Meryem Bouchemi1, Christophe Roubeix2, Karima Kessal2, Luisa Riancho2, Anne-Laure Raveu2, Hayet Soualmia3, Christophe Baudouin4, Françoise Brignole-Baudouin5. 1. Research Laboratory LR99ES11, Department of Biochemistry, Rabta Hospital, Tunis, Tunisia. Electronic address: bouchemimeryem@yahoo.fr. 2. INSERM, U968, Paris F-75012, France; UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris F-75012, France; CNRS, UMR_7210, Paris F-75012, France. 3. Research Laboratory LR99ES11, Department of Biochemistry, Rabta Hospital, Tunis, Tunisia; El Manar University, Higher Institute of Medical Technologies, Tunis, Tunisia. 4. INSERM, U968, Paris F-75012, France; UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris F-75012, France; CNRS, UMR_7210, Paris F-75012, France; Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS, CIC 503, Paris F-75012, France; Hop Ambroise Pare, AP HP, Dept Ophthalmology, F-92100 Boulogne, France; Univ Versailles St Quentin En Yvelines, F-78180 Montigny-Le-Bretonneux, France. 5. INSERM, U968, Paris F-75012, France; UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris F-75012, France; CNRS, UMR_7210, Paris F-75012, France; Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS, CIC 503, Paris F-75012, France; Univ Paris Descartes, Sorbonne Paris Cité, Paris F-75006, France; Faculté de Pharmacie de Paris, Univ Paris Descartes, Sorbonne Paris Cité, Paris, 17 F-75006, France.
Abstract
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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