3D Bioprinting using UNIversal Orthogonal Network (UNION) Bioinks.

Three-dimensional (3D) bioprinting is a promising technology to produce tissue-like structures, but a lack of diversity in bioinks is a major limitation. Ideally each cell type would be printed in its own customizable bioink. To fulfill this need for a universally applicable bioink strategy, we developed a versatile, bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers. We term this family of materials UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin-like protein (ELP), and polyethylene glycol (PEG) were each used as backbone polymers to create inks with storage moduli spanning 200 to 10,000 Pa. Because UNION bioinks are crosslinked by a common chemistry, multiple materials can be printed together to form a unified, cohesive structure. This approach is compatible with any support bath that enables diffusion of UNION crosslinkers. Both matrix-adherent human corneal mesenchymal stromal cells and non-matrix-adherent human induced pluripotent stem cell-derived neural progenitor spheroids were printed with UNION bioinks. The cells retained high viability and expressed characteristic phenotypic markers after printing. Thus, UNION bioinks are a versatile strategy to expand the toolkit of customizable materials available for 3D bioprinting.

Effect of mesenchymal stromal cells encapsulated within polyethylene glycol-collagen hydrogels formed in situ on alkali-burned corneas in an ex vivo organ culture model.

BACKGROUND AIMS: Corneal inflammation after alkali burns often results in vision loss due to corneal opacification and neovascularization. Mesenchymal stem cells (MSCs) and their secreted factors (secretome) have been studied for their anti-inflammatory and anti-angiogenic properties with encouraging results. However, topical instillation of MSCs or their secretome is often accompanied by issues related to delivery or rapid washout. Polyethylene glycol (PEG) and collagen are well-known biomaterials used extensively in scaffolds for tissue engineering. To effectively suppress alkaline burn-induced corneal injury, the authors proposed encapsulating MSCs within collagen gels cross-linked with multi-functional PEG-succinimidyl esters as a means to deliver the secretome of immobilized MSCs. METHODS: Human MSCs were added to a neutralized collagen solution and mixed with a solution of four-arm PEG-N-hydroxysuccinimide. An ex vivo organ culture was conducted using rabbit corneas injured by alkali burn. MSCs were encapsulated within PEG-collagen hydrogels and injected onto the wounded cornea immediately following alkali burn and washing. Photographs of the ocular surface were taken over a period of 7 days after the alkali burn and processed for immunohistochemical evaluation. Samples were split into three groups: injury without treatment, MSCs alone, and MSCs encapsulated within PEG-collagen hydrogels. RESULTS: All corneas in ex vivo organ culture lost their transparency immediately after alkali burn, and only the groups treated with MSCs and MSCs encapsulated within PEG-collagen hydrogels recovered some transparency after 7 days. Immunohistochemical analysis revealed increased expression of vimentin in the anterior corneal stroma of the group without treatment indicative of fibrotic healing, whereas less stromal vimentin was detected in the group containing MSCs encapsulated within the PEG-collagen hydrogels. CONCLUSIONS: PEG-collagen hydrogels enable the encapsulation of viable MSCs capable of releasing secreted factors onto the ocular surface. Encapsulating MSCs within PEG-collagen hydrogels may be a promising method for delivering their therapeutic benefits in cases of ocular inflammatory diseases, such as alkali burn injuries.

Detection of Optic Disc Abnormalities in Color Fundus Photographs Using Deep Learning.

