Microfluidic Long-Term Gradient Generator with Axon Separation Prototyped by 185 nm Diffused Light Photolithography of SU-8 Photoresist.

We have developed a cast microfluidic chip for concentration gradient generation that contains a thin (~5 µm² cross-sectional area) microchannel. The diffusion of diffused 185 nm ultraviolet (UV) light from an inexpensive low-pressure mercury lamp exposed a layer of the SU-8 photoresist from the backside and successfully patterned durable 2 µm-high microchannel mold features with smooth bell-shaped sidewalls. The thin channel had appropriate flow resistance and simultaneously satisfied both the rapid introduction of test substance and long-term maintenance of gradients. The average height and width at the half height of the channel, defined by a 2 µm-wide line mask pattern, were 2.00 ± 0.19 µm, and 2.14 ± 0.89 µm, respectively. We were able to maintain the concentration gradient of Alexa Fluor 488 fluorescent dye inside or at the exit of the thin microchannel in an H-shaped microfluidic configuration for at least 48 h. We also demonstrated the cultivation of chick embryo dorsal root ganglion neuronal cells for 96 h, and the directional elongation of axons under a nerve growth factor concentration gradient.