Our protocol recognizes the need to better recapitulate the human blood-brain barrier in vitro for the study of complex cellular mechanisms that affect post-stroke recovery. Our triple model exhibits robust integrity which is partly related to large compartmenting of well inserts, allowing us to decrease the change frequency and avoid disruption of populations. Our model is a great tool for studying the molecular and cellular changes that occur at the blood-brain barrier during and after ischemic stroke.
Our protocol allows for the discovery of novel targets and therapeutics to improve post-stroke recovery. Current treatments for ischemic stroke are time sensitive and consequentially are not always effective. Begin by cultivating HBVP in T75 culture flasks with an activated surface for cell adhesion within a 5%carbon dioxide incubator at 37 degrees Celsius until confluent.
Once confluence is reached, aspirate the old pericyte medium and wash the cells with five milliliters of warm Dulbecco's PBS. Aspirate the Dulbecco's PBS and detach the cells from the flask using a combination of four milliliters of warm trypsin EDTA solution and one milliliter of Dulbecco's PBS. Incubate the flask for five minutes at 37 degree Celsius in a carbon dioxide incubator.
View under a microscope to confirm whether the cells are detached from the flask. Add five milliliters of warm pericyte medium containing 2%FBS to the flask and transfer the detached cells to a 50 milliliter centrifuge tube. Centrifuge the cell suspension for three minutes at 200 g allowing the cells to form a pellet in the bottom of the tube.
Aspirate the medium from the tube ensuring the cell pellet remains intact. Re-suspend the cell pellet in pericyte medium. Calculate the amount of medium depending on the confluence of the cells and the number of well inserts needed.
Take 10 microliters of the re-suspended cells, place them into a cell counting slide, and count the number of cells. Determine the cell density and seed 300, 000 cells per insert in one milliliter of pericyte medium onto the abluminal side of the well inserts. Cultivate primary human astrocytes using the astrocyte medium containing 2%FBS as described in the text manuscript.
Determine the cell density and seed 300, 000 cells per well onto the bottom of the tissue culture six-well plates. Cover the plate to prevent evaporation and incubate overnight. Similarly, cultivate HBMEC in tissue culture dishes using complete classic medium containing 10%FBS as described in the text manuscript.
Take out the tissue culture six-well plates containing astrocytes and the well inserts containing pericytes from the incubator. Aspirate the astrocyte medium from the tissue culture six-well plates and add one milliliter of pericyte medium and one milliliter of astrocyte medium to each well. Aspirate the pericyte medium from the well inserts and place them into the tissue culture six-well plates containing the seeded astrocytes.
Seed HBMEC at a density of 300, 000 cells per well in two milliliters of complete classic medium onto the apical side of the same well inserts. Wash the cells thrice with Dulbecco's PBS. For triple cell cultures subjected to oxygen-glucose deprivation, add glucose-free medium to both the apical and basolateral compartments.
Replace the culture medium with fresh medium and normoxic control cell cultures. Place control triple cultures in the 5%carbon dioxide incubator at 37 degrees Celsius. Place a Petri dish containing 20 milliliters of sterile water in the hypoxia incubator chamber to provide adequate humidification of the cultures.
Open the chamber by releasing the ring clamp, then arrange the cell cultures on the shelves. Seal the chamber by securing the ring clamp. Open the inlet and outlet ports of the chamber and attach the tubing from the top of the chamber's flow meter.
Attach the tubing from the bottom of the flow meter to the gas tank via an air filter. Open the tank flow control valve by turning it counterclockwise to allow minimum gas flow. Slowly open the pressure regulator valve by turning clockwise.
Flush the chamber with the gas mixture for five minutes at a flow rate of 20 liters per minute. Disconnect the chamber from the gas source and close both white plastic clamps firmly. Turn off the tank flow control valve by turning clockwise.
Close the pressure regulator valve by turning counterclockwise. Place the hypoxia chamber in a conventional incubator set at 37 degrees Celsius for four hours. Place the sterilized TEER instrument into the biosafety cabinet and plug the electrodes into the epithelial volt ohm meter.
