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Engineering physiologically relevant in vitro models of human organs remains a fundamental challenge. Despite significant strides made within the field, many promising organ‐on‐a‐chip models fall short in recapitulating cellular interactions with neighboring cell types, surrounding extracellular matrix (ECM), and exposure to soluble cues due, in part, to the formation of artificial structures that obstruct >50% of the surface area of the ECM. Here, a 3D cell culture platform based upon hydrophobic patterning of hydrogels that is capable of precisely generating a 3D ECM within a microfluidic channel with an interaction area >95% is reported. In this study, for demonstrative purposes, type I collagen (COL1), Matrigel (MAT), COL1/MAT mixture, hyaluronic acid, and cell‐laden MAT are formed in the device. Three potential applications are demonstrated, including creating a 3D endothelium model, studying the interstitial …