<p>Spatial manipulation of flow gradients and chemical microenvironments is essential for understanding fundamental biological mechanisms and investigating therapeutic responses in adherent cells. Convection-dominated gradient generators in microfluidic devices enable tunable chemical and shear stress gradients across large cell culture areas. However, most concentration generators are irreversibly sealed and operate in a narrow range of shear stresses, which restricts access to the cells after treatment and the physiological relevance of the flow conditions. Here, we present a reversibly sealable microfluidic platform that enables spatiotemporally controlled delivery of multiple small molecules to mammalian cells grown on large glass coverslips. Our device generates a relatively wide range of shear stresses and robust, spatially predictable chemical gradients across centimeter-scale areas and provides optical access compatible with live-cell imaging; it operates in the Stokes and laminar flow regimes. A mechanical sandwich clamp enables leak-free perfusion into the cell culture chamber and access to the cells after treatment. We experimentally and numerically demonstrate the ability to modulate the amount of mixing between co-flowing streams of small molecules. We verify the uptake of fluorophores across a monolayer of cells and assess their viability after perfusion and removal from the device. This platform provides a versatile and reusable approach for studying cellular responses to microenvironmental gradients in varied physiologically relevant shear stress conditions.</p>