investigation. MethodsThe human epithelial corneal cell line (HCE-2) was cultured in vitro, treated with AgNPs, and subjected to UV. The cell’s viability, migration, calcium concentration, and expression/protein level of selected proteins were investigated by appropriate methods including cytotoxicity tests, “wound healing” assay, Fluo8/Fura2 AM staining, qRT-PCR, and western blot. ResultsIncubation of human corneal cells (HCE-2) with AgNP did not affect cells viability but limited cells migration and resulted in altered calcium homeostasis, decreased the presence of ATP-activated P2X7, P2Y2 receptors, and enhanced the expression of PACAP. Furthermore, AgNPs pretreatment helped restrain some of the deleterious effects of UV irradiation. Interestingly, AgNPs had no impact on the protein level of ACE2, which is important in light of potential SARS-CoV-2 entrance through the cornea. ConclusionsSilver nanoparticles are safe for corneal epithelial cells in vitro.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.