Assessing Interneuron Response to Chemoattractive Cues from Periventricular Endothelial Cells

Fix a three-compartment culture insert in a polymer-coated dish for cell attachment.

Invert the dish, mark the middle compartment, and reorient it. Add a medium containing interneurons to this compartment.

Add a medium containing human periventricular endothelial cells, or PVECs, brain vessel cells, to one of the outer compartments.

In another outer compartment, add a medium containing non-periventricular endothelial cells as a control.

Add a co-culture medium to the dish to prevent polymer drying. Incubate for cell adherence.

Disassemble the insert, creating rectangular patches of cells with small cell-free gaps between each cell type.

Refresh with the co-culture medium and incubate.

Endothelial cells trigger interneurons to migrate into cell-free spaces.

Meanwhile, PVECs release chemical attractants that enhance the migration of interneurons toward the PVEC-containing patch.

Higher interneuron migration toward the PVEC patch than the control cell patch confirms the interneuron response to chemoattractive cues from the PVECs.

To perform the chemoattraction assay, place a three-well culture insert in the center of a poly-L-ornithine and laminin-coated 35-millimeter dish. Turn the dish upside down, and mark the boundary around the middle compartment of the insert using a permanent marker with an ultra-fine tip.

Seed 30,000 GABAergic interneurons in 70 microliters of neuronal medium in the middle compartment. Then, seed 10,000 periventricular endothelial cells, and 10,000 control endothelial cells in 70 microliters of their respective medium in the two outer compartments.

Add 1 milliliter of co-culture medium along the side of the dish to prevent the coating on the dish from drying. After 48 hours, remove the insert, and incubate the cells at 37 degrees Celsius and 5% carbon dioxide for 36 hours.