Abstract
Abstract: :
Purpose: Present prosthetic devices stimulate neurons electrically with limited spatial control and without cell type specificity. Our purpose is to explore whether neuronal growth can be directed to a chip where focal neurotransmitter stimulation would provide a more physiologic and neuron-specific transfer of information. Methods: Microlithographic fabrication techniques from the computer chip industry were used (i) to stamp microcircuit-like patterns of biomolecules onto a surface with 5-µm resolution and (ii) to create microapertures that connect to a microfluidic channel system. Small pulses of neurotransmitters (i.e., artificial synapse) are delivered underneath single cells. Standard photolithographic techniques were used to fabricate photoresist coated silicon substrates (molds) for microstamp fabrication. Microapertures were made in silicon nitride and the microfluidic channels were fabricated from a PDMS matrix. Neurites from isolated rat retinal ganglion cells (RGCs) and PC-12 cells were cultured on the patterns of growth modulating factors. RGCs were purified by sequential immunopanning to greater than 99.5% purity from P7 Sprague-Dawley rats. Approximately 50,000 RGCs were seeded onto the patterned surfaces. RGCs were cultured at 37 ºC and 6.5% CO2 in 2 mL of serum-free medium (Neurobasal with supplements). Dynamic fluorescence measurements of the calcium indictor, fluo-4, were used to measure activity of RGC and PC-12 cells on the chip. Results: RGC and PC-12 neurites were directed by surface micropatterns of laminin to grow toward focal stimulation sites. Cells and their neurites that were directed to grow over 5-µm apertures connected to a microfluidics system could be stimulated with pulsed neurotransmitters. Transmitter stimulation caused a calcium increase along the neurite and in the cell soma, indicating transmission of signal to the cell soma. With the appropriate flow rate and concentration, no stimulation effects were found outside a 5-µm radius from the apertures. RGC and PC-12 cells were stable over these apertures, and did not detach with the picoliter volumes delivered. Conclusion: The ability to direct the growth of RGCs, and to use a microfabricated neurotransmitter delivery system, demonstrates the feasibility of a visual prosthesis interface based on direct neuronal stimulation with physiologically appropriate neurotransmitters.
Keywords: 308 age-related macular degeneration • 553 regeneration • 415 ganglion cells