In order to develop novel drugs and compounds that target tumors in the central nervous system, the accessibility and passage through the blood-brain barrier is highly important. Although the cytotoxicity of the compounds can easily be measured by incubating the cells with the therapeutic molecules, it doesn't really tell anything about the actual delivery to the tumor site in the brain. To address this issue, we developed the following protocol that describes how to recreate the blood-brain tumor barrier in vitro on a dish.
The usage of immortalized cells of murine or human origin makes this assay very flexible. It can also be easily scaled up to screen dozens of compounds with high reproducibility. In addition, this method allows both quantification and visualization of the passage and targeting ability of the compounds before proceeding with preclinical models.
Therefore, our method partially replaces the animal models by using the pre-established cell lines. Inclusion of patient-derived brain tumor cells assays the patient heterogeneity, while the use of immortalized cells to establish the blood-brain barrier ensures the reproducibility of the results. Altogether, this blood-brain barrier model offers a really interesting tool to assay the drug diffusion or the delivery of drug vehicles.
Visual demonstration of the method is critical because seeding of the cells at the opposite side of the insert require manipulation that is not oftenly described in other protocols using similar setup. For instance, obtaining the adhesion of astrocytes when the insert is placed upside down highly benefits from visual representation. Under a sterile cell culture hood, carefully wash the cultured astrocytes with five milliliters of sterile PBS.
Use a vacuum pump to gently discard the PBS, and add two milliliters of cell dissociation reagent for five 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 a sterile serological pipette to transfer the detached cells from the vessel to a sterile 15-milliliter tube.
Centrifuge at 250 times g for three 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 six-well plate.
When the centrifugation is complete, carefully discard the supernatant from the cell suspension, and resuspend the astrocyte pellet in one milliliter of ABM-plus by gently pipetting the pellet on the tube's 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 the 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 six-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 six-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 five milliliters of sterile PBS. Use a vacuum pump to gently discard the PBS, and add two milliliters of cell dissociation reagent for five 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 times g for three minutes at room temperature.
After this, discard the supernatant, and resuspend the endothelial cell pellet in one 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 six-well plate by transferring three milliliters of pre-warmed 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 five 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 six-well plate containing two 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 times g for three minutes. Discard the supernatant, and gently resuspend the spheres in one 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 three 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 sides 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. Collect the samples over time for direct quantification as previously described.
First, collect 100 microliters of the medium from both the blood and the brain sides of the BBTB mimic, and transfer each to a separate flat-bottomed, black, 96-well plate for subsequent fluorescence measurements. Next, prepare a 50-micromolar solution of sodium fluorescein in EBM-minus, making sure to prepare 2.5 milliliters per well. Pre-warm this solution to 37 degrees Celsius, and replace the media from the blood side of the inserts with media containing this sodium fluorescein solution.
Start a timer as soon as the medium is replaced. Carefully collect 100 microliters of media from both the blood and brain sides of the inserts at five minutes, 30 minutes, 60 minutes, and 120 minutes. Transfer each sample to separate wells of the appropriate black, 96-well plate.
Use a plate reader to quantify the fluorescence from the collected samples. First, dilute the lysosome fluorescent dye to the appropriate working concentration in pre-warmed medium. Add this to the cells, and incubate at 37 degrees Celsius with 5%carbon dioxide for 45 minutes.
Then, rinse the cells three times with ice-cold PBS. Discard the PBS, and add three milliliters of ice-cold 4%paraformaldehyde to the wells and 2.5 milliliters to the insert. Incubate on ice for 10 minutes.
After this, discard the paraformaldehyde, and rinse the cells three times with PBS. Remove the PBS, and counterstain the cell nuclei using a DAPI solution at a final concentration of one microgram per milliliter in distilled water. Incubate at room temperature for seven minutes.
Then, remove the DAPI, and wash the membranes three times with distilled water. Carefully cut the membrane, remove the distilled water, and place it on a drop of mounting medium on a glass microscope slide. Add another drop of mounting medium on the other side of the membrane, and carefully cover it with a borosilicate cover glass.
In this study, endothelial cells were co-cultured with astrocytes to form a blood-brain tumor barrier-like interface in an in vitro setup. Confocal imaging of the murine BBTB mimic shows the expression and cellular localization of the tight junction proteins zonula occludens-1 and claudin-5 in bEND3. Contacts between the endothelial cells and astrocytes clearly induces the relocation of these two proteins to the endothelial cell-to-cell contacts compared to the bEND3 monocultures.
Using immunofluorescence staining to visualize the GFAP-expressing astrocytes at the brain side of the membrane, it is possible to observe and study the astrocytic processes and end-feet contacting the endothelial cells through the membrane. To compare the permeability values of the in vitro BBTB mimics with the in vitro blood-brain barrier, the real-time diffusion of sodium fluorescein is imaged through a cranial window implanted in nude mice. Using a fluorescence stereo microscope, sodium fluorescein diffusion from the blood vessel capillaries deriving from the main pial blood vessels is recorded before, during, and after systemic injection of the probe.
The transcytosis of nanoparticles that target patient-derived glioblastoma spheres is then compared to illustrate how this BBTB mimic can be used to visualize the passage of compounds from the blood side to the brain side. The NP110-associated fluorescent signal co-localizes with the lysosomes in the endothelial cells, astrocytes, and tumor cells. In addition, NP110s are detected in between the endothelial cells and astrocytes, passing through the membrane pores of the insert.
The passage of NP110s is quantified by measuring the fluorescence from samples collected from both the blood and brain side, and these values are compared to those determined nanoparticles that are 350 nanometers. As can be seen, only NP110s are able to cross the BBTB mimics, while NP350 remained on the blood side, resulting in lower permeability values for these nanoparticles. Handle the insert with care, and cautiously spread the astrocyte cell suspension on the brain side of the insert without direct contact of the membrane.
Any damage of the membrane will result in leak. This method can be adapted to answer the question of the brain delivery of peripherally administered payloads. By using different inserts with larger membrane pores, it's also possible to study cell extravasation.
This technique can be modified by using different cell types to mimic other cellular barriers. It also paves the way to a more responsible and ethical research, aiming to reduce the number of laboratory animals used for anti-cancer drug validation. Please remember that paraformaldehyde is a very toxic chemical and should be handled accordingly.
Also, be careful while using a sharp scalpel to extract the membranes from the inserts.
