Efficient alignment of primary CNS neurites using structurally engineered surfaces and biochemical cues

Abstract

Tissue engineering strategies for the central nervous system (CNS) have been largely hampered by the complexity of neural cell interactions and limited ability to control functional circuit formation. Although cultures of primary CNS neurons give key insight into an in vivo state, these cells are extremely sensitive to local micro-environments and are therefore often replaced with cell lines. Here we aimed to combine primary CNS neurons with surface nano- and micro-topography, and biochemical cues, to direct neurite outgrowth. Neurons were cultured on nano-fibers and micro-grooves either coated with poly-L-lysine and laminin (PLL–LN) or pre-seeded with naturally supporting astrocyte cells. Developing neurites extended parallel to PLL–LN coated topography, significantly more on micro-grooved than nano-fiber substrata. Astrocytes were found to direct neurite alignment to a greater extent compared to structured surface cues, highlighting the importance for biochemical signalling and cellular architecture. Equally neuron–neuron interactions strongly influenced neurite outgrowth. On micro-structured surfaces neurite orientation was regulated by contact guidance cues at the edges of grooves. All of our findings show that we can control the behaviour of primary CNS neurons in vitro using surface engineering approaches. This will allow us to establish neuronal circuitry, to model neurodegenerative diseases and advance regenerative medicine strategies.