The microfluidic device composed of three main compartments was designed using CAD (computer-assisted design) on a photolithography mask, which consisted of a central chamber for an ECM-like hydrogel (diameter, 5 mm) and a pair of perfusable channels flanking the center chamber (connected to the chamber with a range of pillars that allow the generation of stable gradients by diffusion).

The microfluidic device in PDMS (Dow Corning) elastomer was fabricated by replica molding from an SU-8 (MicroChem)/silicon master based on a ratio of 10:1 to curing agent. The master mold was produced by ultraviolet (UV) lithography. After pouring PDMS on a mold, the mixture was degassed in a vacuum chamber for air bubble removal and cured at 80°C for 1 hour in the oven. The devices were then cut out by a razor blade; the fluidic connection ports were punched, and bonding to a glass slide was done after oxygen plasma of both surfaces and conformal contact (Harrick Plasma cleaner/sterilizer, 4 mbar, 120 s). Assembly quality control and characterization were done by optical 3D profilometry (VK-X250, KEYENCE, Germany).

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