Reversibly-sealable microfluidic platform for multi-molecule gradient delivery to large adherent cell cultures

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.