In vitro physiological relevant model of human trabecular cells to define key events related to the onset of open angle glaucoma

Abstract: Glaucoma is a neurodegenerative disease identified as the second cause of blindness in the world. Oxidative attack induces a loss of trabecular meshwork cells (TM) and an impairment of its functionality, triggering the cascade that leads to glaucoma. Actually therapies can slow down intraocular pression, a disease hallmark, without prevent blindness. This makes urgent to set a physiologically-relevant model based on human TM, defining key events of disease, and checking the effectiveness of targeted therapies for treatment and prevention of glaucoma (1-5). So, we developed three different in vitro models of human TM, to check which could better mimic the glaucoma onset: conventional 2D and innovative 3D static and biodynamic models. Human TM were grown in standard conditions for 2D model; 3D-TM cultures, set up in Matrigel, were maintained in single flow bioreactor, LiveBox1 (LB1, IV-Tech srl, Italy) in static model, and for biodynamic model LiveBox1 was connected with a peristaltic pump (Live Flow, IV-Tech srl) with constant flow rate (6,7). To mimic acute stress TM were exposed to single H2O2 treatment (500-750 uM,) for 2 hour followed by 22 hours of recovery, whilst chronic stress was simulated by repeated H2O2 exposures with recovery phases in between, until 7 and 15 days (2D and both 3D models, respectively). At each checkpoint time, several biomarkers were analysed. Proliferation/metabolic state, by Alamar blue assay (8), evidenced a better healthy state of 3D vs 2D, confirmed by confocal imaging. Static and dynamic 3D-TM showed an efficient response to stress, in terms of NF-kB activation, mostly in biodynamic models; PARP cleavage, apoptotic marker, increased more in 2D than in 3D static cultures, and surprising resulted undetectable in biodynamic. The observed changes in homeostatic responses in 3D biodynamic model highlight the dynamic benefit in simulating a chronic stress, and is the starting point for glaucoma studies.