Profuse and selective growth in vitro of rat spinal axons on a micro-patterned poly (ethylene imine) grid

To distribute neurites (axons) along a surface and to guide them towards specific point targets we cultured spinal cord explants on coverslips printed with a micro-patterned grid of poly (ethylene imine) (PEI) lanes. The grid was prepared by micro-contact printing with silicone stamps. Spinal cord explants were resected from neonatal rats. A tiny bunch of glass filaments was used to ensure adhesion of the explant to the coverslip. One end of the bunch was glued to the coverslip while the other end pressed the explant firmly onto the coverslip. Spinal cord explants cultured in a collagen matrix, on a coverslip stamped with poly D-lysine (PDL), or on a coverslip uniformly coated with PEI or PDL were used as controls. Cultures were maintained for 6 days in vitro (div). Outgrowth from the explant was observed using phase contrast microscopy. None of the explants detached from the coverslip. Neurites emerged randomly from the explants, but upon crossing one of the grid lanes they subsequently followed the grid pattern. The outgrowing neurites were guided by the PEI lanes of the grid and reached lengths of up to 2400 μm. After 6 div no signs of degeneration were observed in the outgrowth of explants cultured on a stamped coverslip or on a homogeneously coated coverslip, while degeneration did appear in the explants cultured in collagen after 4 div. Compared to control explants cultured on PDL (either on stamped or on uniformly coated coverslips), explants cultured on a micro-patterned PEI grid grew more profuse (more and longer neurites). Because these cultures can be easily manipulated, this paradigm is ideally suitable for studies of neuronal networks and for studies that necessitate the guidance of neurites towards a specific target (in culture), for instance the electrodes of a multi-electrode array in a culture dish.