Generation of stable soluble-factor gradients in microfluidic devices enables studies of various cellular events such as chemotaxis and differentiation. However, many gradient devices directly expose cells to constant fluid flow and that can induce undesired responses from cells due to shear stress and/or wash out of cell-secreted molecules. Although there have been devices with flow-free gradients, they typically generate only a single condition and/or have a decaying gradient profile that does not accommodate long-term experiments. Here we describe a microdevice that generates several chemical gradient conditions on a single platform in flow-free microchambers which facilitates steady-state gradient profiles. The device contains embedded normally-closed valves that enable fast and uniform seeding of cells to all microchambers simultaneously. A network of microchannels distributes desired solutions from easy-access open reservoirs to a single output port, enabling a simple setup for inducing flow in the device. Embedded porous filters, sandwiched between the microchannel networks and cell microchambers, enable diffusion of biomolecules but inhibit any bulk flow over the cells.

Here we describe a simple yet efficient approach to making through-holes in a bound polydimethylsiloxane membrane for use in 3D microfluidic applications. Localized tearing of an elastomeric membrane is achieved by ripping an elastomeric stamp that is bound to the membrane by posts at desired regions. The tears in the membrane are confined by the underlying channel architecture of the substrate to which the membrane is bound. By varying the membrane thickness and channel dimensions, holes of different sizes can be obtained. This high-throughput method of generating through-holes will enable the design of complex microfluidic devices that require crossing of channel networks.