Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier

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 homogenously 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 disk 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.