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Peer-reviewed articles


Jin,Y., Dixon,B., Jones,L., Gorbet,M. The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro International Journal of Molecular Sciences 2021;22(23):12899 [ Show Abstract ]

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.


Jin,Y., Jones,L., Gorbet,M. Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation Scientific report 2020;10(1):19690 [ Show Abstract ]

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.


Gorbet,M., Peterson,R., McCanna,D., Woods,C., Jones,L., Fonn,D. Human corneal epithelial cell shedding and fluorescein staining in response to silicone hydrogel lenses and contact lens disinfecting solutions Current eye research 2014;39(3):245-256 [ Show Abstract ]

Purpose: A pilot study was conducted to evaluate human corneal epithelial cell shedding in response to wearing a silicone hydrogel contact lens/solution combination inducing corneal staining. The nature of ex vivo collected cells staining with fluorescein was also examined. Methods: A contralateral eye study was conducted in which up to eight participants were unilaterally exposed to a multipurpose contact lens solution/silicone hydrogel lens combination previously shown to induce corneal staining (renu® fresh™ and balafilcon A; test eye), with the other eye using a combination of balafilcon A soaked in a hydrogen peroxide care system (Clear Care®; control eye). Lenses were worn for 2, 4 or 6 hours. Corneal staining was graded after lens removal. The Ocular Surface Cell Collection Apparatus was used to collect cells from the cornea and the contact lens. Results: In the test eye, maximum solution-induced corneal staining (SICS) was observed after 2 hours of lens wear (reducing significantly by 4 hours; p < 0.001). There were significantly more cells collected from the test eye after 4 hours of lens wear when compared to the control eye and the collection from the test eye after 2 hours (for both; n = 5; p < 0.001). The total cell yield at 4 hours was 813 ± 333 and 455 ± 218 for the test and control eyes, respectively (N = 5, triplicate, p = 0.003). A number of cells were observed to have taken up the fluorescein dye from the initial fluorescein instillation. Confocal microscopy of fluorescein-stained cells revealed that fluorescein was present throughout the cell cytoplasm and was retained in the cells for many hours after recovery from the corneal surface. Conclusion: This pilot study indicates that increased epithelial cell shedding was associated with a lens-solution combination which induces SICS. Our data provides insight into the transient nature of the SICS reaction and the nature of fluorescein staining observed in SICS. © 2014 Informa Healthcare USA, Inc.

Mohammadi,S., Jones,L., Gorbet,M. Extended latanoprost release from commercial contact lenses: In vitro studies using corneal models PLOS ONE 2014;9(9):e106653 [ Show Abstract ]

In this study, we compared, for the first time, the release of a 432 kDa prostaglandin analogue drug, Latanoprost, from commercially available contact lenses using in vitro models with corneal epithelial cells. Conventional polyHEMA-based and silicone hydrogel soft contact lenses were soaked in drug solution ( solution in phosphate buffered saline). The drug release from the contact lens material and its diffusion through three in vitro models was studied. The three in vitro models consisted of a polyethylene terephthalate (PET) membrane without corneal epithelial cells, a PET membrane with a monolayer of human corneal epithelial cells (HCEC), and a PET membrane with stratified HCEC. In the cell-based in vitro corneal epithelium models, a zero order release was obtained with the silicone hydrogel materials (linear for the duration of the experiment) whereby, after 48 hours, between 4 to 6  of latanoprost (an amount well within the range of the prescribed daily dose for glaucoma patients) was released. In the absence of cells, a significantly lower amount of drug, between 0.3 to 0.5 , was released, (). The difference observed in release from the hydrogel lens materials in the presence and absence of cells emphasizes the importance of using an in vitrocorneal model that is more representative of the physiological conditions in the eye to more adequately characterize ophthalmic drug delivery materials. Our results demonstrate how in vitro models with corneal epithelial cells may allow better prediction of in vivo release. It also highlights the potential of drug-soaked silicone hydrogel contact lens materials for drug delivery purposes.


Gorbet,M. B., Tanti,N. C., Crockett,B., Mansour,L., Jones,L. Effect of contact lens material on cytotoxicity potential of multipurpose solutions using human corneal epithelial cells Molecular Vision 2011;173458-3467 [ Show Abstract ]

