Guided axon outgrowth of neurons by molecular gradients generated from femtosecond laser-fabricated micro-holes

Abstract

ObjectiveTransplantation of scaffold-embedded guided neurons has been reported to increase neuronal regeneration following brain injury. However, precise axonal integration between host and transplant neurons to form functional synapses remains a major problem. Thus, a high-precision tool to actuate neuronal axon outgrowth in real-time conditions is required to attain robust axon regeneration. This study aims to establish a microfluidic platform for precise and real-time axon outgrowth guidance. Methods: A microfluidic device with a 4 μm thick thin-glass sheet as the neuron culture substrate is fabricated. Surface of the glass sheet is chemically modified to facilitate neuron attachment. Femtosecond (fs) laser is used to engrave the glass sheet to achieve micro-holes, where netrin-1 is released for directing the movement of the neuronal axon. Results: Numerical simulation and experimental data demonstrate that netrin-1 gradient is formed after it passes through the micro-hole. The neuronal response results show the outgrowth rate of the axon is significantly increased by netrin-1 gradient. Furthermore, a majority of neuronal axons exhibit guided outgrowth characterized by positive turning angles of axon displacement in the direction of netrin-1 gradients. Conclusion: Integrating fs laser and microfluidic device facilitates controlled and instantaneous axon outgrowth in a non-invasive manner. Significance: The developed real-time microfluidic platform shows potential in the application for on-site neuronal transplantation, which is significant for the treatment of a range of neurological disorders and injuries.