In vitro BBB penetration of NPs

In vitro Nanoparticle Permeability Assay 

Defrosting and Maintenance of Cultures

1. Coat T-75 flasks (CellTreat, 229341) with 1% gelatin (porcine skin type A; Sigma, G1890; 5-6 mL for T-75 flask) and incubate them at 37°C for 15 minutes. 
2. After 15 minutes, aspirate gelatin and add 10 mL media (Dulbecco's Modified Eagle's Medium (DMEM; Gibco, 11965118) with 10% fetal bovine serum (FBS; Gibco, 26140079)) per flask.
3. Thaw bEnd.3 cells (ATCC® CRL-2299™) and seed them as 0.5-1 x 10⁶ bEnd.3 cells/flasks in total volume of 15 mL.
4.  Grow cells at 37°C, 5% CO₂. Replace the media with fresh media every 2-3 days.

Cell Seeding into Transwell^® inserts

1. When the cells reach 80-90% confluency, start permeability experiments.
2. On the day of cell seeding to Transwell^® inserts (Corning, 3401), remove growth factor reduced (GFR) Matrigel^® (Corning, 354230) aliquots from freezer and thaw them on ice.

CAUTION: Matrigel^® jellifies quickly at room temperature. Prepare Matrigel aliquots using pre-chilled pipette tips and pre-chilled microcentrifuge tubes prior to experiments. Work on ice and avoid multiple freeze-thaw cycles.

1. Dilute GFR Matrigel^® in cold DMEM (without FBS and antibiotics) and coat 12 mm polycarbonate Transwell^® inserts with 2% GFR Matrigel^®.
2. Incubate Transwell^® inserts at 37°C for 1 hour.

CAUTION: Cells must be seeded immediately at the end of this period. 

1. Remove the media from T-75 flasks by aspiration. 
2. Wash cells with DPBS (Ca²⁺/Mg²⁺ free; Gibco, 14190359) and remove the solution by aspiration. 
3. Pipette 3 mL pre-warmed TrypLE^TM Express (Gibco; 12604-021) or 0.05% Trypsin/EDTA (Gibco; 15400-054) solution into T-75 flasks. 
4. Swirl the flask to ensure the solution to cover all the cells. 
5. Monitor the trypsinization process at room temperature under microscope. 
6. Release the rounded cells from the culture surface by hitting the side of the flask against your palm until most of the cells are detached. 
7. If you are using Trypsin, pipette 6 mL Trypsin Neutralizing solution (Lonza, CC-5002) or 10% FBS-containing DMEM to the flask to inhibit tryptic activity. If you are using TrypLE^TM Express, use DMEM to dilute the enzyme. 
8. Transfer the cell suspension into a 15-mL centrifuge tube (CellTreat, 229411) and centrifuge at 250×g for 5 minutes to pellet the cells. 
9. Aspirate the supernatant from the tube without disturbing the cell pellet.
10. Resuspend the cells in 2 mL DMEM (with FBS and P/S) by gently pipetting the cells up and down and count the cells.
11. Aspirate Matrigel^® in Transwell^® inserts and wash inserts 1X with DMEM (without FBS and antibiotics). 
12. Plate cells immediately into coated inserts at a density of 80,000 cells/insert in 500 mL.
13. Add 1.5 mL medium to the basolateral side and replace media in apical/basolateral sides every 2-3 days. 
14. Monitor Transendothelial Electrical Resistance (TEER) of endothelial cells using an EVOM-2 Volt/Ohm meter (World Precision Instruments). 
15. Once TEER reaches ≥50 ohm*cm² (~ after 1 week), perform permeability experiments. 

Nanoparticle Permeability Experiments

1. Prior to experiments, prepare a new 12-well plate with 1.5 mL DMEM/well (without serum and antibiotic solution).

CAUTION: It is recommended to use a phenol red-free medium (Gibco, 21063029) to prevent problems with fluorescence measurements. If phenol red-free medium is not commercially available, prepare calibration curves using labelled nanoparticles in phenol red-containing media to see the effect of phenol red in the measurements. Alternatively, HEPES-buffered Ringer’s solution can be used for nanoparticle permeability experiments. 

1. Dilute fluorescence-labelled nanoparticles in DMEM (or HEPES-buffered Ringer’s solution – See Step 24) at the desired concentration. 

CAUTION: The concentration range for different nanoparticle formulations must be determined prior to experiments. Calibration curves or prior in vitro experiments can help to find the best concentration range for each nanoparticle formulation.

1. Transfer Transwell^® inserts containing cells into the new 12-well plate prepared on Step 24.

CAUTION: Include at least 2 empty filters (per nanoparticle formulation) without cells, but with Matrigel coating, to calculate maximum (100%) nanoparticle penetration.

1. Aspirate the media from the apical part of the inserts and add 500 mL nanoparticle solution to the apical compartment in DMEM (or HEPES-buffered Ringer’s solution). Collect aliquots from apical and basolateral sides at time zero.

CAUTION: If you aim to find the effect of serum on nanoparticle penetration through blood-brain barrier, dilute nanoparticles in DMEM with mouse serum (Sigma, M5905) and incubate 30 min before the permeability experiments. 

1. Keep Transwell^® plates on an orbital shaker, at 37°C, 5% CO₂.
2. After 4 h, withdraw Transwell^® inserts from the receiver compartment. 
3. Collect aliquots from the apical and basolateral compartments and measure volumes in these compartments for the penetration and mass balance calculations. 
4. Prepare different concentrations of nanoparticle formulations in the solution you use in permeability studies (See Steps 24 and 25) to plot calibration curves. 
5. Quantify fluorescence by a plate reader (Infinite Pro 200, Tecan) using proper excitation/emission values for your fluorescent nanoparticles. Use at least three inserts with cells in each permeability measurement. Use empty filters without cells to determine the maximum nanoparticle penetration (100% penetration) in vitro.
6. Use calibration curves to calculate concentration of nanoparticles in apical and basolateral sides. 
7. Using volume values in basolateral/apical sides at the end of the experiment, calculate the amount (mass) of the nanoparticles in both compartments. 
8. To assess possible adsorption to plastics and non-specific binding to cells, calculate mass balance (%) by dividing the amount of nanoparticle recovered in both compartments at the end of the experiment to the total amount added in the donor compartment at time zero. Mass balance value should be between 80% and 120% for reliable calculations.
9. Calculate amount of nanoparticles penetrated to the basolateral compartment in empty filters (without cells; See Step 26). Consider this value as 100% penetration (penetration through filters, without endothelial cell barrier).
10. Normalize the data (% penetration) for different conditions using the value from empty filters (100% penetration).