We present a method for depositing cells in the microchambers of a sealed microfluidic device and establishing flow across the chambers independently and serially. The device comprises a transparent poly(dimethylsiloxane) (PDMS) microfluidic network (MFN) having 2 cell chambers with a volume of 0.49 μL, 6 microchannels for servicing the chambers, and 1 microchannel linking both chambers. The MFN is sealed with a Si chip having 6 vias and ports that can be left open or connected to high-precision pumps. Liquids are drawn through each chamber in parallel or sequentially at flow rates from 0.1 to 10 μL min -1. Plugs of liquid as small as 0.5 μL can be passed in one chamber within 5 s to 5 min. Plugs of liquid can also be introduced into a chamber for residence times of up to 30 min. By injecting different liquids into 3 ports, 3 adjacent laminar streams of liquid can be drawn inside one chamber with lateral concentration gradients between the streams ranging from 20 to 500 μm. The flexibility of this device for depositing cells and exposing them to liquids in parallel or serially is illustrated by depositing two types of cells, murine N9 microglia and human SH-S5Y5 neuroblastoma. Microfluidic communication between the chambers is illustrated by stimulating N9 microglia using ATP to induce these cells to release plasma membrane vesicles. The vesicles are drawn through the second chamber containing neuroblastoma and collected in a port of the device for off-chip analysis using confocal fluorescence microscopy. Cells in the MFN can also be fixed using a solution of formaldehyde for further analysis after disassembly of the MFN and Si lid. This microfluidic device offers a simple, flexible, and powerful method for depositing two cell populations in separate chambers and may help investigating pathways between the cells populations. © 2009 Springer Science+Business Media, LLC.