Blood-Brain Tumor Barrier or BBTB Model: An Adaption of the Blood-Brain Barrier or BBB Model to Evaluate Delivery of Therapeutics to the Tumor

Under a sterile cell culture hood, carefully wash the cultured astrocytes with 5 milliliters of sterile PBS. Use a vacuum pump to gently discard the PBS and add 2 milliliters of cell dissociation reagent for 5 minutes to detach the cells.

Then, add 10 milliliters of sterile complete astrocyte cell culture medium to the vessel to inhibit the activity of the cell dissociation reagent. Use sterile serological pipette to transfer the detached cells from the vessel to a sterile 15-milliliter tube.

Centrifuge at 250 x g for 3 minutes at room temperature. Meanwhile, set out the inserts. Use sterile forceps to place the inserts with the brain side up on the lid of a sterile 6-well plate.

When the centrifugation is complete, carefully discard the supernatant from the cell suspension and resuspend the astrocyte pellet in 1 milliliter of ABM plus by gently pipetting the pellet on the tubes wall up to five times.

Then, count the cells and adjust the cell suspension density to 150,000 cells in 400 microliters of ABM plus per insert. Place this cell suspension into the middle of the brain side of the insert's membrane and carefully spread it using capillary force with a sterile pipette tip.

With the brain side of the inserts still up, place the 6-well plate back on the inserts. Place the plate and inserts with the brain side up into an incubator at 37 degrees Celsius and 5% carbon dioxide to allow for cell adhesion.

After this, revert the 6-well plate back to its regular position with the inserts now blood side up. Add medium and continue the incubation as outlined in the text protocol. Under a sterile cell culture hood, carefully wash the cultured endothelial cells with 5 milliliters of sterile PBS.

Use a vacuum pump to gently discard the PBS and add 2 milliliters of cell dissociation reagent for 5 minutes to detach the cells. Then, add 10 milliliters of sterile complete endothelial cell culture medium to the vessel to inhibit the activity of the cell dissociation reagent.

Use a sterile serological pipette to transfer the detached cells from the vessel to a sterile 15-milliliter tube. Centrifuge at 250 x g for 3 minutes at room temperature. After this, discard the supernatant and resuspend the endothelial cell pellet in 1 milliliter of EBM plus by slowly pipetting the cell suspension on the tube's wall up to five times.

Count the cells and adjust the cell suspension density to 200,000 cells in 2.5 milliliters of endothelial cell culture, devoid of serum and vascular endothelial growth factor-A per insert. Retrieve the plate containing the inserts.

Carefully discard the medium from the blood side and replace it with the 2.5 milliliters of endothelial cell suspension. Then, return the plate to the incubator and leave it overnight to allow the endothelial cells to adhere to the membrane.

The next day, prepare a 6-well plate by transferring 3 milliliters of prewarmed ABM minus to each well. Using sterile forceps, handle the inserts to carefully discard the endothelial complete medium from the blood side and place the insert into the new plate containing ABM minus. Then, add 2.5 milliliters of EBM minus.

Leave the inserts in the incubator with minimal physical disturbance or temperature variation for 5 days. On the day of the transfer, replace the medium. For immunofluorescence imaging, place up to four round sterile borosilicate coverslips into each well of a 6-well plate, containing 2 milliliters of poly-D-lysine. Incubate at room temperature for 30 minutes.

Meanwhile, use a sterile serological pipette to carefully transfer the tumor spheres from the cell culture vessel to a 15-milliliter sterile tube. Centrifuge the tumor spheres at 250 x g for 3 minutes. Discard the supernatant and gently resuspend the spheres in 1 milliliter of GBM minus.

Count the cells and adjust the cell density to approximately 10,000 spheres or 100,000 cells per milliliter of GBM minus. Next, discard the poly-D-lysine from the wells and rinse the wells three times with sterile PBS. Seed each well of the plate with 3 milliliters of the tumor spheroid suspension and transfer the inserts with the BBTB mimic onto the tumor cell suspension.

Incubate overnight at 37 degrees Celsius with 5% carbon dioxide to allow the blood and brain tumor sites of the assay to come to equilibrium. The next day, replace the media in the blood side with EBM minus supplemented with the molecules, drugs, or nanoparticles of interest.