BACKGROUND: To date, deep learning-based detection of optic disc abnormalities in color fundus photographs has mostly been limited to the field of glaucoma. However, many life-threatening systemic and neurological conditions can manifest as optic disc abnormalities. In this study, we aimed to extend the application of deep learning (DL) in optic disc analyses to detect a spectrum of nonglaucomatous optic neuropathies. METHODS: Using transfer learning, we trained a ResNet-152 deep convolutional neural network (DCNN) to distinguish between normal and abnormal optic discs in color fundus photographs (CFPs). Our training data set included 944 deidentified CFPs (abnormal 364; normal 580). Our testing data set included 151 deidentified CFPs (abnormal 71; normal 80). Both the training and testing data sets contained a wide range of optic disc abnormalities, including but not limited to ischemic optic neuropathy, atrophy, compressive optic neuropathy, hereditary optic neuropathy, hypoplasia, papilledema, and toxic optic neuropathy. The standard measures of performance (sensitivity, specificity, and area under the curve of the receiver operating characteristic curve (AUC-ROC)) were used for evaluation. RESULTS: During the 10-fold cross-validation test, our DCNN for distinguishing between normal and abnormal optic discs achieved the following mean performance: AUC-ROC 0.99 (95 CI: 0.98-0.99), sensitivity 94% (95 CI: 91%-97%), and specificity 96% (95 CI: 93%-99%). When evaluated against the external testing data set, our model achieved the following mean performance: AUC-ROC 0.87, sensitivity 90%, and specificity 69%. CONCLUSION: In summary, we have developed a deep learning algorithm that is capable of detecting a spectrum of optic disc abnormalities in color fundus photographs, with a focus on neuro-ophthalmological etiologies. As the next step, we plan to validate our algorithm prospectively as a focused screening tool in the emergency department, which if successful could be beneficial because current practice pattern and training predict a shortage of neuro-ophthalmologists and ophthalmologists in general in the near future.

In situ-forming hyaluronic acid hydrogel through visible light-induced thiol-ene reaction.

Here we present hyaluronic acid (HA) hydrogels crosslinked via thiol-ene reaction initiated by visible blue light exposure in the presence of riboflavin phosphate (RFP). The gelation procedure is rapid and proceeds as effectively with exposure to blue light as it does with UV light. We successfully initiated the thiol-ene reaction by RFP with blue light, which triggered gelation that proceeds over about 5 min at 36 °C after an initial small change in modulus upon light exposure. Gel transparency was also evaluated, and the HA gel exhibited over 80% transmittance in the visible spectrum. The degradation and protein release kinetics of the photo-crosslinked HA hydrogel are also presented. The capacity of blue light to initiate thiol-ene reaction was equal to or more effective than UV light of the same energy. The cytocompatibility of hydrogels was evaluated using corneal fibroblasts, and the light-induced fabrication procedure and resultant gel materials did not affect cell viability. The results indicate that an RFP-based, BL-initiated photo-reaction to gelate HA may be an effective and promising modality for applications where in situ gelation is desired.

Immobilization of Growth Factors to Collagen Surfaces Using Pulsed Visible Light.

In the treatment of traumatic injuries, burns, and ulcers of the eye, inadequate epithelial tissue healing remains a major challenge. Wound healing is a complex process involving the temporal and spatial interplay between cells and their extracellular milieu. It can be impaired by a variety of causes including infection, poor circulation, loss of critical cells, and/or proteins, and a deficiency in normal neural signaling (e.g., neurotrophic ulcers). Ocular anatomy is particularly vulnerable to lasting morbidity from delayed healing, whether it be scarring or perforation of the cornea, destruction of the conjunctival mucous membrane, or cicatricial changes to the eyelids and surrounding skin. Therefore, there is a major clinical need for new modalities for controlling and accelerating wound healing, particularly in the eye. Collagen matrices have long been explored as scaffolds to support cell growth as both two-dimensional coatings and substrates, as well as three-dimensional matrices. Meanwhile, the immobilization of growth factors to various substrates has also been extensively studied as a way to promote enhanced cellular adhesion and proliferation. Herein we present a new strategy for photochemically immobilizing growth factors to collagen using riboflavin as a photosensitizer and exposure to visible light (∼458 nm). Epidermal growth factor (EGF) was successfully bound to collagen-coated surfaces as well as directly to endogenous collagen from porcine corneas. The initial concentration of riboflavin and EGF as well as the blue light exposure time were keys to the successful binding of growth factors to these surfaces. The photocrosslinking reaction increased EGF residence time on collagen surfaces over 7 days. EGF activity was maintained after the photocrosslinking reaction with a short duration of pulsed blue light exposure. Bound EGF accelerated in vitro corneal epithelial cell proliferation and migration and maintained normal cell phenotype. Additionally, the treated surfaces were cytocompatible, and the photocrosslinking reaction was proven to be safe, preserving nearly 100% cell viability. These results suggest that this general approach is safe and versatile may be used for targeting and immobilizing bioactive factors onto collagen matrices in a variety of applications, including in the presence of live, seeded cells or in vivo onto endogenous extracellular matrix collagen.

