We describe the development of experimental platforms to quantify the regeneration of injured central nervous system (CNS) neurons by combining engineering technologies and primary neuronal cultures. Although the regeneration of CNS neurons is an important area of research, there are no currently available methods to screen for drugs. Conventional tissue culture based on Petri dish does not provide controlled microenvironment for the neurons and only provide qualitative information. In this review, we introduced the recent advances to generate in vitro model system that is capable of mimicking the niche of CNS injury and regeneration and also of testing candidate drugs. We reconstructed the microenvironment of the regeneration of CNS neurons after injury to provide as in vivo like model system where the soluble and surface bounded inhibitors for regeneration are presented in physiologically relevant manner using microfluidics and surface patterning methods. The ability to control factors and also to monitor them using live cell imaging allowed us to develop quantitative assays that can be used to compare various drug candidates and also to understand the basic mechanism behind nerve regeneration after injury.

Neurons, one of the most polarized types of cells, are typically composed of cell bodies (soma), dendrites, and axons. Many events such as electric signal transmission, axonal transport, and local protein synthesis occur in the axon, so that a method for isolating axons from somata and dendrites is required for systematically investigating these axonal events. Based on a previously developed neuron culture method for isolating and directing the growth of central nervous system axons without introducing neutrophins, we report three modified microfluidic platforms: (1) for performing biochemical analysis of the pure axonal fraction, (2) for culturing tissue explants, and (3) a design that allows high content assay on same group of cells. The key feature of these newly developed platforms is that the devices incorporate a number of microgrooves for isolating axons from the cell body. They utilize an open cellculture area, unlike the enclosed channels of the previous design. This design has extended the axonal channel so that a sufficient amount of pure axonal fraction can be obtained to perform biochemical analysis. The design also addresses the drawback of the previous neuron culture device, which was not adaptable for culturing thick neuronal tissues such as brain explants, neurospheres, and embryoid bodies, which are essential model tissues in neuroscience research. The design has an open cellculture area in the center and four enclosed channels around open area, and is suitable for multiple drug screening assays.

This paper describes simple photolithography-based methods to fabricate multi-level three-dimensional (3D) microstructures without repeated processes using flexible and transparent film substrates such as polyethylene terephthalate (PET).

Mitochondria, synaptic vesicles, and other cytoplasmic constituents have to travel long distance along the axons from cell bodies to nerve terminals. Interruption of this axonal transport may contribute to many neurodegenerative diseases including Alzheimer's disease (AD). It has been recently shown that exposure of cultured neurons to β-amyloid (Aβ) resulted in severe impairment of mitochondrial transport. This Letter describes an integrated microfluidic platform that establishes surface patterned and compartmentalized culture of neurons for studying the effect of Aβ on mitochondria trafficking in full length of axons. We have successfully quantified the trafficking of fluorescently labeled mitochondria in distal and proximal axons using image processing. Selective treatment of Aβ in the somal or axonal compartments resulted in considerable decrease in mitochondria movement in a location dependent manner such that mitochondria trafficking slowed down more significantly proximal to the location of Aβ exposure. Furthermore, this result suggests a promising application of microfluidic technology for investigating the dysfunction of axonal transport related to neurodegenerative diseases.

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