A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

Abstract

Dry eye disease (DED) affects more than 100 million people worldwide, causing significant patient discomfort and imposing a multi-billion-dollar burden on global health care systems. In DED patients, the natural biolubrication process that facilitates pain-free blinking goes awry due to an imbalance of lipids, aqueous medium, and mucins in the tear film, resulting in ocular surface damage. Identifying strategies to reduce adhesion and shear stresses between the ocular surface and the conjunctival cells lining the inside of the eyelid during blink cycles is a promising approach to improve the signs and symptoms of DED. However, current preclinical models for screening ocular lubricants rely on scarce, heterogeneous tissue samples or model substrates that do not capture the complex biochemical and biophysical cues present at the ocular surface. To recapitulate the hierarchical architecture and phenotype of the ocular interface for preclinical drug screening, we developed an in vitro mucin-deficient DED model platform that mimics the complexity of the ocular interface and investigated its utility in biolubrication, antiadhesion, and barrier protection studies using recombinant human lubricin, a promising investigational therapy for DED. The biomimetic platform recapitulated the pathological changes in biolubrication, adhesion, and barrier functionality often observed in mucin-deficient DED patients and demonstrated that recombinant human lubricin can reverse the damage induced by mucin loss in a dose- and conformation-dependent manner. Taken together, these results highlight the potential of the platformand recombinant human lubricinin advancing the standard of care for mucin-deficient DED patients.

The Role of Membrane‐Tethered Mucins in Axial Epithelial Adhesion in Controlled Normal Stress Environments

“…Both cell lines express mucins at their apical surfaces, albeit with different expression levels and distributions. [ 12,15 ] To establish the corneal/conjunctival epithelial interfaces, HCjE cells were cultured on circular collagen‐coated glass coverslips glued on custom‐designed frames (referred to as top plates, TP, Experimental Section), and hTCEpi cells were cultured on collagen‐coated glass‐bottom culture dishes (referred to as bottom plates, BP, Experimental Section). To measure the distance between cell layers, diffraction‐limited fluorescent beads were adsorbed to the glass surfaces via van der Waals forces prior to cell seeding.…”

“…The gap distance was determined via laser scanning microscopy. Upon reaching confluency (>70%), these cell types can be differentiated into stratified epithelia expressing membrane‐tethered mucins (Figure S1, Supporting Information, [ 12,14,16 ] and Figure 1A). High calcium and serum content in the culture medium induces the stratification process, which is marked by the expression of membrane‐associated mucins on the epithelia.…”

“…The model system we employ here accurately captures many important elements of the contact/interaction between the (human/mammalian) corneal surface and the conjunctiva (eyelid). As previously established, [ 12,14 ] the model permits the assessment of the collective behavior of cells (in a stratified layer) on a parallel and flat geometry with mucin expression in the native environment. Furthermore, this environment critically depends on the level of mucin coverage.…”

“…Stratified ocular epithelial surfaces offer a flattened morphology and a homogeneous mucin expression. [ 12 ] To quantify the contribution of mucins to axial adhesion, the integrity of the mucin layer was perturbed by inducing mucin deficiency with a recombinant protease, StcE. [ 13 ] StcE is a bacterial protease from Escherichia coli that cleaves glycoproteins that contain mucin‐like domains.…”

Effect of Recombinant Human Lubricin on Model Tear Film Stability

“…Previous work has shown that rh -lubricin is able to adsorb onto and form a viscous, hydrated layer on hydrophilic glass surfaces, 11 as is the case during our heating step. Other in vitro models incorporating corneal and conjunctival epithelial cells also show that both topical treatment using native human lubricin (Seo et al, 2019) 23 and the incubation of cells in solution with rh -lubricin 21 resulted in the adherence of lubricin to the epithelial surface. We demonstrated that adsorbed rh- lubricin alone was not sufficient to maintain a fully wetted area when rh- lubricin was absent from the model tear film.…”