SMARTPHONE-BASED DILATED FUNDUS PHOTOGRAPHY AND NEAR VISUAL ACUITY TESTING AS INEXPENSIVE SCREENING TOOLS TO DETECT REFERRAL WARRANTED DIABETIC EYE DISEASE.

PURPOSE: To compare clinical assessment of diabetic eye disease by standard dilated examination with data gathered using a smartphone-based store-and-forward teleophthalmology platform. METHODS: 100 eyes of 50 adult patients with diabetes from a health care safety-net ophthalmology clinic. All patients underwent comprehensive ophthalmic examination. Concurrently, a smartphone was used to estimate near visual acuity and capture anterior and dilated posterior segment photographs, which underwent masked, standardized review. Quantitative comparison of clinic and smartphone-based data using descriptive, kappa, Bland-Altman, and receiver operating characteristic analyses was performed. RESULTS: Smartphone visual acuity was successfully measured in all eyes. Anterior and posterior segment photography was of sufficient quality to grade in 96 and 98 eyes, respectively. There was good correlation between clinical Snellen and smartphone visual acuity measurements (rho = 0.91). Smartphone-acquired fundus photographs demonstrated 91% sensitivity and 99% specificity to detect moderate nonproliferative and worse diabetic retinopathy, with good agreement between clinic and photograph grades (kappa = 0.91 ± 0.1, P < 0.001; AUROC = 0.97, 95% confidence interval, 0.93-1). CONCLUSION: The authors report a smartphone-based telemedicine system that demonstrated sensitivity and specificity to detect referral-warranted diabetic eye disease as a proof-of-concept. Additional studies are warranted to evaluate this approach to expanding screening for diabetic retinopathy.

Design and ex vivo development of a suprachoroidal spacer implant to treat glaucoma.

Glaucoma is a leading cause of visual impairment and blindness in the United States and worldwide. Elevated intraocular pressure (IOP) has been identified as the only modifiable risk factor in glaucoma, and there exists a need for a glaucoma procedure that is safe, efficacious, and can be performed in the outpatient clinic setting. Suprachoroidal expansion has been explored as a method to lower IOP previously. The purpose of this work was to design a monolithic hydrogel implant that would not clear or degrade to potentially achieve long term (possibly permanent) IOP reduction. Here, we developed and showed ex vivo testing of a novel photo-crosslinked polyethylene glycol (PEG) suprachoroidal spacer implant delivered via a custom-designed injector system. We optimized the composition, shape, and mechanics of the implant to be suitable for implantation with the suprachoroidal space. We developed a microneedle injector system to deliver this implant. We showed precise control over implant location and volume occupied within the suprachoroidal space. Further preclinical testing is needed to demonstrate efficacy.

Quantifying particle concentration via AI-enhanced optical coherence tomography.

Efficient and robust quantification of the number of nanoparticles in solution is not only essential but also insufficient in nanotechnology and biomedical research. This paper proposes to use optical coherence tomography (OCT) to quantify the number of gold nanorods, which exemplify the nanoparticles with high light scattering signals. Additionally, we have developed an AI-enhanced OCT image processing to improve the accuracy and robustness of the quantification result.

Electrospun Nanofiber Membrane for Cultured Corneal Endothelial Cell Transplantation.

The corneal endothelium, comprising densely packed corneal endothelial cells (CECs) adhering to Descemet's membrane (DM), plays a critical role in maintaining corneal transparency by regulating water and ion movement. CECs have limited regenerative capacity within the body, and globally, there is a shortage of donor corneas to replace damaged corneal endothelia. The development of a carrier for cultured CECs may address this worldwide clinical need. In this study we successfully manufactured a gelatin nanofiber membrane (gelNF membrane) using electrospinning, followed by crosslinking with glutaraldehyde (GA). The fabricated gelNF membrane exhibited approximately 80% transparency compared with glass and maintained a thickness of 20 µm. The gelNF membrane demonstrated desirable permeability and degradability for a Descemet's membrane analog. Importantly, CECs cultured on the gelNF membrane at high densities showed no cytotoxic effects, and the expression of key CEC functional biomarkers was verified. To assess the potential of this gelNF membrane as a carrier for cultured CEC transplantation, we used it to conduct Descemet's membrane endothelial keratoplasty (DMEK) on rabbit eyes. The outcomes suggest this gelNF membrane holds promise as a suitable carrier for cultured CEC transplantation, offering advantages in terms of transparency, permeability, and sufficient mechanical properties required for successful transplantation.

