How Microfluidics Can Help to Understand and Promote Nerve Healing after Injury: A Neurobiological Microfluidic Device with Electrophysiological Functionality

Abstract

We present a microfluidic device that has been designed to study the neuroregeneration of nerve tissue after injury. Reconnection between neurons of the peripheral nervous system after injury or dissection is essential for regaining the full motoric and sensory capabilities. The neuroregeneration of damaged axons and the functional reconnection to a target cell is a complex processes, where (1) chemical guidance and (2) topographic guidance of the neurite growth cone play a crucial role. To identify new chemical guidance substances and to understand how geometry guides neurite growth, we have developed a microfluidic in-vitro system for studying neuronal cell cultures. The microchannels act as artificial guidance conduits for the growth of nerve processes (dendrites, axons), also named neurites in the following text. The microstructured system consists of a microelectrode array with sub-10 µm microelectrodes for recording neuronal signals plus a microfluidic attachement. This microfluidic attachment has 2 macrochannels with a width above 100 µm that can accommodate entire cells. Both macrochannels are connected by up to 60 microchannels which are 100 to 500 µm long but only 10 µm wide and 2 µm high, so that cell somata cannot enter the microchannels while cell processes (axons, dendrites) are small enough to enter the channel. A neuronal culture of mouse superior cervical ganglion (SCG) is seeded in the source macrochannel, while the target macrochannel stays cell-free and is filled cell medium with neurotrophic factors added. These soluble neurotrophic factors act as chemical guidance cues for the neurites and have been under debate as enhancers of axon regeneration. In our microfluidic system the neurites can reach the target macrochannel by growing through the small microchannels that are too small for entire cell somata. This topographic guidance provided by the microchannels mimics’ topographic guidance of the endoneurial channel formed by Schwann cells for a reconnecting neuron. We will present the design considerations as well as the actual fabrication of this microfluidic system on the microelectrode system. Simulations on the diffusion of chemical entities through the microchannels will be presented. Finally, images of the neurites grown through the microchannels will be shown and prove the research concept. Neurotrophic factors are also under debate for accelerating the functional recovery, i.e. the recovery of the neuron to propagate action potentials along the neurite. With our microfluidic setup the microelectrodes of the microelectrode array have been aligned such that every microchannel contains at least one (up to four) microelectrodes. By implementing microelectrodes inside of the microfluidic channels also the electrophysiological activity of the reconnecting neurites can be monitored. This electrical activity of neurons is a measure for the health status of a neuron. With the microelectrodes providing electrochemical sensor functionality to the microfluidic channels, this important question could be answered. We will present the effect, that various gradients of neurotrophic factors have on the growth and electrical activity of growing neurons. Finally, a recommendation will be given on the optimum concentration for the addition of nerve growth factor in order to promote a faster growth of electrophysiologicallly active neurites. The potential to use this microfluidic platform for identifying new nerve growth promotors and for quantifying the effects will be discussed. Concepts for advancing this microfluidic system further as cell culture reactors as well as for other interdisciplinary areas including neurobiological and medical applications will be presented. Figure 1