Source: Le Joncour, V. et al. Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish. J. Vis. Exp. (2019)
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.
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.
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.
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.
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).
The authors have nothing to disclose.
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 |