Sterilize the electrodes in 30 milliliters of 70%isopropyl alcohol solution for a minimum of 30 minutes. Turn on the TEER instrument and set the function to ohms. Remove the electrodes from the 70%isopropyl alcohol solution and place them in 20 milliliters of Dulbecco's PBS for a minimum of 30 minutes until the digital readout on the TEER device reads zero ohms.
Insert the long prong of the electrode through one of the three openings in the well insert hangar of the blank well insert control lowering it until it touches the bottom of the well. Ensure that the short prong is resting above the apical culture on the bottom of the well insert. Wait until the digital readout values on the TEER instrument level off before recording the value.
Place the electrodes back into Dulbecco's PBS to wash them between measurements. Continue to collect all the TEER measurements for two more blank well insert controls. Collect the TEER measurements of the sample plates as demonstrated earlier.
Once all the measurements have been taken place the electrode back into the 70%isopropyl alcohol solution for 30 minutes. Then disconnect the electrodes from the TEER instrument and allow them to air dry. Calculate the TEER values.
Aspirate the medium from the basolateral compartment and replace it with two milliliters of phenol red free endothelial cell growth medium in the triple cell culture blood-brain barrier model. Wash the cells in the apical compartment twice with HBSS. Add one milliliter of the FITC dextran solution in the apical compartment and cover the plate with aluminum foil, then place the plate in the incubator set at 37 degrees Celsius with 5%carbon dioxide for one hour.
Take 100 microliters of medium from the basolateral compartments and transfer it into a black 96-well plate. Measure the fluorescence using a microplate reader with the excitation and the emission wavelengths set to 480 and 530 nanometers, respectively. Seed 200 microliters of HBMEC, primary astrocytes, and HBVP in the center of poly-D-lysine coated 35 millimeter glass bottom dishes at a density of 150, 000 cells per dish.
Before seeding HBMEC, coat the bottom of the dishes with the attachment factor. Allow the cells to attach to the glass surface by leaving them overnight at 37 degree Celsius in a carbon dioxide incubator. Once confluence is reached, discard the culture medium and add two milliliters of pre-warmed glucose-free medium for oxygen-glucose deprivation or normal medium for controls.
After oxygen-glucose deprivation treatment, replace the medium with the imaging optimized medium in all dishes, then perform fluorescence live-cell imaging with the GFP filter and top stage confocal microscope incubator as described in the text manuscript. The barrier integrity of the endothelial monolayer in the indicated blood-brain barrier model configurations was assessed by determining TEER before and after oxygen-glucose deprivations, as well as after 24 hours of re-oxygenation. For four hours the oxygen-glucose deprivation caused a significant decrease in TEER values only in HBMEC monoculture and the co-culture model with HBMEC and HBVP.
These decreased levels reached the baseline levels following 24 hours of re-oxygenation. The TEER values in the triple co-culture model were significantly higher than those in the double co-culture controls or monoculture control immediately after oxygen-glucose deprivation. The paracellular permeability of endothelial monolayers to 20 kilodalton molecular mass, FITC dextran, was drastically increased in the HBMEC monoculture and to a lesser extent in the co-culture model with HBMEC and HBVP as compared to normoxic controls.
Furthermore, 20 kilodalton FITC dextran permeability levels were the lowest in the triple brain-blood barrier model among all models under normoxic and oxygen-glucose deprivation conditions. No changes were observed in 70 kilodalton, FITC dextran flux across endothelial monolayers in any of the models. All primary human types exposed to oxygen-glucose deprivation exhibited strong green fluorescence, proving that the imaged live cells were hypoxic.
After seeding human brain vascular pericytes on the abluminal side of well inserts, it is very important to cover them with the flip plate to prevent evaporation. In our model we use a relatively small, 0.4 micrometer pore diameter of polyester well inserts making it suitable for the screening of drug candidates to any disease that requires crossing the blood-brain barrier for treatment. This technique provides an excellent opportunity to visualize modification of proteins and transporters if needed.
