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.

Begin by cultivating HBVP in T75 culture flasks with an activated surface for cell adhesion within a 5% carbon dioxide incubator at 37 degrees Celsius until confluent. Once confluence is reached, aspirate the old parasite medium and wash the cells with 5 milliliters of warm Dulbecco's PBS. Aspirate the Dulbecco's PBS and detach the cells from the flask, using a combination of 4 milliliters of warm trypsin-EDTA solution and 1 milliliter of Dulbecco's PBS.

Incubate the flask for 5 minutes at 37 degrees Celsius in a carbon dioxide incubator. View under a microscope to confirm whether the cells are detached from the flask. Add 5 milliliters of warm parasite medium, containing 2% FBS to the flask, and transfer the detached cells to a 50-milliliter centrifuge tube. Centrifuge the cell suspension for three minutes at 200 g, allowing the cells to form a pellet in the bottom of the tube.

Aspirate the medium from the tube, ensuring the cell pellet remains intact. Resuspend the cell pellet in parasite medium. Calculate the amount of medium, depending on the confluence of the cells and the number of well inserts needed. Take 10 microliters of the resuspended cells. Place them into a cell-counting slide, and count the number of cells.

Determine the cell density and seed 300,000 cells per insert in 1 milliliter of parasite medium onto the luminal side of the well inserts. Cultivate primary human astrocytes using the astrocyte medium, containing 2% FBS, as described in the text manuscript. Determine the cell density and seed 300,000 cells per well onto the bottom of the tissue culture 6-well plates. Cover the plate to prevent evaporation, and incubate overnight.

Take out the tissue culture 6-well plates containing astrocytes in the well inserts containing pericytes from the incubator. Aspirate the astrocyte medium from the tissue culture 6-well plates, and add 1 milliliter of parasite medium and 1 milliliter of astrocyte medium to each well.

Aspirate the pericyte medium from the well inserts, and place them into the tissue culture 6-well plates containing the seeded astrocytes. Seed HBMEC at a density of 300,000 cells per well in 2 milliliters of complete classic medium onto the apical side of the same well inserts.