Pupil size and LASIK: a review.

PURPOSE: To provide a literature review on the evidence both for and against pupil size as an independent predictor of adverse visual outcomes after LASIK. METHODS: Peer-reviewed publications on the effect of pupil size on LASIK outcomes since 2002 are reviewed. Particular attention was paid to the following attributes of each publication: type of study, number of patients or eyes, mean age, mean level of myopia, mean pupil size, testing conditions, ablation zone diameter, presence or absence of blend zones, and mean follow-up period. RESULTS: Among the 19 studies examined, none correlates a persistent relationship between pupil size and night vision complaints (NVCs) beyond 3 months when LASIK was performed with a 6.0-mm optical zone or larger ablation. The studies that did explicitly determine a correlation either included some or all patients with ablation zones smaller than 6.0 mm or did not specify ablation diameter at all. Among the studies that had drawn more mixed conclusions, the studies either covered short follow-up intervals (1 to 3 months) or showed a progressive improvement in NVCs over time in a relatively small patient cohort. CONCLUSIONS: As keratorefractive technology continues to evolve, the role of pupil size warrants further investigation; however, based on the literature reviewed herein, modern LASIK has negated the role of the low light pupil in predicting adverse visual outcomes after LASIK outside of the early postoperative period.

In vivo biocompatibility of two PEG/PAA interpenetrating polymer networks as corneal inlays following deep stromal pocket implantation.

This study compared the effects of implanting two interpenetrating polymer networks (IPNs) into rabbit corneas. The first (Implant 1) was based on PEG-diacrylate, the second (Implant 2) was based on PEG-diacrylamide. There were inserted into deep stromal pockets created using a manual surgical technique for either 3 or 6 months. The implanted corneas were compared with normal and sham-operated corneas through slit lamp observation, anterior segment optical coherence tomography, in vivo confocal scanning and histological examination. Corneas with Implant 1 (based on PEG-diacrylate) developed diffuse haze, ulcers and opacities within 3 months, while corneas with Implant 2 (based on PEG-diacrylamide) remained clear at 6 months. They also exhibited normal numbers of epithelial cell layers, without any immune cell infiltration, inflammation, oedema or neovascularisation at post-operative 6 month. Morphological studies showed transient epithelial layer thinning over the hydrogel inserted area and elevated keratocyte activity at 3 months; however, the epithelium thickness and keratocyte morphology were improved at 6 months. Implant 2 exhibited superior in vivo biocompatibility and higher optical clarity than Implant 1. PEG-diacrylamide-based IPN hydrogel is therefore a potential candidate for corneal inlays to correct refractive error.

In Situ-forming Collagen Hydrogels Crosslinked by Multifunctional Polyethylene Glycol as a Matrix Therapy for Corneal Defects: 2-Month Follow-up In Vivo.

PURPOSE: We recently showed that in situ-forming collagen gels crosslinked through multifunctional polyethylene glycol (PEG) supported corneal epithelialization 7 days after treatment of lamellar keratectomy wounds. In this study, we aimed to evaluate the longer-term regenerative effects of this gel in animals. METHOD: Corneal wound healing was assessed 60 days after lamellar keratectomy and gel treatment using slitlamp examination, optical coherence tomography (OCT), pachymetry, corneal topography, an ocular response analyzer, and tonometry. The corneas were evaluated for the presence of beta-tubulin, cytokeratin 3, zonula occludens-1, and alpha smooth muscle actin (SMA) markers. Gene expression of aldehyde dehydrogenase 3A1 (ALDH3A1), cluster of differentiation 31, CD163, alpha-SMA, hepatocyte growth factor, and fibroblast growth factor 2 (FGF-2) and protein expression of CD44 and collagen VI were evaluated. RESULTS: Intraocular pressure, corneal thickness, and hysteresis for the corneas treated with collagen-PEG gels did not significantly change compared with the saline group. However, placido disk topography revealed greater regularity of the central cornea in the gel-treated group compared to the saline group. The gel-treated group exhibited a lower degree of epithelial hyperplasia than the saline group. Immunohistochemical and gene expression analysis showed that the gel-treated corneas exhibited lower alpha-SMA expression compared with the saline group. CD163 and CD44 were found to be elevated in the saline-treated group compared with normal corneas. CONCLUSIONS: The in situ-forming collagen-PEG gel promoted epithelialization that improved central corneal topography, epithelial layer morphology, and reduced expression of fibrotic and inflammatory biomarkers after 60 days compared to the saline group.