Purpose: Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) contact lenses. This in vitro study was undertaken to identify the potential for interaction between MPS, SiHy surface treatments, and lens materials, which may lead to changes in the response of human corneal epithelial cells (HCEC) to MPS-soaked lenses. Methods: The MPS tested were renu fresh (formerly known as ReNu MultiPlus; ReNu), OptiFree Express (OFX), OptiFree RepleniSH, SoloCare Aqua, and Complete Moisture Plus. The SiHy materials evaluated were lotrafilcon A, lotrafilcon B, comfilcon A, galyfilcon A, and balafilcon A (BA). MPS-soaked lenses were placed on top of adherent HCEC. The effect of MPS dilutions (0.1 to 10% final concentration in medium) was also characterized. Cell viability, adhesion phenotype and caspase activation were studied after 24-h cell exposure. OFX released from lenses was determined using UV absorbance. Results: A significant reduction in viability (between 30 to 50%) was observed with cells exposed to lenses soaked in ReNu and OFX. A significant downregulation of α3 and β1 integrins, with integrin expression ranging from 60% to 75% of control (cells with no lens), was also observed with OFX and ReNu-soaked lenses. With the exception of BA, all other lenses soaked in OFX resulted in significant caspase activation, whereby over 18% of cells stained positive for caspases. Minimal caspase activation was observed in cells exposed to ReNu and Solo soaked lenses. For both OFX and ReNu, exposing cells to at least a 5% dilution had a significant effect on viability and integrin expression. While Complete and Solo did not lead to reduction in viability, cells exposed to a 10% dilution showed reduced integrin expression down to less than 70% of control value. Comparing cell response to diluted MPS solutions and various MPS-soaked lenses showed that it is not possible to reliably use cell response to MPS dilution alone to assess MPS biocompatibility. Conclusions: Our results demonstrate that the reaction of HCEC to MPS are affected by the type of lenses the MPS is released from and may potentially be influenced by the surface treatment (or lack of it) of SiHy materials. © 2011 Molecular Vision.

Tanti,N. C., Jones,L., Gorbet,M. B. Impact of multipurpose solutions released from contact lenses on corneal cells Optometry and Vision Science 2011;88(4):483-492 [ Show Abstract ]

Purpose. To assess, in vitro, the effect of the release of contact lens multipurpose solutions (MPS) from two silicone hydrogel lenses on human corneal epithelial cells. Methods. A monolayer of immortalized human corneal epithelial cells was seeded in a 24-well plate in keratinocyte serum-free medium. Lotrafilcon A (LA) and balafilcon A (BA) lenses were placed on top of the adherent cells for 8 and 24 h, after being soaked in MPS, borate-buffered (Unisol) or phosphate-buffered saline overnight. Cells were assayed for viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or for α3, β1, and β4 integrin expression and caspase activation by flow cytometry. Results. After 8 h, LA lenses soaked in Unisol, Opti-Free Express (OFX), and ReNu MultiPlus (ReNu) showed decrease in cell viability. LA and BA soaked in Complete Moisture Plus (Complete) had similar viability at around 85% of control. After 24 h, a further decrease in viability was observed with all MPS-soaked lenses; LA soaked in OFX significantly reduced viability compared with Unisol-soaked lenses. In addition, reduced levels of integrin expression for lenses soaked in OFX and ReNu, and for BA soaked in Complete were observed. At 24 h, only LA soaked in OFX led to an increase in caspase activation. Conclusions. Our results indicate an increase in cytotoxicity with borate-based MPS solutions in vitro when compared with both phosphate-buffered saline and borate-exposed lenses, suggesting that biocides and/or additives play a role in the observed cell reaction. Moreover, the mechanism of in vitro solution-induced toxicity appeared to be mediated by lens type, suggesting differences in the preferential adsorption/release profile of certain compounds. Copyright © 2011 American Academy of Optometry.


Gorbet,M. B., Tanti,N. C., Jones,L., Sheardown,H. Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions Molecular Vision 2010;16272-282 [ Show Abstract ]

Purpose: Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. Methods: An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2-hydroxyethylmethacrylate (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Results: Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of β1 and α3 integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Conclusions: Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells. © 2010 Molecular Vision.

Scientific Presentations


Mohammadi S, Jones L, Gorbet M. Investigation of Latanoprost Release from Contact Lens Materials in the presence of cells and under in vitro Tear Replenishment Invest Ophthalmol Vis Sci 2018;E-Abstract 1770 [ Show Abstract ]

Purpose: To effectively mimic the ocular environment for biocompatibility and drug delivery studies, the limited volume and replenishment of the tear film and the presence of cells should be considered. By using an in vitro corneal epithelial cell model combined with a tear replenishment (TR) method, this study aimed to investigate the delivery of Latanoprost by commercially available contact lenses (CL) and compare the dynamic release results to non-replenished (immersion) conditions.

Methods: To enable in vitro “lens wear”, cell culture inserts were formed to recreate the geometry of the cornea prior to seeding of human corneal epithelial cells. The curved cornea models (CCMs) were ready after 7 and 14 days for mono-layer and stratified models respectively. The in vitro TR was achieved via intermittent flow of a tear solution analogue over the CCMs at a rate of 1mL/hour. Three CLs (balafilcon A; senofilcon A; etafilcon A), were soaked for 24hrs in the glaucoma drug, Latanoprost, at concentrations of 123 µg/mL (high doping; HD) and 9.5 µg/mL (low doping; LD). Lenses were then onlayed on the CCMs (n=3) and drug concentration was determined on the basal (diffusion/transport) and apical (supernatant) sides after 1, 4, 8 and 12hrs.

Results: A zero-order kinetic was observed for basal drug concentration over the 12hr period. The drug release and diffusion through the CCMs was significantly higher in etafilcon A (p<0.001). Also for HD lenses, the overall release was comparable to the prescribed daily dose. The apical release of the drug was significantly lower for replenished vs. immersed CCMs (p<0.001) in HD lenses. In contrast to stratified CCMs, cell viability was significantly lower under replenished conditions compared to immersed mono-layer CCMs (p=0.002). The release ratio of HD to LD lenses were in agreement to the doping ratio.

