Keywords:femtosecond laser, microfluidic device, bio-application
Cell transplantation is the current gold standard for the treatment of brain damage. Neuronal integration post-transplantation between donor and host has always been the biggest challenge. Misalignment of neuronal integration site and timing could lead to severe side effects. Therefore, guided neurite growth, both spatially and temporally, is necessary for achieving functional brain recovery. Femtosecond laser has been used for precision spatial neurite guidance. The exclusive feature of femtosecond laser allows real-time manipulation during cell culture. Therefore, not only spatial but also temporal control of neuronal manipulation could be attained. In this work, we developed a real-time manipulation system utilizing femtosecond laser for guiding spatiotemporal arrangements of PC12 cells by nerve growth factor (NGF) stimulation in a microfluidic device. The microfluidic device consisted of four layers: PDMS chamber, thin-glass (thickness, 4 μm), PDMS channel, and glass substrate. PC12 cell line was cultured on the thin-glass. NGF (100 ng/ml) was introduced into the PDMS channel. After 1 DIV, femtosecond Ti:Sapphire laser amplifier (800 nm, 150 fs, 1 kHz, 250 nJ/pulse) was focused through a 10x objective (NA = 0.25) on the thin-glass to fabricate the micro-holes. The femtosecond laser irradiation within 2-8 s created micro-holes with diameter of 0.3-1.5 μm. From the micro-holes, NGF was released and induced the neurite growth of PC12 cells toward micro-holes. Interestingly, two distinct neurite elongations were observed at different cells-to-micro-holes distances. Longer neurite extension was observed at cells positioned far (420–840 μm), rather than at close (0–420 μm) distance from the micro-holes. This result suggests that two regions of the NGF gradient were generated, indicating the slow release of NGF through the micro-holes. Combined, these results demonstrate that our method allows control of multiple neuronal arrangements with directional neurite growth.