Development of biocompatible Parylene neurocages for action potential stimulation and recording

Abstract

Neurons, and the neural networks they form, are at the heart of our biological and cognitive functions. Traditional in vitro techniques for studying neural networks use two-dimensional multi-electrode arrays. While furthering the study of neural networks, the inherent mobility of the neurons and the lack of specificity between neurons and electrodes can limit the use of these arrays. Initial work, the neuro-well, eliminated these problems by physically trapping individual neurons in wells. While neural networks were formed and action potentials recorded with arrays of neuro-wells, the bulk micromachining techniques required a complex fabrication process, with limited scalability and a low yield, thus inhibiting their further development. Parylene neurocages counteract these difficulties by using surface micromachined structures to trap neurons in close proximity to electrodes, without inhibiting their growth. The use of surface micromachining techniques minimizes the fabrication and scaling complexities, improving the device yield. The neurocages can be fabricated on either glass or silicon substrates, with a variety of electrical insulation materials, including Parylene and silicon-nitride. Parylene is a biocompatible polymer that is non-toxic, extremely inert, and resistant to moisture and most chemicals. Its conformal deposition makes it easy to fabricate 3D structures like the neurocage. Parylene is transparent, allowing the neurons to be easily seen. Individual neurons are placed into the neurocages, either manually with a pressure-driven micropipette or automatically with a laser tweezers system. The neurocages have openings to allow the neurites to extend out of the neurocages and form synaptic connections with their neighboring neurons. Each neurocage has its own electrode, which is platinized to increase its capacitance. Successful growth of neural networks has been achieved using arrays of neurocages with Parylene and silicon-nitride insulation on both silicon and glass substrates. These neurocages have a long-term cell survival rate of ~ 50% after 3 weeks and have proven 99% effective in trapping neurons. The neurons inside the neurocages have been successfully stimulated, with both current and voltage pulses. Action potentials, both spontaneous and resulting from a current stimulus, have been recorded from neurons comprising the neural networks.