Overview
In this video, we describe a comprehensive protocol to establish an in vitro model mimicking the blood-brain tumor barrier. The model holds promise to understand the delivery of therapeutic agents for the treatment of brain tumors.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Establishment of the BBTB Mimics
NOTE: Cell culture medium and supplements are detailed in the Table of Materials.
- Preparation of Astrocytes
NOTE: The following volumes are suitable for a 10 cm Petri dish or a T75 cell culture flask.- Under a sterile cell culture hood, carefully wash the cultured astrocytes with 5 mL of sterile phosphate-buffered saline (PBS). Gently discard the PBS using a vacuum pump and add 2 mL of the cell dissociation reagent for 5 min (at 37 °C, see the Table of Materials) to detach the cells. Check the cell detachment under the microscope. Do not exceed 5 min of incubation to limit the stress on the cells.
- Add 10 mL of sterile complete astrocyte cell culture medium (ABM+) 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 mL tube. Centrifuge the cell suspension for 3 min at 250 rcf (acceleration: 9 rcf/s, deceleration: 5 rcf/s) at room temperature (RT).
- Meanwhile, prepare the inserts (see the Table of Materials): Using sterile forceps, place the inserts with the brain side up (Figure 1A) on the lid of a sterile 6-well plate (Figure 1B). Verify beforehand that the plate can be placed upside down on the inserts without touching or moving the inserts during the process.
NOTE: The proper placement of the inserts allows the entrapment of the astrocyte suspension in-between the membrane and the bottom of the well. - Once centrifuged, carefully discard the supernatant from the cell suspension; resuspend the astrocyte pellet in 1 mL of ABM+ by gently resuspending the pellet on the tube's wall up to 5x. Avoid excessive pipetting of the cells to limit the stress on the cells. Count the cells and adjust the cell suspension density to 1.5 x 105 cells in 400 µL of ABM+/insert.
- Place the cell suspension in the middle of the brain-side of the insert's membrane (Figure 1B) and, very carefully, spread it by using capillary force with a sterile pipette tip. Avoid direct contact as the membrane is particularly fragile.
- With the brain side of the inserts still up, place the 6-well plate back on the inserts. This ensures that the cell suspension is trapped between the membrane and the actual bottom of the well (Figure 1C). Avoid air bubbles in the cell suspension, as it will prevent the homogeneous spread of the astrocytes on the membrane.
- Place the plate and inserts, with the brain side up, in the incubator (at 37 °C with 5% CO2) to allow the cell adhesion for a minimum of 2 h (murine immortalized astrocytes) and up to 6 h (human primary astrocytes).
NOTE: As the inserts are maintained upside down, visualization of the cell adhesion is not possible under a microscope. It is, therefore, recommended to seed a separate regular cell culture vessel and control the cell adhesion in the vessel over time. Careful manipulation of the membrane is a must as the results will be unreliable when membranes are damaged. - At the end of the incubation time, verify the absence of cell suspension leaks outside of the seeding area and discard the inserts if they are leaky. Revert the 6-well plate to its regular position, with inserts that will now have the blood side up (Figure 1A). Add 2.6 mL of ABM+ to each well. Pour 2.5 mL of complete astrocyte medium into each insert and place the plate in the incubator (at 37 °C with 5% CO2).
2. Preparation of Endothelial Cells
NOTE: For the murine brain microvascular endothelial cells (bEND3), the cells must reach 100% confluence to ensure maximal cell-cell contacts triggering the optimal tight junction protein expression on the day of the experiment. This does not apply for the human umbilical vein endothelial cells (HuAR2T) as the presence of astrocytes is required for a tight junction protein expression for these cells.
- Proceed as previously described for the astrocytes for cell dissociation. Once centrifuged, carefully discard the supernatant; resuspend the endothelial cell pellet in 1 mL of complete endothelial cell culture medium (EBM+) by slowly pipetting the cell suspension on the tube's wall up to 5x. Avoid excessive pipetting of the cells to limit the stress on the cells. Count the cells and adjust the cell suspension density to 2 x 105 cells in 2.5 mL/insert of endothelial cell culture medium devoid of serum (EBM-) and vascular endothelial growth factor-A (VEGF-A).
- Take out the plate containing the inserts, carefully discard the medium from the blood side, and replace it with 2.5 mL of the endothelial cell suspension. Return the plate to the incubator (at 37 °C with 5% CO2) and leave it overnight for the endothelial cells to adhere to the membrane.
- The next day, prepare a sterile 6-well plate by transferring 3 mL of prewarmed serum-free astrocyte medium (ABM-) to each well. By handling the inserts with sterile forceps, carefully discard the endothelial complete medium from the blood side, place the insert in the new plate containing ABM-, and add 2.5 mL of EBM-.
NOTE: The use of EBM- is critical for the establishment of the endothelial barrier. - Leave the inserts in the incubator (at 37 °C with 5% CO2) with minimal physical disturbance and temperature variations for 5 days, allowing the production of the endothelial basement membrane, astrocyte contacts with endothelial cells, and eventually, the BBTB mimic formation. Replace the medium on the day of the transfer on glioma cell cultures.
3. Preparation of Glioma Cells
NOTE: Although patient-derived glioblastoma spheres are used here, the following protocol can be easily adapted for adherent, commercially available glioblastoma cells such as U-87MG.
- Optionally, for immunofluorescence imaging, place up to four round sterile borosilicate coverslips (ø 0.9 cm) per well in a 6-well plate containing 2 mL of poly-D-lysine (0.01%). Incubate at room temperature for 30 min.
- Meanwhile, carefully transfer the tumor spheres from the cell culture vessel to a 15 mL sterile tube using a sterile serological pipette. Centrifuge the tumor spheres for 3 min at 250 rcf.
