Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model

Abstract

Intraocular pressure (IOP) is mostly regulated by aqueous humor outflow through the human trabecular meshwork (HTM) and represents the only modifiable risk factor of glaucoma. The lack of IOP-modulating therapeutics that targets HTM underscores the need of engineering HTM for understanding the outflow physiology and glaucoma pathology in vitro. Using a 3D HTM model that allows for regulation of outflow in response to a pharmacologic steroid, a fibrotic state has been induced resembling that of glaucomatous HTM. This disease model exhibits HTM marker expression, ECM overproduction, impaired HTM cell phagocytic activity and outflow resistance, which represent characteristics found in steroid-induced glaucoma. In particular, steroid-induced ECM alterations in the glaucomatous model can be modified by a ROCK inhibitor. Altogether, this work presents a novel in vitro disease model that allows for physiological and pathological studies pertaining to regulating outflow, leading to improved understanding of steroid-induced glaucoma and accelerated discovery of new therapeutic targets.

Model systems for the study of steroid-induced IOP elevation

“…Initial attempts to utilize commercially available mixed cellulose ester (HATF) filters for growing the TM cells were only met with limited success (Perkins et al, 1988) although HTM cells grown on such filters responded to steroid treatment by changing transendothelial flow (Underwood et al, 1999). Similarly polyester trans-well inserts do not provide optimal support (Torrejon et al, 2015) as cells tend to orient on these membranes randomly and membranes provide significant resistance to flow because of low porosity. More recently, trans-well inserts have also been used to culture HSC cells for physiological studies (Pedrigi et al, 2011).…”

“…More importantly cells grown on SU-8 respond to steroid (prednisolone acetate) in a physiologic manner by decreasing simulated outflow facility and decreasing their phagocytic activity. These changes were also associated with an increase in production of various ECM components (Torrejon et al, 2015). …”

“…Importantly, culturing of HTM cells and HSC cells on SU-8 membranes has allowed the development of basal to basal co-cultures of the two cell types, thus creating a 3D biomimetic culture of the JCT area (Torrejon, 2015b). These cultures are responsive to steroid treatment and thus represent an ideal in-vitro system for studies in the pathogenesis of steroid-induced IOP elevation as they allow for modeling of the complex interactions between cells in the outflow pathways.…”

Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

“…Eliminating or reducing the number of host TM cells are also useful. In a recent study, an ex vivo 3D bioengineered TM scaffold repopulated by human primary TM cells was developed, but without the distinct layers of juxtacanalicular, corneoscleral and uveal TM (Torrejon et al, 2016). Transgenic (Tg-MYOC Y437H (Zhu et al, 2016) ) and laser photocoagulation mouse models (Yun et al, 2014) have also been used or proposed for TM transplantation, respectively.…”

Biomaterials for Regenerative Medicine Approaches for the Anterior Segment of the Eye

“…Photolithography has been used to create an in vitro scaffold for human trabecular meshwork cells with features that are 7 µm wide, about 5 µm high, and 12 µm apart out of SU-8 2010 photoresist to mimic the outflow environment in vivo. [59] These were coated with gelatin and seeded with human trabecular meshwork cells, which after culturing for 14 d formed a 40 µm thick trabecular meshwork structure. This was demonstrated to behave as expected in response to steroids and therefore could act as a useful in vitro 3D model for glaucoma drug development.…”