Abstract
It has been recently known that not only the presence of inhibitory molecules associated with myelin but also the reduced growth capability of the axons limit mature central nervous system (CNS) axonal regeneration after injury. Conventional axon growth studies are typically conducted using multi-well cell culture plates that are very difficult to use for investigating localized effects of drugs and limited to low throughput. Unfortunately, there is currently no other in vitro tool that allows investigating localized axonal responses to biomolecules in high-throughput for screening potential drugs that might promote axonal growth. We have developed a compartmentalized neuron culture platform enabling localized biomolecular treatments in parallel to axons that are physically and fluidically isolated from their neuronal somata. The 24 axon compartments in the developed platform are designed to perform four sets of six different localized biomolecular treatments simultaneously on a single device. In addition, the novel microfluidic configuration allows culture medium of 24 axon compartments to be replenished altogether by a single aspiration process, making high-throughput drug screening a reality.
Highlights
Damages in central nervous system (CNS) often result in permanent deficit and it has been long thought that adult mammalian CNS cannot be functionally recovered after injury, unlike peripheral nervous system (PNS)
In order to understand the relationship between the intrinsic growth capability and the regeneration mechanism as well as to discover potential growth factors or drugs that promote CNS axon growth/regeneration, an in vitro tool that enables precise and localized chemical microenvironments control with higher efficiency is critically needed
Isolated axons can be locally treated with various biomolecules in the absence of neuronal somata or dendrites for analyzing the effects of treatment on axonal growth. This enables localized biomolecular treatments to axons and allows isolated axons to be quantitatively analyzed even at commonly used in vitro cell culture densities by measuring the area of the axon compartment covered with axons [27]
Summary
Damages in central nervous system (CNS) often result in permanent deficit and it has been long thought that adult mammalian CNS cannot be functionally recovered after injury, unlike peripheral nervous system (PNS). Some of recent research has shown that manipulation of axonal intrinsic growth control pathways promotes CNS axon regeneration while neutralization of commonly known axon inhibiting extrinsic factors results in only limited axon regeneration in vivo [3,4,5]. This clearly indicates that both the extrinsic inhibitory factors as well as the intrinsic growth capability of the neurons contribute to the regeneration of CNS axons. A neuron culture platform capable of localized biomolecular treatments with higher throughput that can culture neurons at commonly used cell culture densities will be a powerful tool for studying in vitro CNS axon growth toward screening of potential drugs that can promote regeneration/growth. The novel microfluidic design of the device minimizes direct labor input toward a real high-throughput screening, where an only single aspiration step can replenish culture medium or biomolecular treatments of 24 compartments altogether
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