Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules

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)

This video describes a permeability assay to evaluate the permeability of in vitro blood-brain barrier using sodium fluorescein molecules.

Protocol

Measurement of the BBTB Mimic Permeability Passive diffusion of the small-molecular-weight fluorescent dye sodium-fluorescein (Na-Fl) over time from the blood to the brain side of the inserts allows the calculation of the permeability values according to the following formula: Here, dFwell is the fluorescence value measured in the well at a certain time point minus the cell culture medium …

Materials

Material and Reagent
10 mL serological pipet	ThermoFisher Scientific	170361
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
Basal Medium Eagle	ThermoFisher Scientific	21010046	BME-1
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
Greiner CELLSTAR 96 well plates S	Sigma-Aldrich	Greiner 655090	Black polystyrene wells flat bottom (with micro-clear bottom)
Fluorescein sodium salt	Sigma-Aldrich	F6377
FLUOstar Omega microplate reader	BMG Labtech
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