Effect of benzalkonium chloride on trabecular meshwork cells in a new in vitro 3D trabecular meshwork model for glaucoma

Abstract

PurposeTo validate a new culture model of primary human trabecular meshwork cells (p-hTMCs) using Matrigel®, in order to mimic in vitro 3D–TM organization, and to investigate the proinflammatory effect of benzalkonium chloride (BAK) in 3D p-hTMC cultures. Methodsp-hTMCs, seeded onto Matrigel®-coated inserts were stimulated with BAK (10−4%), dexamethasone (DEX) (10−6M) or transforming growth factor-beta 2 (TGF-β2) (5ng/ml) for 48h and observed with confocal microscopy. The BAK effect at 10−4% or 5.10−3% on the gene expressions of interleukin-6 (IL-6), interleukin-8 (IL-8) and matrix metalloproteinase (MMP-9) was investigated using qRT-PCR in 2D and 3D p-hTMC cultures. Resultsp-hTMCs seeded in Matrigel® were able to organize themselves in a 3D-spatial conformation in the different conditions tested with cross-linked actin network (CLAN) formation in presence of DEX or TGF-β2 and intercellular space contraction with TGF-β2. IL-6 and IL-8 gene expressions increased in presence of BAK in 2D and in 3D p-hTMC cultures. BAK 10−4% only showed a tendency to stimulate MMP-9 expression in p-hTMCs after 24h-recovery. ConclusionsWe investigated this new 3D–TM in vitro model in Matrigel® matrix for pathophysiological and toxicological purposes. It appears as a new promising tool for a better understanding of TM behavior in physiological and stress conditions, as well as toxicological evaluations of antiglaucoma eyedrops and preservatives.