Generating an Ultra-Low-Density Neuronal Culture Using a High-Density Neuronal Feeder Layer

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.

In this procedure, place 24-well plates with etched bottoms for high-density neurons in the culture hood. Remove 300 microliters of the preconditioned medium by vacuum. Then, plate 0.6 milliliters of high-density cell suspension at 250,000 neurons per milliliter.

Place the other 224-well plates with coverslips in the culture hood. Remove 300 microliters of the preconditioned medium by vacuum, before plating 0.6 milliliters of low-density cell suspension at 10,000 neurons per milliliter on the coverslips. Afterward, return all for 24-well plates to the incubator, and incubate for two hours. After that, flip the low-density coverslips with adhering neurons to the high-density culture, and make sure the neurons are facing each other. Subsequently, return the two plates with coculture to the incubator.

Feed the co-cultures by adding 300 microliters of fresh feed medium at DIV 5. Following this initial feeding, add 300 microliters of fresh feed medium without cytosine arabinoside to each well every week. Should the culture be kept for more than one month, replace half of the medium with fresh feed medium every week, beyond one-month time-point.