Microfluidic device fabrication

A three-layer device was designed in AutoCAD that consisted of interaction channels, growth chambers, and main channels. The microfluidic master was pattered in three stages of photolithography using a micropattern generator (Heidelberg Instruments μPG 101). Unlike centrifuges, the spin coater protocol used for microfabrication is specified by an rpm. For the first layer, the silicon wafer was baked for 10 min at 200 °C and spin-coated at 4000 r.p.m. using SU-8 2000.5 (MicroChem) to generate 0.5 μm height. This layer was exposed to the interaction channels at 58 mW with a 47% dwell time using a 4 mm writehead, followed by a post-exposure bake for 30 min at 95 °C. The second layer was spin-coated at 3000 r.p.m. using a 26 : 1 mixture of SU-8 2000.5 to SU-8 3005, to produce 1.5 μm height. After aligning to the first layer, the wafer was exposed to the second patterning layer (growth chambers). Following an additional post-exposure bake, a third layer of SU-8 3025 photoresist was spin-coated at 3000 r.p.m. to generate 25 μm height. The wafer was exposed to the final layer consisting of the main channels, resistors, and inlets. Following a final post-exposure bake, the features were developed using SU-8 developer (MicroChem). The master was treated overnight with vapor phase (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane (Gelest) at room temperature. To fabricate each device, a 7 : 1 mixture of polydimethylsiloxane (Sylgard 184) to curing agent (Sylgard 184) was used to coat the master. After curing overnight at 100 °C, inlet and outlet holes were punched using a biopsy corer (WellTech). The surfaces were exposed to air plasma (Harrick Plasma PCD-32G) for 23 s to ionize the surface of the device to bond to the glass coverslips (ThermoFisher). Finally, the surfaces were bonded and baked for 1 hr at 100 °C to seal the device channels. For each experiment, the microfluidic device was flushed with 0.5% Tween 20 (Sigma-Aldrich) to prevent cells from adhering to the device. To load the cells into the growth chambers, a vacuum pressure of 330 mm Hg was applied.