Co-culture with trabecular meshwork cells promotes barrier function in an organ-on-chip model of Schlemm’s canal inner endothelial wall

Abstract

Purpose : Platforms for glaucoma drug screening rely on reconstituting in vitro the inner wall endothelium of Schlemm’s canal (SC), the only continuous barrier to aqueous humour outflow. As SC and trabecular meshwork (TM) cell interactions regulate outflow in vivo, we hypothesize that co-culture with TM cells will promote SC barrier function within our existing organ-on-chip model of the inner wall (Wheeler et al., ARVO 2023).

Methods : The model comprises a microfluidic device that constrains a hydrogel between two media channels. Primary human SC cells were seeded on the hydrogel surface, while primary human TM cells were seeded within a 3D collagen I hydrogel (7mg/ml). The model was cultured for 5 days with or without TM cells, either under static conditions or with flow conditioning by perfusion at 1 cmH₂O in the basal-to-apical (BA) or apical-to-basal (AB) direction for 2-3 hrs/day. SC and TM cells were labelled with cell tracker green and red, respectively, and SC cell migration into the hydrogel was imaged by confocal microscopy. Giant vacuole-like structures (GVLs) were imaged under BA perfusion (0.5cmH₂O) in calcein-AM stained SC cells after 9 days of flow conditioning.

Results : Under static conditions, the presence of TM cells reduced SC cell migration into the hydrogel after 5 days [82±69µm vs. 176±63µm, N=4 vs. 6 chips, p=0.005]. Flow conditioning led to a more elongated and branching TM cell morphology relative to static culture. GVLs were observed in SC cells after 9 days of BA flow conditioning with TM cells. GVLs were 23.5±16.7µm in diameter [N=14; Figure] and were highly dynamic, exhibiting recurrent growth, collapse and occasional fusion. Without TM cells, SC-cell seeded hydrogels detached from the microchannel walls by 9 days, leading to chip failure.

Conclusions : As GVLs are unlikely to form in the absence of a hydraulically resistive endothelial barrier, flow conditioning and co-culture with TM cells appears necessary to reconstitute inner wall barrier function in vitro. More faithful in vitro models of the inner wall would underpin future screening platforms for glaucoma drug discovery and development.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

A: Confocal slice of SC/TM cells (green) on a collagen hydrogel (red) under BA pressure gradient. White arrows: GVLs within an SC cell. B: SC cells with flat morphology, migrating into hydrogel without TM cells or pressure gradient.