Abstract
Purpose :
The inner wall endothelium of Schlemm’s Canal (SC) contributes to the generation of aqueous humour outflow resistance, which increases in glaucoma to cause elevated IOP. To study outflow resistance generation in vitro, we aim to develop an organ-on-chip model of SC. However, cultured SC cells often migrate into 3D hydrogels. In this project, we optimise microfluidic design, hydrogel composition and crosslinking, and seeding density to achieve a 2D SC cell monolayer on a 3D hydrogel within a microfluidic device.
Methods :
SC cells (SC89b) were cultured on hydrogels of rat tail collagen I (Thermo Fisher, 10224442) within two microfluidic designs: a commercial device that constrains the hydrogel between micropillars (Aim Biotech, idenTx 3) and a custom device that constrains the hydrogel between partial walls. Two collagen concentrations were tested (2 mg/ml and 8 mg/ml), with or without 0.01% riboflavin crosslinker (activated by blue light (470 nm, 8 Watts/cm2, 3 min)). SC cells were seeded at 1.0 or 1.7x106 cells/cm2 and imaged 24 or 72 hrs post-seeding by confocal microscopy. We measured the maximum penetration depth of SC cells into the hydrogel using ImageJ, with statistics performed using a 2-tailed Student’s t-test.
Results :
SC cells seeded within the custom microfluidic device with partial walls exhibited a more continuous cell layer, whereas the cell layer in the commercial device was frequently disrupted near the micropillars. Crosslinked hydrogels adhered better to the microchannel walls than hydrogels without crosslinker, which visibly detached. Increasing the cell seeding density tended to increase the penetration depth of SC cells into the hydrogel (143 ± 48 vs. 295 ± 18 µm [mean±SEM]; N = 3; p = 0.09). Increasing the collagen concentration at the lower seeding density significantly reduced penetration depth at 24 hrs (173 ± 41 vs. 10 ± 10 µm; N = 3, p = 0.05) and prevented migration entirely in 2 of 3 samples for at least 36 hrs.
Conclusions :
By tailoring microfluidic design, hydrogel composition, crosslinking and seeding density, it is possible to generate a SC endothelial monolayer that mimics the 2D morphology of the inner wall in situ. This design will facilitate imaging of SC cell biomechanics under physiological flows to better investigate the mechanism of outflow resistance generation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.