Investigating the coupled effects of stiffness and stretch on the trabecular meshwork cells using a hydrogel-integrated microfluidic system.

Glaucoma is characterized by progressive stiffening of the trabecular meshwork (TM), which is associated with elevated intraocular pressure and tissue dysfunction. Although substrate stiffness and mechanical stretch both regulate TM homeostasis, their combined effects remain poorly understood. Here, a hydrogel-integrated microfluidic platform is presented that enables simultaneous control of substrate stiffness, via tunable gelatin methacryloyl (GelMA) hydrogels, and equibiaxial quasi-static stretch, via hydraulic actuation. Finite element analysis validates the applied strain field, and optimized crosslinking ensures structural stability. As an initial demonstration, primary normal TM (nTM) and glaucomatous TM (gTM) cells were cultured under coupled stiffness and stretch conditions. The nTM cells modulated α-smooth muscle actin (α-SMA), myocilin (MYOC), matrix metalloproteinase-2 (MMP2), and collagen type I (COL1) with the imposed mechanical cues, whereas the gTM cells showed elevated α-SMA, reduced mechanical regulation of MMP2, and diminished stretch-mediated COL1 suppression, while retaining stiffness-dependent FAK phosphorylation and MYOC responses. These differences emerged primarily under combined stiff and stretched conditions, indicating that coupled mechanical cues can reveal candidate disease-relevant phenotypes not apparent under either cue alone. The platform resolves stiffness- and stretch-dependent responses in TM cells and provides a scalable basis for validating these responses across additional donors.