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

• Published in: Toxicology in vitro 2017-06, Vol.41, p.21-29
• Main Authors: Bouchemi, Meryem, Roubeix, Christophe, Kessal, Karima, Riancho, Luisa, Raveu, Anne-Laure, Soualmia, Hayet, Baudouin, Christophe, Brignole-Baudouin, Françoise
• Format: Article
• Language: English
• Subjects: Actin; Benzalkonium chloride; Benzalkonium Compounds - toxicity; Cell culture; Cell Culture Techniques; Cells; Cells, Cultured; Collagen; Confocal microscopy; Conformation; Contraction; Crosslinking; Cytokines; Cytoskeleton - drug effects; Dexamethasone; Dexamethasone - toxicity; Drug Combinations; Gelatinase B; Gene Expression - drug effects; Glaucoma; Growth factors; Humans; Inflammation; Inserts; Interleukin 6; Interleukin 8; Interleukin-6 - genetics; Interleukin-8 - genetics; Laminin; Life Sciences; Matrigel; Matrix metalloproteinase; Matrix Metalloproteinase 9 - genetics; Metalloproteinase; Microscopy; Ophthalmic Solutions - toxicity; Preservatives; Preservatives, Pharmaceutical - toxicity; Proteoglycans; Three dimensional models; Three-dimensional culture; Trabecular meshwork; Trabecular Meshwork - cytology; Transforming Growth Factor beta2 - toxicity; Transforming growth factor-b1
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2017.02.006

To 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. p-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. p-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. We 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. •Matrigel® was used to develop a 3D-phTMCs culture.•3D-phTMCs treated with TGF-β2 and DEX may be used as glaucoma in vitro models.•The effect of BAK, an eyedrops preservative, was investigated in 3D-phTMC culture compared to 2D culture.•Gene expression of IL-6 and IL-8 increased under the effect of BAK.