Title: Advanced 3D human-based in vitro models to identify glaucoma-specific molecular markers and to study the pathological crosstalk leading to neuroinflammation
Abstract:
Retinal cell death is a common pathological hallmark of several neurodegenerative diseases, including glaucoma, diabetic retinopathy, and age-related macular degeneration. Among these, Primary Open-Angle Glaucoma (POAG) represents the most prevalent form and stands as the second leading cause of irreversible blindness worldwide. POAG is primarily characterized by elevated Intraocular Pressure (IOP), which triggers progressive injury to the optic nerve and the subsequent loss of Retinal Ganglion Cells (RGCs). Developing novel neuroprotective strategies and accurately assessing the efficacy and adverse effects of potential treatments heavily relies on robust, reproducible experimental models.
We developed an innovative, human-based in vitro 3D model of the trabecular meshwork utilizing HTMCs embedded in a Matrigel® scaffold and cultured within a dynamic perfusion bioreactor. To mimic the progression from early to advanced POAG stages, the platform was subjected to prolonged OS and to Higher Pressure (HP) flow, achieved using a calibrated plunger at the outlet pipe. Characterization of adaptive and degenerative responses involved assessments of F-actin expression, cell viability, mitochondrial function, proinflammatory cytokine secretion, and apoptotic signaling pathways. OS showed a pivotal role in HTMC degeneration, compared to the increase in Pressure alone, while, in combined OS and HP exposure, HP exacerbates OS effects like in advanced glaucoma stage.
This 3D-HTMC millifluidic platform has proved to be a useful tool to test the effectiveness of commercial eye drop formulations, with different target of therapeutic actions. The downstream effects on the retina were then assessed by treating human 3D neuron-like cells with conditioned medium from stressed HTMC, and it was observed an impairment of neural cell behavior by way of a cellular cross-talk.
Following this, to develop a more reliable model of retinopathy affecting glaucoma, our aim is to enhance the millifluidic platform by adding Retinal Organoids (RO) derived from human Induced Pluripotent Stem Cells. RO, isolated and cultured at least for 35 days, showed a different regulation of more than 200 retinal proteins, and at the end-point of procedure, two RGC subtypes were identified among nearly 40 different types of cells arranged in a complex manner. Proteomic analysis evidenced more than 7000 proteins and 2000 of them were expressed, at different time-points and levels.
Preliminary data seem to be a valuable starting point to study the pathological crosstalk leading to neuroinflammation and pathophysiological changes affecting the retina. We do hope that these promising results will lead us to propose this model in the near future as a useful tool for testing other neuroprotective strategies.
These controlled platforms minimize confounding systemic variables, align with the 3Rs principles (Replacement, Reduction, and Refinement), and provide a highly rigorous framework for screening novel therapeutic compounds. Ultimately, these models bridge the gap between basic research and translational clinical strategies for glaucoma and related optic neuropathies.