Collagen Gels Crosslinked by Photoactivation of Riboflavin for the Repair and Regeneration of Corneal Defects.

Bioengineered corneal tissue is a promising therapeutic modality for the treatment of corneal blindness as a substitute for cadaveric graft tissue. In this study, we fabricated a collagen gel using ultraviolet-A (UV-A) light and riboflavin as a photosensitizer (PhotoCol-RB) as an in situ-forming matrix to fill corneal wounds and create a cohesive interface between the crosslinked gel and adjacent collagen. The PhotoCol-RB gels supported corneal epithelialization and exhibited higher transparency compared to physically crosslinked collagen. We showed that different riboflavin concentrations yielded gels with different mechanical and biological properties. In vitro experiments using human corneal epithelial cells (hCECs) showed that hCECs are able to proliferate on the gel and express corneal cell markers such as cytokeratin 12 (CK12) and tight junctions (ZO-1). Using an ex vivo burst assay, we also showed that the PhotoCol-RB gels are able to seal corneal perforations. Ex vivo organ culture of the gels filling lamellar keratectomy wounds showed that the epithelium that regenerated over the PhotoCol-RB gels formed a multilayer compared to just a double layer for those that grew over physically cross-linked collagen. These gels can be formed either in situ directly on the wound site to conform to the geometry of a defect, or can be preformed and then applied to the corneal wound. Our results indicate that PhotoCol-RB gels merit further investigation as a way to stabilize and repair deep and perforating corneal wounds.

Embedded 3D Bioprinting of Collagen Inks into Microgel Baths to Control Hydrogel Microstructure and Cell Spreading.

Microextrusion-based 3D bioprinting into support baths has emerged as a promising technique to pattern soft biomaterials into complex, macroscopic structures. It is hypothesized that interactions between inks and support baths, which are often composed of granular microgels, can be modulated to control the microscopic structure within these macroscopic-printed constructs. Using printed collagen bioinks crosslinked either through physical self-assembly or bioorthogonal covalent chemistry, it is demonstrated that microscopic porosity is introduced into collagen inks printed into microgel support baths but not bulk gel support baths. The overall porosity is governed by the ratio between the ink's shear viscosity and the microgel support bath's zero-shear viscosity. By adjusting the flow rate during extrusion, the ink's shear viscosity is modulated, thus controlling the extent of microscopic porosity independent of the ink composition. For covalently crosslinked collagen, printing into support baths comprised of gelatin microgels (15-50 µm) results in large pores (≈40 µm) that allow human corneal mesenchymal stromal cells (MSCs) to readily spread, while control samples of cast collagen or collagen printed in non-granular support baths do not allow cell spreading. Taken together, these data demonstrate a new method to impart controlled microscale porosity into 3D printed hydrogels using granular microgel support baths.

Artificial Intelligence Improves Patient Follow-Up in a Diabetic Retinopathy Screening Program.

Purpose: We examine the rate of and reasons for follow-up in an Artificial Intelligence (AI)-based workflow for diabetic retinopathy (DR) screening relative to two human-based workflows. Patients and Methods: A DR screening program initiated September 2019 between one institution and its affiliated primary care and endocrinology clinics screened 2243 adult patients with type 1 or 2 diabetes without a diagnosis of DR in the previous year in the San Francisco Bay Area. For patients who screened positive for more-than-mild-DR (MTMDR), rates of follow-up were calculated under a store-and-forward human-based DR workflow ("Human Workflow"), an AI-based workflow involving IDx-DR ("AI Workflow"), and a two-step hybrid workflow ("AI-Human Hybrid Workflow"). The AI Workflow provided results within 48 hours, whereas the other workflows took up to 7 days. Patients were surveyed by phone about follow-up decisions. Results: Under the AI Workflow, 279 patients screened positive for MTMDR. Of these, 69.2% followed up with an ophthalmologist within 90 days. Altogether 70.5% (N=48) of patients who followed up chose their location based on primary care referral. Among the subset of patients that were seen in person at the university eye institute under the Human Workflow and AI-Human Hybrid Workflow, 12.0% (N=14/117) and 11.7% (N=12/103) of patients with a referrable screening result followed up compared to 35.5% of patients under the AI Workflow (N=99/279; χ2df=2 = 36.70, p < 0.00000001). Conclusion: Ophthalmology follow-up after a positive DR screening result is approximately three-fold higher under the AI Workflow than either the Human Workflow or AI-Human Hybrid Workflow. Improved follow-up behavior may be due to the decreased time to screening result.

