Mucin‐Like Glycoproteins Modulate Interfacial Properties of a Mimetic Ocular Epithelial Surface

Abstract Dry eye disease (DED) has high personal and societal costs, but its pathology remains elusive due to intertwined biophysical and biochemical processes at the ocular surface. Specifically, mucin deficiency is reported in a subset of DED patients, but its effects on ocular interfacial properties remain unclear. Herein a novel in vitro mucin‐deficient mimetic ocular surface (Mu‐DeMOS) with a controllable amount of membrane‐tethered mucin molecules is developed to represent the diseased ocular surfaces. Contact angle goniometry on mimetic ocular surfaces reveals that high surface roughness, but not the presence of hydrophilic mucin molecules, delivers constant hydration over native ocular surface epithelia. Live‐cell rheometry confirms that the presence of mucin‐like glycoproteins on ocular epithelial cells reduces shear adhesive strength at cellular interfaces. Together, optimal surface roughness and surface chemistry facilitate sustainable lubrication for healthy ocular surfaces, while an imbalance between them contributes to lubrication‐related dysfunction at diseased ocular epithelial surfaces. Furthermore, the restoration of low adhesive strength at Mu‐DeMOS interfaces through a mucin‐like glycoprotein, recombinant human lubricin, suggests that increased frictional damage at mucin‐deficient cellular surfaces may be reversible. More broadly, these results demonstrate that Mu‐DeMOS is a promising platform for drug screening assays and fundamental studies on ocular physiology.

(Bruker, Santa Barbara, CA). The cells were cultured on 50-mm glass bottom petri dishes pretreated with collagen. A force volume of 10*10 μm (4*4 pixels) was acquired for each cell. The Hertzian contact model was used to extract the Young's modulus from the force curves by the NanoScope Analysis 1.9 software. The AFM was mounted on top of a Zeiss Axio Observer Z1 inverted epifluorescence microscope outfitted with a 20X objective.
Live Cell Rheometer (LCR) The basic design of the LCR has been described previously. [33][34][35] Some modifications have been implemented to improve the throughput of the instrument.
Sample plate preparation To prepare the bottom plate, a 3-mm deep opening was created by a ranch on the side of a 35-mm glass-bottom petri dish (Cellvis). To assemble the top plate, a 12-mm circle coverslip was glued with Norland NOA60 optical adhesives onto a 3D-printed handler for the force sensor to approach. The handlers were printed either by an Ultimaker 2 with polylactic-acid or through Shapeways 3D printing service with versatile plastics.
Cell culture Bottom and top plates were coated with rat-tail Type I collagen at 5 μg/cm 2 for 30 minutes at 37 o C. hTCEpi cells were seeded onto the bottom plates at 350,000 cells/ml and HCjE were seeded onto the top plates at 200,000 cells/ml in the growth medium (GM). The top plates were kept in 24-well plastic tissue culture plate with the coverslip side facing up.
After the cells reached confluence, the GM was replaced with the stratification medium. LCR experiments were done on the seventh day of stratification. To generate mucin-deficient dryeye model, cells were treated with 0.5 μg/ml StcE in stratification medium overnight.
Adhesion assay On the day of the experiment, the cell culture was washed with PBS three times, and switched to CO 2 -independent medium supplemented with L-glutamine, 10% fetal bovine serum, 10 ng/mL EGF, and 1% penicillin-streptomycin. To test the lubrication effect of lubricin, stock solutions of lubricin (2.20 mg/mL amino acid concentration, 4.40 mg/mL total protein concentration) were added into full CO 2 -independent medium to a desired final concentration. As control groups, bovine serum albumin was reconstituted into 5 mg/ml stock solution in PBS and then added into full CO 2 -independent medium to a desired final concentration.
To start the adhesion assay, the top plates were gently placed onto the bottom plates. The assembled plates were kept at 37 o C and ambient CO 2 level for 2 hours before the step strain experiments.
Step strain experiment The experiment was controlled by a customized MATLAB code. After the force sensor was brought in contact with the top plate, a user-defined step motion was applied through the micromanipulator. A DAQ board (DAQ USB6008, National Instruments) collected the voltage readings as a function of time from the force sensor which were converted to force levels, , using a known conversion factor given by the manufacturer.
The nominal contact area, , was determined as the area of a 12-mm circle coverslip. The shear stress exerted on the corneal epithelial cell monolayers was defined as shear force over area, or . The gap, , was determined in gap height measurement (see below). The shear strain exerted on the corneal epithelial cell monolayer was defined as the shear distance over the gap height in the normal direction, or .
The apparent modulus as a function of time was defined as shear stress over shear strain and shown in Eq 1. An F-test on the variance of sum of squared residuals has shown that the twocomponent exponential function performed statistically better than the one-component exponential function, but was not outperformed by the three-component exponential function.
The peak modulus was defined as the first modulus value after the step strain was applied.
The plateau modulus was extracted from the two-component exponential fitting, .
Statistical analysis was performed using a two-tail Welch's t-test.