Developments in micro-manufacture as well as biofabrication technologies are driving our ability to create complex tissue models such as ‘ organ-on-a-chip ’ devices. The complexity of neural tissue, however, requires precisely specific cellular connectivity across many neuronal populations, and thus there have been limited reports of complex ‘ brain-on-a-chip ’ technologies modelling specific cellular circuit function. Here we describe the development of a model of in vitro brain circuitry designed to accurately reproduce part of the complex circuitry involved in neurodegenerative diseases; using segregated co-culture of specific basal ganglia (BG) neuronal subtypes to model central nervous system circuitry. Lithographic methods and chemical modification were used to form structured micro-channels, which were populated by specifically cultured neuronal sub-types to represent parts of the inter-communicating neural circuit. Cell morphological assessment and immunostaining showed connectivity, which was supported by electrophysiology measurements. Electrical activity of cells was measured using patch-clamp, showing voltage dependant Na + and K + currents, and blocking of Na + current by TTX, and calcium imaging showing TTX-sensitive slow Ca 2+ oscillations resulting from action potentials. Monitoring cells across connected ports post-TTX addition demonstrated both upstream and downstream changes in activity, indicating network connectivity. The model developed herein provides a platform technology that could be used to better understand neurological function and dysfunction, contributing to a growing urgency for better treatments of neurodegenerative disease. We anticipate the use of this advancing technology for the assessment of pharmaceutical and cellular therapies as a means of pre-clinical assessment, and further for the advancement of neural engineering approaches for tissue engineering.
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.
Restoration of function following damage to the central nervous system (CNS) is severely restricted by several factors. These include the hindrance of axonal regeneration imposed by glial scars resulting from inflammatory response to damage, and limited axonal outgrowth toward target tissue. Strategies for promoting CNS functional regeneration include the use of nanotechnology. Due to their structural similarity, synthetic nanofibers could play an important role in regeneration of CNS neural tissue toward restoration of function following injury. Two-dimensional nanofibrous scaffolds have been used to provide contact guidance for developing brain and spinal cord neurites, particularly from neurons cultured in vitro. Three-dimensional nanofibrous scaffolds have been used, both in vitro and in vivo, for creating cell adhesion permissive milieu, in addition to contact guidance or structural bridges for axons, to control reconnection in brain and spinal cord injury models. It is postulated that nanofibrous scaffolds made from biodegradable and biocompatible materials can become powerful structural bridges for both guiding the outgrowth of neurites and rebuilding glial circuitry over the "lesion gaps" resulting from injury in the CNS.
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