Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

Our goal is to determine the molecular and cellular mechanisms that mediate Alzheimer's disease pathogenesis to uncover therapeutic and diagnostic opportunities. Currently, the molecular pathways and mechanisms involved in the onset and development of Alzheimer's disease are poorly understood. This is due to mouse models and other in vitro Alzheimer's disease models failing to recapitulate unique human biology that is crucial to the development of the disease.

We have developed an in vitro model of the blood-brain barrier that can be applied to study cerebrovascular pathologies associated with Alzheimer's disease, dementia, and other forms of neurodegeneration. This model has been employed to show that APOE4, the strongest risk factor for Alzheimer's disease, acts in part through a parasite-specific pathway to increase pathogenic amyloid burden. Using induced pluripotent stem cells, we can construct human brain tissue in vitro that can be used to investigate disease pathology and drug screenings.

Such an in vitro blood-brain barrier model enables personalized therapeutic and diagnostic approaches. Our 3D in vitro blood-brain barrier model demonstrates physiologically relevant interactions including vascular tube formation and localization of astrocyte end-feet with vasculature. It is also capable of modeling disease relevant phenotypes, including amyloid pathology.

Finally, the use of patient-derived induced pluripotent stem cells allows the study of any desired genetic background. Using the in vitro blood-brain barrier model, we discovered pathways that mediate pathological cerebrovascular amyloid accumulation. This led us to explore therapeutic strategies that can reverse this pathology in aged APOE4 mice, which we are currently following up on.

Begin by preparing the differentiated endothelial cells, parasites, and astrocytes for assembling the iBBB model. After aspirating the cell culture medium from the differentiated endothelial cells, wash the cells with PBS once, then add a protease collagenase mixture to the washed cells and dissociate the cells at 37 degrees Celsius for five minutes. Transfer the protease collagenase mix with the dissociated cells into a conical tube containing hECSR, maintaining a one-to-three ratio of the mix to hECSR.

Then centrifuge the cells at 300 G for three minutes and aspirate the supernatant before resuspending the resultant cell pellet in the hECSR. Dissociate the differentiated parasites with the protease collagenase mixture at 37 degrees Celsius for five minutes. Add the appropriate amount of N2B27 in a conical tube and transfer the dissociated cells in the protease collagenase mix to it, maintaining a one-to-three ratio of the mix to N2B27.

Spin down the cells at 300 G for three minutes. After aspirating the supernatant, resuspend the cell pellet in N2B27. Dissociate the differentiated astrocytes with the protease collagenase mixture at 37 degrees Celsius for five minutes.

Transfer the dissociated cells in the protease collagenase mix to a conical tube containing complete astrocyte medium at a one-to-three ratio of the mix to the medium. After centrifuging the cells at 300 G for three minutes, aspirate the supernatant and resuspend the cell pellet in complete astrocyte medium. The cells are now ready for assembling the iBBB.

Assemble the human pluripotent stem cell-derived blood-brain barrier, or iBBB, using the dissociated endothelial cells, parasites, and astrocytes. Combine the three types of cells in a conical tube while including 10%more than the calculated number of cells to account for pipetting errors. Spin down the cell mixture at 300 G for three minutes.

Aspirate the supernatant, leaving the cell pellet with approximately 50 microliters of the medium. Using a P200 pipette, gently resuspend the cell pellet in the residual medium to create a single cell slurry. Place the tube containing the resultant cell slurry on ice and add a sufficient amount of reduced GF basement membrane matrix to it per the desired number of iBBBs.

Mix the cells homogeneously while not introducing any air bubbles. Pipette 50 microliters of the resultant cell matrix mixture into a well of a 48-well or 96-well glass bottom culture dish and distribute it evenly throughout the bottom of the dish. Incubate the dish at 37 degrees Celsius for 30 to 40 minutes to polymerize the matrix while encapsulating the cells.

Finally, add 500 microliters of supplemented astrocyte medium to the well and maintain the iBBBs in culture. For a 96-well plate, add 100 to 150 microliters of medium to the well until they are sufficiently developed for downstream assays, which typically takes two weeks. The properly formed iBBB appeared as a solidified single translucent disc under a brightfield microscope.

After 24 hours, evenly distributed single cells were identified. After two weeks, more distinct structures, although difficult to define, were visible.