Lymphatic vessels play a critical role in maintaining fluid balance throughout the body, and the eye is no exception. Similar to systemic lymphatics, which remove excess fluid from tissues to maintain homeostasis, specialized lymphatic-like channels in the eye are responsible for draining aqueous humor—the clear fluid that circulates within the anterior chamber and helps maintain intraocular pressure (IOP). Disruption of this drainage system leads to fluid accumulation, elevated IOP, and, ultimately, glaucomatous damage to the optic nerve. Glaucoma remains a major public health challenge, affecting 60 million people globally and often progressing silently until significant vision loss occurs. Despite its prevalence, many of the cellular and molecular mechanisms underlying impaired aqueous humor drainage remain poorly understood, in part due to limitations of conventional experimental models.
To address these challenges, we engineered an “eye lymphatics-on-a-chip” platform that faithfully recapitulates the human aqueous humor drainage pathway, including Schlemm’s canal (SC) and the surrounding trabecular meshwork (TM). This microphysiological system allows precise control of cellular microenvironments, fluid flow, and biochemical signaling, enabling mechanistic studies of ocular fluid dynamics in a human-relevant context. Using this platform, we were able to investigate how dysfunction in the trabecular meshwork compromises Schlemm’s canal function, a process central to steroid-induced glaucoma—a condition in which corticosteroid treatments inadvertently raise IOP and increase glaucoma risk.
Schlemm’s canal endothelial cells are lymphatic-like, expressing markers such as Prox1 and VEGFR3, and respond to the lymphangiogenic factor VEGFC. Based on this, we first tested human lymphatic endothelial cells (LECs) within the SC channel as a proof of concept. Remarkably, LECs closely mimicked SC cell behavior in our system, validating their utility as a surrogate for primary SC cells in glaucoma modeling. These initial experiments were subsequently confirmed with primary human SC endothelial cells, ensuring that our observations were physiologically relevant and robust.
An important “behind-the-scenes” lesson emerged during model development. Initially, we tested monocultures of either SC or TM cells, but neither responded to dexamethasone (DEX), the steroid commonly used to induce ocular hypertension in experimental models. While this lack of response was initially disappointing, it provided a critical insight: modeling steroid-induced glaucoma requires both cell types in proximity. Only in coculture did we observe reduced aqueous humor outflow and changes in endothelial junction structure, demonstrating that interactions between TM cells and SC cells are essential to recapitulate disease mechanisms. This finding underscores the importance of minimal cellular conditions for accurately modeling complex tissue-level physiology and highlights a key advantage of organ-on-chip systems over traditional monoculture approaches.
Our device also reproduces physiologically relevant aqueous humor outflow velocities, an essential feature for mimicking in vivo conditions. The measured average flow velocity in the chip ranged between 1.5–2.0 μm/s, which aligns closely with estimates from human eyes. Based on published anatomical data, the average width of Schlemm’s canal is approximately 233 μm, and its perimeter is roughly 38 mm, giving a total outflow surface area of ~8.85 mm². Given a typical human aqueous humor flow rate of 1.5–3.0 μL/min (0.025–0.05 μL/s), the calculated physiological outflow velocity is approximately 2.8–5.7 μm/s. Our chip reproduces this range, demonstrating that the platform can mimic fluid dynamics at physiologically relevant scales, a critical feature for studying glaucoma pathophysiology.
Using this system, we identified a previously unrecognized signaling mechanism involving ALK5 and VEGFC that contributes to impaired fluid drainage and elevated IOP under steroid treatment. These findings were validated in vivo using mouse models, through both pharmacological interventions and genetic modifications. Importantly, these results suggest two potential therapeutic strategies for protecting or restoring aqueous humor outflow: blocking ALK5 activity or supplementing VEGFC in the eye alongside steroid treatment. This discovery opens new avenues for the development of targeted therapies to prevent steroid-induced glaucoma, a condition that currently lacks precise interventions.
Looking forward, our platform provides a unique opportunity to investigate additional genetic and molecular targets relevant to glaucoma beyond steroid-induced forms. Many genes implicated in glaucoma affect multiple cell types, making it difficult to dissect their individual contributions using conventional animal models. In contrast, our system allows for precise genetic manipulation of each cell type separately, followed by combination in the device to study their interactions. This modularity enables a deeper understanding of the diverse mechanisms underlying various forms of glaucoma and may accelerate the identification of novel therapeutic targets.
The broader significance of this work is reinforced by recent priorities set by the NIH and FDA, which emphasize the importance of New Approach Methods (NAMs) for generating human-relevant data in biomedical research. Our “eye lymphatics-on-a-chip” represents a NAM that not only captures human ocular physiology more faithfully than traditional models but also allows for mechanistic and translational studies that were previously challenging or impossible.
In summary, our study demonstrates that a human eye lymphatics-on-a-chip platform can accurately model aqueous humor drainage, recapitulate steroid-induced glaucoma, and reveal previously unknown mechanisms regulating intraocular pressure. By enabling controlled coculture of TM and lymphatic-like SC cells, reproducing physiological flow rates, and supporting precise genetic and pharmacological manipulations, this system provides a powerful tool for glaucoma research. Beyond glaucoma, this platform may serve as a model for studying other ocular diseases involving fluid regulation, offering a new paradigm for human-relevant eye research and therapeutic development.