- Discard the supernatant, gently resuspend the spheres in 1 mL of bFGF/EGF-free (GBM-) glioma cell medium and count the cells. Adjust the cell density to approximately 104 spheres/mL (105 cells/mL) in GBM-.
- Discard the poly-D-lysine from the wells and rinse them 3x with sterile PBS. Seed the plate with 3 mL/well of the tumor spheroid suspension and transfer the inserts with the BBB mimic on the tumor cell suspension.
- Incubate overnight (at 37 °C with 5% CO2) to allow equilibrium between the blood and the brain tumor sides of the assay. On the next day, replace the media in the blood side with EBM- supplemented with the molecules/drugs/nanoparticles of interest. Samples are collected over time for direct quantification as described in the previous section. Cells are fixed at a precise time point for fluorescence imaging.
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Representative Results
Figure 1: Description of the blood-brain tumor-barrier (BBTB) model. (A) Schematic representation of the locations of different cell types. (B) Illustration of the insert placement on the 6-well plate cover and the seeding technique for the astrocytes on the brain side of the insert’s membrane. (C) Illustration of the 6-well plate placement allowing the astrocyte adhesion. (D) Immunofluorescence micrographs of the tight junction proteins zonula occludens-1 (ZO-1, upper row, red) and claudin-5 (lower row, green). The protein expression is compared to the murine brain microvascular endothelial cells (bEND3) cultured on the blood side of the BBTB alone as a monoculture (left column) or with murine immortalized HIFko astrocytes (right column). Cell nuclei are counterstained with DAPI (blue). (E) Immunofluorescence micrograph showing the glial fibrillary acidic protein (GFAP, red) in HIFko astrocytes cultured at the brain side of the BBTB. The high-magnification image shows astrocyte processes and end-feet (arrows) contacting the endothelial cells through the membrane pores (right panel). The identity of the HIFko astrocytes was verified by immunofluorescence staining of the simian virus 40 large T antigen (SV40 large T, green) used for the immortalization of the cells. Endothelial cells express neither the GFAP nor the SV40 large T and, therefore, can be partially observed through the transparent, opposite side of the membrane as DAPI-only stained cells (dashed lines). Cell nuclei are counterstained with DAPI (blue).
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Materials
Name | Company | Catalog Number | Comments |
Cells | |||
bEND3 | ATCC | CRL-2299 | Cultured in: DMEM (1g/L glucose) supplemented with 10% FBS- 5 mL L-glutamine and 5 mL penicillin/streptomycin |
HIFko immortalized mouse astrocytes | Isolated in Dr. Gabriele Bergers Lab | https://doi.org/10.1016/ S1535-6108(03)00194-6 | Cultured in: BME-1 supplemented with 5% FBS- 5 mL 1 M HEPES-5 mL 100 mM sodium pyruvate- 3 g D-glucose and 5 mL penicillin/ streptomycin |
HuAR2T | Isolated in Dr. Dagmar Wirth Lab | https://doi.org/10.1089/ten.tea.2009.0184 | Cultured in: EBM-2 with SupplementMix normal human primary astrocytes Lonza CC-2565 Cultured in: ABM with SingleQuots |
Material and Reagents | |||
100 mm x17 mm Dish Nunclon Delta | ThermoFisher Scientific | 150350 | |
10 mL serological pipet | ThermoFisher Scientific | 170361 | |
15 mL Conical Sterile Polypropylene Centrifuge Tubes | ThermoFisher Scientific | 339650 | |
ABM Basal Medium 500 mL | Lonza | CC-3187 | For primary human astrocytes. ABM+: contains all the additives from the supplement mix. ABM-:all the additives except for rhEGF and FBS |
Accutase Cell Detachment Solution | Corning | 25-058-CI | |
AGM SingleQuots Supplements and Growth Factors | Lonza | CC-4123 | |
B27 supplement | Gibco | 17504-044 | For both GBM + and - medium |
Basal Medium Eagle | ThermoFisher Scientific | 21010046 | BME-1 |
Corning Costar TC-Treated 6-Well Plates | Sigma-Aldrich | CLS3506 | |
Corning Transwell polyester membrane cell culture inserts | Sigma-Aldrich | CLS3452 | |
D-glucose | Sigma-Aldrich | G8270 | Dissolve in 50 mL of BME-1 and sterile filter before adding to the medium |
Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham | Gibco | 21331-020 | Specific to the culture of the patient-derived spheres isolated in our lab; may vary for other glioma cell lines |
EBM-2 growth Medium SupplementMix | PromoCell | c-39216 | EBM+: contains all the additives from the supplement mix. EBM-:all the additives except for VEGF-A and FBS |
Endothelial Basal Medium 2 (EBM-2) | PromoCell | c-22211 | EBM+: contains all the additives from the supplement mix. EBM-:all the additives except for VEGF-A and FBS |
Fetal Bovine Serum (FBS), qualified, heat inactivated, E.U.-approved South America Origin | ThermoFisher Scientific | 10500056 | |
Fluorescein sodium salt | Sigma-Aldrich | F6377 | |
Greiner CELLSTAR 96 well plates | Sigma-Aldrich | Greiner 655090 | Black polystyrene wells flat bottom (with micro-clear bottom) |
Menzel-Gläser 0.9 cm round borosilicate Cover Slips | Thermo Scientific | 10313573 | |
PBS tablets | Medicago | 09-9400-100 | One tablet per liter of dH2O; then sterilize the solution by autoclaving |
Poly-D-lysine hydrobromide | Sigma-Aldrich | P6407 | |
Recombinant Human EGF | Peprotech | GMP100-15 | For GBM+ medium |
Recombinant Human FGF-basic (154 a.a.) | Peprotech | 100-18B | For GBM+ medium |