Long-term Culture of a Single Neuron using a Microwell with a Porous SiN Membrane

For long-term culture of a single neuron, many astrocytes and a single neuron were co-cultured on both sides of a 1 µm thick silicon nitride membrane which has many microholes of 3 µm in diameter. After astrocytes were cultured on the back side of the membrane for 1 week, a neuron was seeded on the front side of the membrane of a microwell bottom and cultured for 5 weeks. By fluorescently staining VGLUT1 (vesicular glutamate transporter 1) which is localized at a presynaptic terminal, we found that the number of synapses formed by a single neuron drastically increases within the first 2 weeks and keeps high values for weeks afterward.