Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Start by adding pericytes to the underside of the well-insert.

Cover the well-insert with a culture plate and incubate.

Pericytes attach to the membrane of the well-insert, forming a single-cell layer or a monolayer.

Take another culture plate.

Add human astrocytes to the well in a suitable medium. Then, incubate the plate.

Astrocytes attach to the bottom of the well and form a monolayer.

Discard the medium and add fresh astrocyte and pericyte medium.

Now, take the culture plate containing the well-insert with the pericyte layer.

Invert the plate and transfer the well-insert to the well containing the astrocyte layer.

Add the human brain endothelial cells to the upper side of the well-insert and incubate.

The endothelial cells attach to the well insert, forming a triple cell culture system that resembles the blood-brain barrier, a crucial interface separating the bloodstream and the brain.