Generating an Ultra-Low-Density Neuronal Culture Using a High-Density Neuronal Feeder Layer
Transcription
Begin with two multi-well plates: one with an etched bottom coated with a polymer and the other featuring a polymer-coated coverslip.
Seed a high-density suspension of the embryonic neurons into the plate with the etched bottom. Add an ultra-low-density neuron suspension to the coverslip containing well.
Incubate to allow neuronal attachment to the polymers.
Remove the coverslip and place it over the high-density neuron culture, enabling the neurons to face each other.
The etched surface with elevated structures creates a micro-space that separates neurons of different densities.
The high-density culture acts as a feeder layer and secretes the neural growth factor.
Due to the confined diffusion space, the growth factors accumulate around the neurons, facilitating their growth and the development of neuronal projections.
Introduce a neurobasal medium with cytosine arabinoside that selectively inhibits the growth of the non-neuronal cells.
Regularly replenish with a fresh neurobasal medium to maintain the neuronal culture.
