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

Abstract

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.

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.

1. Preparation of Astrocytes
   NOTE: The following volumes are suitable for a 10 cm Petri dish or a T75 cell culture flask.
   1. Under a sterile cell culture hood, carefully wash the cultured astrocytes with 5 mL of sterile phosphate-buffered saline (....

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 th.......

Materials

List of materials used in this article

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