Establishing a Co-Culture of Interneurons with Periventricular Endothelial Cells

Take a culture dish coated with a cell culture substrate to facilitate cell adhesion.

The dish contains a one-well culture insert without a base attached to its center, creating a defined compartment. The outline of this compartment is marked on the dish to observe cell migration.

Introduce a cell suspension into the compartment that contains periventricular endothelial cells or PVECs, cells lining the blood vessels in the brain, and interneurons that migrate along the PVECs during brain development.

Add a co-culture medium to the dish to maintain moisture, and incubate to facilitate the cells adhering to the substrate.

Remove the insert to eliminate the physical barrier; refresh the medium, and incubate again.

The PVECs proliferate, expanding the endothelial cell network beyond the insert boundary.

The PVECs secrete factors that act as guidance cues, directing the interneurons to migrate along the PVEC network.

The co-culture exhibiting interneuron migration is ready for analysis.

To perform the co-culture assay, suspend 30,000 GABAergic interneurons and 30,000 human periventricular endothelial cells in 70 microliters of co-culture medium. Then, seed this cell solution inside a one-well insert compartment. After 48 hours, remove the insert. Incubate the co-culture at 37 degrees Celsius and 5% carbon dioxide for five days.