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

Protocol

1. Measurement of the BBTB Mimic Permeability
   

   1. 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 autofluorescence value, dT is the time in seconds, A is the surface of the barrier in square centimeters, and dFinsert is the fluorescence value measured in the insert at the same time point minus the medium autofluorescence value).
   2. Collect 100 µL of the medium from both the blood and the brain sides of the BBTB mimic and transfer each of them to a separate flat-bottomed, black 96-well plate for subsequent fluorescence measurements. Use the plain media as the blank to correct the autofluorescence.
   3. Prepare 2.5 mL per well of the Na-Fl (50 µM) in EBM-. Prewarm the Na-Fl solution to 37 °C. Replace the media from the blood side of the inserts with the media containing Na-Fl. Start a timer as soon as the medium is replaced.
   4. Carefully collect 100 µL of media from both the blood and the brain side of the inserts at 5, 30, 60, and 120 min. Transfer each sample to separate wells of the black 96-well plate.
   5. Accordingly, replace the collected media from the inserts to maintain the volume balance between both sides. Place the inserts back in the incubator between each sample collection to minimize the temperature variations.
   6. Quantify the fluorescence from the collected samples, using a plate reader with the filter set on 480/560 nm (excitation and emission, respectively).
      NOTE: Fluorescence from the brain side is nearly undetectable at the 5 min time point. High values compared to the blank indicate a leak of/damage to the insert's membrane or the barrier; therefore, exclude these from further analyses. Expected Na-Fl permeability values for the BBTB should be in the 10–5 to 10–6 cm/s range (Table 1).

Table 1: Values of the sodium-fluorescein (Na-Fl) permeability (in centimeters per second) determined in vitro in the indicated co-culture systems and in vivo in NMRI nude mice. Data from a representative experiment (n = 3 mice).

Murine BBTB mimic	bEND3	bEND3+HIFko As	bEND3+GB	bEND3+HIFko As+GB	In vivo
Permeability (10–6 cm/s)	27.63	6.74	26.8	10.83	5.57
SD (10–6 cm/s)	3.45	3.01	7.99	2.65	2.19
Human BBTB mimic	HuAR2T	HuAR2T+hIAs	HuAR2T+GB	HuAR2T+hIAs+GB
Permeability (10–6 cm/s)	104.92	47.4	89.08	48.24
SD (10–6 cm/s)	27.1	14.32	10.21	13.07

Materials

Name	Company	Catalog Number	Comments
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