Large-scale microfluidic gradient arrays reveal axon guidance behaviors in hippocampal neurons

High-throughput quantitative approaches to study axon growth behaviors have remained a challenge.We have developed a 1024-chamber microfluidic gradient generator array that enables large-scale investigations of axon guidance and growth dynamics from individual primary mammalian neurons,which are exposed to gradients of diffusible molecules.Our microfluidic method(a)generates statistically rich data sets,(b)produces a stable,reproducible gradient with negligible shear stresses on the culture surface,(c)is amenable to the long-term culture of primary neurons without any unconventional protocol,and(d)eliminates the confounding influence of cell-secreted factors.Using this platform,we demonstrate that hippocampal axon guidance in response to a netrin-1 gradient is concentration-dependent—attractive at higher concentrations and repulsive at lower concentrations.We also show that the turning of the growth cone depends on the angle of incidence of the gradient.Our study highlights the potential of microfluidic devices in producing large amounts of data from morphogen and chemokine gradients that play essential roles not only in axonal navigation but also in stem cell differentiation,cell migration,and immune response.