Back-to-back Layered Co-culture on a SiN Porous Membrane for Stable Measurement of Neuronal Extracellular Potentials

Abstract

We developed a novel technique for measuring neuronal extracellular potentials in a neuron-astrocyte co-culture system using a SiN (silicon nitride) membrane having microelectrodes and microholes. An 8×8 microelectrode array was formed on one side of a 1 µm-thick SiN membrane, and multiple microholes of 5 µm in diameter were opened through the membrane. Then, neurons were cultured on the microelectrodes after astrocytes were cultured on the back side of the membrane. The neuronal spikes measured in this novel back-to-back co-culture method were compared with those in the conventional method in which both of neurons and astrocytes were co-cultured on the microelectrodes. In the conventional method, formation and enlargement of cellular aggregates made neurons out of touch with microelectrodes, and posed measurement of a small number of spikes. On the other hand, our novel method permitted the firm contact between neurons and microelectrodes and the cell-to-cell interaction through microholes, and therefore neuronal spikes increased drastically with culture time, which led to stable measurement of neuronal extracellular potentials.