Conclusions: The Latanoprost release studies under dynamic conditions further confirmed the significant role that cells play in the release of that drug from a CL material. These results also demonstrated yet another important role that a dynamic release model will have in predicting the amount of drug that can be absorbed through the cornea or lost from a CL into the tear film/lacrimal system. Recreating the microfluidics of the TR in vitro can contribute to a better understanding of interactions between the ocular surface and drug-delivery CL.


Gorbet M, Toameh D, Zhang J, Phan C-M, Walther H, Jones L. Development of a dynamic co-culture ocular cell in vitro model for ocular biocompatibility testing Invest Ophthalmol Vis Sci 2017;E-Abstract 4727


Mohammadi S, Jones L, Gorbet M. Ocular drug release models: comparisons between in vitro cell, dynamic release, and fixed volume models 10th World Biomaterials Congress, Montreal, 2016


Flanagan J, Stavropoulos A, Luensmann D, Postnikoff C, Gorbet M. Comparison of the Cellular Response to Overnight Contact Lens Wear on the Sclera and Cornea Invest Ophthalmol Vis Sci 2013;54:E-abstract 5654

Gorbet M, Luensmann D, Jones L. The response of tear film neutrophils to occasional overnight lens wear Invest Ophthalmol Vis Sci 2013;54: E-Abstract 2069

Mohammadi S, Jones L, Gorbet M. Investigation of Latanoprost release from contact lens materials using in vitro cell models Invest Ophthalmol Vis Sci 2013;54: E-Abstract 5472

Mohammadi S, Jones L, Gorbet M. In vitro cell models for drug release studies from contact lenses Canadian Optometry Schools Research Conference, Waterloo, Canada, 2013


Gorbet M, Cira D, Peterson R, Woods C, Luensmann D. The acute effect of benzalkonium chloride and sodium fluorescein on epithelial cells collected from the human ocular surface Contact Lens & Anterior Eye 2012;35, S1:e22-e23

Gorbet M, Luensmann D, Luck S, Jones L. Response Of Tear Film Neutrophils To Different Stimuli Invest Ophthalmol Vis Sci 2012;53:ARVO E-Abstract 5271

Gorbet M, Mohammadi S, Jones L. Investigation of drug-release materials using in vitro cell models 2020 NSERC ophthalmic materials conference, Burlington, Canada, 2012

Mohammadi S, Gorbet M. Design and Development of an In Vitro Tear Replenishment System Invest Ophthalmol Vis Sci 2012;53:ARVO E-Abstract 6109

Situ P, McCanna D, Gorbet M, Jones L. Confocal Imaging Of Hyper-reflective Corneal Epithelial Cells During And After Contact Lens Wear Invest Ophthalmol Vis Sci 2012;53:ARVO E-Abstract 4698


Gorbet M, Tanti N, Jones L. Impact of contact lens solution/material combinations on corneal epithelial cells in vitro International Society for Contact Lens Research (Napa Valley, California), 2011


Gorbet M, Tanti N, Crockett B, Jones L. Impact of lens material on in vitro cytotoxicity potential of multipurpose solutions on human corneal epithelial cells Invest Ophthalmol Vis Sci 2010;51:E-Abstract 3415

Srinivasan S, Martell E, Heynen M, Luensmann D, Cira D, Gorbet M, Jones L. Ocular surface sampling techniques 7th Canadian University Conference in Optometry (Montreal, Canada), 2010

Srinivasan S, Martell E, Heynen M, Luensmann D, Cira D, Gorbet M, Jones L. Ocular surface sampling techniques 20:20 National Science and Engineering Council Network meeting (Horseshoe Valley, Ontario, Canada), 2010


Peterson R, Gorbet M, Woods CA, Fonn D. The transient nature of solution induced corneal staining Optom Vis Sci 2009;86:E-abstract 90816

Tanti N, Jones L, Gorbet M. Effect of MPS released from silicone hydrogel lenses on corneal epithelial cell adhesion phenotype and apoptotic pathways in vitro Invest Ophthalmol Vis Sci 2009;50:E-abstract 4614


Gorbet M, Jones L, Sheardown H. The adverse effect of borate buffered versus phosphate buffered packaging solutions on corneal cellular viability and integrin expression Contact Lens & Anterior Eye 2008;31, 6:290-291

Tanti N, Gorbet M, Jones L. In vitro analysis of the potential cytotoxicity of contact lenses soaked in multi-purpose solutions Contact Lens & Anterior Eye 2008;31, 5:250-251


Gorbet M, Duan X, Jones L, Sheardown H. Activation of human corneal epithelial cells on collagen gels Invest Ophthalmol Vis Sci 2007;48: E-abstract 1891

Gorbet M, Jones L, Sheardown H. Corneal epithelial cell viability and integrin expression is affected by contact lens packaging solutions ISCLR meeting, (Whistler, Canada), 2007