Co-Culture of Schwann Cells with DRG Neurons on a Pre-Stretched Membrane

Take a silicon chamber attached to a matrix-coated, stretched silicon membrane.

Add dorsal root ganglia or DRG cell suspension and incubate.

The matrix promotes cell attachment and multiplication, while the stretched surface guides axon alignment.

Add growth inhibitors that eliminate actively dividing glial cells, allowing non-dividing neurons to survive. 

Remove the medium. Add a growth medium and incubate.

The neuron senses an increased membrane stiffness in the stretched direction and orients the axon growth accordingly. 

Add cultured and harvested Schwann cells to the DRG culture and incubate.

The Schwann cells attach to the regenerated axon, insulating the neuron.

This enhances the survival and functionality of neurons, establishing a co-culture.

Prior to seeding the DRG neurons, remove the PLL solution from the chamber. Rinse the PDMS surface with sterile water and air-dry it. After resuspending the cells, let the suspension sit for 1 to 2 minutes to allow the debris to settle to the bottom of the tube. Next, add 1.5 milliliters of cell suspension into each stretch chamber, and incubate it at 37 degrees Celsius with 5% CO₂.

To eliminate glial cells at one day in vitro, add 10 microliters or 15 microliters of the FdU/U mixture stock solution to each well. After 7 hours, replace this medium with fresh standard growth medium, and place the pre-stretched culture device in a 37 degrees Celsius incubator in 5% CO₂. During the cell culture period, change the medium every two days by replacing half the spent medium with fresh medium.

In this procedure, remove the culture medium from the Schwann cells, and add 5 milliliters of 0.05% trypsin-EDTA into the flask. Incubate the cells at 37 degrees Celsius in 5% CO₂ for 2 to 3 minutes. Check the cells under the optical microscope using a 10x objective to see if they lift up from the flask. Then, add 5 milliliters of culture medium to the cells in the flask, and mix.

Subsequently, add the cell suspension to a 15-milliliter centrifuge tube and centrifuge at 20 degrees Celsius for 5 minutes. Then, remove the supernatant and resuspend the pellet in 7 milliliters of standard DOG growth medium. Following this, transfer 10 microliters of the cell suspension into a 0.5-milliliter microcentrifuge tube. Mix it with 10 microliters of trypan blue, and then count the cell number with a hemocytometer, using a 10x objective.

Add the growth medium to dilute the cell suspension to 5,000 cells per milliliter. Next, remove 0.5 milliliters of the medium from the DRG culture in the stretched chamber, and add 0.5 milliliters of Schwann cell suspension to the DRG culture. Culture the cells for one week at 37 degrees Celsius in 5% CO₂. Change the medium every two days by replacing half of the spent medium with fresh standard growth medium.