Establishing an in vitro Biomimetic Human Trabecular Meshwork Hydrogel Model

Abstract

Purpose : The trabecular meshwork (TM) is composed of TM cells and extracellular matrix (ECM). Abnormal TM cell function and ECM remodeling contribute to elevated intraocular pressure in glaucoma. Current in vitro TM models cannot accurately mimic the 3D TM cell-ECM interface. Here, we develop an in vitro biomimetic TM hydrogel using tissue-specific ECM proteins encapsulated with primary human TM (HTM) cells, and test its utility as glaucoma disease model.

Methods : Primary HTM cells were isolated from surgical discard corneal rims and characterized using accepted protocols. TM hydrogels were engineered by mixing HTM cells with functionalized collagen type I, elastin-like polypeptide and hyaluronic acid, and photoinitiator-mediated UV-crosslinking. Live/dead staining was used to investigate HTM cell viability. Construct contraction was determined by longitudinal imaging. Rheology and scanning electron microscopy (SEM) analyses were used to determine TM hydrogel stiffness and microarchitecture. Glaucomatous conditions were induced with dexamethasone (DEX) ± ROCK inhibitor treatment (Y27632). Expression of myocilin and fibronectin, and F-actin organization were determined by immunohistochemistry.

Results : HTM cells showed increased myocilin mRNA (2.1-fold, p<0.01) and protein (82.1%) expression with DEX vs. controls, consistent with reference standards. Live/dead staining revealed high viability of HTM cells after UV crosslinking for 1-30 s (≥90%); 60 s crosslinking resulted in increased cell death vs. all other groups (p<0.05). Rheology testing showed tunable hydrogel stiffness as a function of UV crosslinking time; higher storage vs. loss moduli were noted across groups (p<0.05). SEM analysis revealed a dense microarchitecture with small pores of ≤10 μm, appropriate for a JCT-TM hydrogel model. HTM cells proliferated and contracted the 3D hydrogel network in a time-dependent manner. DEX exposure induced more crosslinked actin networks, and higher myocilin and fibronectin expression vs. controls. DEX-treated TM hydrogels also showed decreased contraction and increased stiffness (p<0.05), which was largely rescued with Y27632 co-treatment. Similar results were observed with TGF-β2 induction/latrunculin B rescue.

Conclusions : We have developed a novel in vitro biomimetic TM hydrogel model with tunable stiffness that allows for detailed investigation of the 3D TM cell-ECM interactions under normal and glaucomatous conditions.

This is a 2020 ARVO Annual Meeting abstract.