A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

Drug delivery to the brain remained a challenge. The development of large panels of new nanomaterials to improve the delivery to the brain highlights the need for real in vitro microsystem to predict brain penetration in the frame of preclinical assays. The main advantage of this technique is to ice the free pain cellular actuals of the blood brain barrier inside the same system allowing cell cell communications required for the induction of the blood brain barrier phenotype.

Moreover, each cell types can be isolated separately for further molecular analysis. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transports, as well as inter and intracellular trafficking. Demonstrating the procedure will be Dr.Clemence Deligne and Dr.Eleonora Rizzi.

Begin by coding the wells with 500 microliters of two micrograms per square centimeter Poly-L-Lysine solution. Incubate overnight or for a minimum of one hour at 37 degrees Celsius and remove the Poly-L-Lysine solution carefully with a glass pipette connected to an aspirating system. Then rinse twice with sterile water.

To collect the astrocytes, first wash the astrocytes once with 10 milliliters of warm 1X PBS CMF. Incubate them for three minutes with 10 milliliters of warm 20%Trypsin EDTA solution at 37 degrees Celsius under 5%carbon dioxide and 21%oxygen. Mechanically detach the cells from the flask and transfer the suspension to a conical tube containing five milliliters of warm non-diluted FCS.

Centrifuge the suspension at 190 times G for five minutes at 20 degree Celsius. Re-suspend the cell pellet in five milliliters of warm astrocyte medium, then dilute 20 microliters of the cell suspension with 80 microliters of 1X PBS CMF. Count the cells using a manual counting chamber under a microscope.

Plate around 40, 000 cells per square centimeter in each Poly-L-Lysine pre-coded well in a volume of 1.5 milliliters of warm astrocyte medium. Place the reverted insert filters at the periphery of a covered 25 millimeters high dish and add 250 microliters of collagen type one solution to the insert filters. Incubate at room temperature for one hour.

Then carefully remove the collagen type one solution with a glass pipette connected to an aspirating system. Wash twice with 250 microliters of DMEM high glucose at room temperature and carefully remove all the solution from the insert filters. Wash the pericytes twice with 10 milliliters of warm 1X PBS CMF and incubate the cells with two milliliters of warm Trypsin.

Observe the cells under the microscope to monitor the action of the Trypsin. When the cells start to detach, remove the Trypsin, then add five milliliters of warm endothelial cell basal medium or ECM to the cells, and start mechanical dissociation. Dilute 20 microliters of the cell suspension with 80 microliters of 1X PBS CMF, and count the cells using a manual counting chamber under a microscope as shown previously.

Seed 44, 500 cells per centimeter square on the pre-coded reverted insert filters in a volume of 250 microliters. Keep the insert filters in a humidified incubator at 37 degrees Celsius for three hours under 5%carbon dioxide and 21%oxygen. Use sterile tweezers to carefully revert the insert filters into a 12 well plate containing 1.5 milliliters of warm ECM per well.

The insert filters are now ready to be coated on the other side. Coat the upper side and the insert filters with 500 microliters of extracellular matrix based hydrogel. Place the insert filters in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide and 21%oxygen for an hour.

Wash them once with 500 microliters of DMEM high glucose at room temperature. Wash the endothelial cell culture once with 10 milliliters of warm 1X PBS CMF and incubate the cells with two milliliters of warm Trypsin. Remove the Trypsin when the cells start to detach, then add five milliliters of warm ECM and start mechanical dissociation.

Dilute 20 microliters of the cell suspension with 80 microliters of 1X PBS CMF and count the cells using a manual counting chamber. Seed the endothelial cells at a density of 71, 500 cells per centimeter square on the pre-coded insert filters in a volume of 500 microliters of warm ECM. Replace the astrocyte medium with 1.5 milliliters of warm ECM per well.

Then transfer the seeded insert filters to the wells containing the astrocytes. Place the tri-culture cell systems in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide and 21%oxygen. Take off the medium carefully from the upper and bottom compartment using a glass pipette connected with an aspiration system.

Replace the medium with warm ECM in a volume of 500 microliters in the upper compartment and 1.5 milliliters in the bottom compartment. Renew the medium every other day until day six by repeating the same procedure. Finally put the cells back into humidified incubator at 37 degree Celsius under 5%carbon dioxide and 21%oxygen.

Immunocytochemistry data confirmed the expression of conventional markers such as platelet derived growth factor receptor beta and Desmine for pericytes, and glial fibrillary acidic protein for astrocytes. Hence, after six days of culture with the pericytes and astrocytes, the monolayer of brain-like endothelial cells or BLECs visualized with the adherent junction staining of Ve-Cadherin displays a continuous localization of TJ proteins CLD5 and Z-01 at the cell borders. Paracellular permeability of BLECS to fluorescent BBB integrity markers, sodium fluorescein and FITC dextran is shown here.

R123 intracellular accumulation in BLECs exhibited a significant increase in the presence of the efflux pump inhibitor Elacridar compared to the control condition with its absence. This indicates the presence of active efflux pump molecules namely P glycoprotein and breast cancer resistance protein or BCRP in the BLEC's. Gene expression of tight junction proteins, transporters, and large molecule receptors normalized by the expression of RPLP0 are shown here.

Here, the values greater than one correspond to higher gene expression in the triple culture model. The red line corresponds to a value of one where the expression level of the two models is equivalent. The representative image shows the protein level of tight junction proteins, transporters, in large molecule receptors normalized by the expression of beta actin.

The transport of fluorescently tagged NIPAM based neutral polymeric nano gels was evaluated. At time zero, polymeric nano gels were placed in the luminal compartment at a concentration of 0.1 milligrams per milliliter. After 24 hours of incubation, 5.82%of the polymeric nano gels were found in the abluminal compartment proving their ability to cross the BLECs.

The correct coating and seeding of the pericytes on the reverted filter is pivotal. Important is the maintenance of the structures sterility and integrity to avoid contamination of the cell culture system.