AI-Human Hybrid Workflow Enhances Teleophthalmology for the Detection of Diabetic Retinopathy.

Objective: Detection of diabetic retinopathy (DR) outside of specialized eye care settings is an important means of access to vision-preserving health maintenance. Remote interpretation of fundus photographs acquired in a primary care or other nonophthalmic setting in a store-and-forward manner is a predominant paradigm of teleophthalmology screening programs. Artificial intelligence (AI)-based image interpretation offers an alternative means of DR detection. IDx-DR (Digital Diagnostics Inc) is a Food and Drug Administration-authorized autonomous testing device for DR. We evaluated the diagnostic performance of IDx-DR compared with human-based teleophthalmology over 2 and a half years. Additionally, we evaluated an AI-human hybrid workflow that combines AI-system evaluation with human expert-based assessment for referable cases. Design: Prospective cohort study and retrospective analysis. Participants: Diabetic patients ≥ 18 years old without a prior DR diagnosis or DR examination in the past year presenting for routine DR screening in a primary care clinic. Methods: Macula-centered and optic nerve-centered fundus photographs were evaluated by an AI algorithm followed by consensus-based overreading by retina specialists at the Stanford Ophthalmic Reading Center. Detection of more-than-mild diabetic retinopathy (MTMDR) was compared with in-person examination by a retina specialist. Main Outcome Measures: Sensitivity, specificity, accuracy, positive predictive value, and gradability achieved by the AI algorithm and retina specialists. Results: The AI algorithm had higher sensitivity (95.5% sensitivity; 95% confidence interval [CI], 86.7%-100%) but lower specificity (60.3% specificity; 95% CI, 47.7%-72.9%) for detection of MTMDR compared with remote image interpretation by retina specialists (69.5% sensitivity; 95% CI, 50.7%-88.3%; 96.9% specificity; 95% CI, 93.5%-100%). Gradability of encounters was also lower for the AI algorithm (62.5%) compared with retina specialists (93.1%). A 2-step AI-human hybrid workflow in which the AI algorithm initially rendered an assessment followed by overread by a retina specialist of MTMDR-positive encounters resulted in a sensitivity of 95.5% (95% CI, 86.7%-100%) and a specificity of 98.2% (95% CI, 94.6%-100%). Similarly, a 2-step overread by retina specialists of AI-ungradable encounters improved gradability from 63.5% to 95.6% of encounters. Conclusions: Implementation of an AI-human hybrid teleophthalmology workflow may both decrease reliance on human specialist effort and improve diagnostic accuracy. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Tear Film Stability as a Function of Tunable Mucin Concentration Attached to Supported Lipid Bilayers.

In this work, we describe the development of a tunable, acellular in vitro model of the mucin layer of the human tear film. First, supported lipid bilayers (SLBs) comprised of the phospholipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and biotinyl cap PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)) are created on the surface of a glass dome with radius of curvature comparable to the human eye. Next, biotinylated bovine submaxillary mucins (BSM) are tethered onto the SLB using streptavidin protein. The mucin presentation can be tuned by altering the concentration of biotinylated BSM, which we confirm using fluorescence microscopy. Due to the optically smooth surface that results, this model is compatible with interferometry for monitoring film thickness. Below a certain level of mucin coverage, we observe short model tear film breakup times, mimicking a deficiency in membrane-associated mucins. In contrast, the breakup time is significantly delayed for SLBs with high mucin coverage. Because no differences in mobility or wettability were observed, we hypothesize that higher mucin coverage provides a thicker hydrated layer that can protect against external disturbances to thin film stability. This advance paves the way for a more physiological, interferometry-based in vitro model for investigating tear film breakup.