Generating a Blood-Brain Barrier Chip Using iPSC-derived Precursor Cells

To begin, bring the dish containing the prepared chips to the biosafety cabinet. Using a P200 pipette, gently wash both channels by adding 200 microliters of neural differentiation medium into the inlet and pulling the liquid from the outlet. Avoiding contact with the ports, carefully aspirate excess media droplets from the surface of the chip.

Gently agitate the cell suspension to ensure a homogeneous cell suspension. To seed the iPSC-derived neural progenitor cells into the top channel to generate the brain side, add a P200 tip containing 30 to 100 microliters of cell suspension at the concentration of 1 x 10⁶ cells per milliliter to the top channel inlet, and gently release the tip from the pipette.

Take an empty P200 pipette to press the plunger, insert into the top channel outlet, and carefully pull the single-cell suspension through the tip. Transfer the chip to the microscope to check the seeding density in homogeneous distribution of cells within the top channel. After confirming the cell density at 20% coverage, immediately place the chips in the incubator for two hours at 37 degrees Celsius.

After that, wash away the cells that do not attach by adding fresh neural differentiation medium into the inlet, and pooling the liquid by a pipette from the outlet. Keep the cells under static conditions at 37 degrees Celsius with a daily neural differentiation medium replacement. After iNPCs have attached or on a subsequent day following seeding, bring the dish containing the prepared chips to the biosafety cabinet. Gently wash the bottom channel with 200 microliters of endothelial cell medium.

Avoiding contact with the ports, carefully aspirate droplets of excess endothelial cell medium from the surface of the chip, making sure to leave medium in both channels. Gently agitate the cell suspension to ensure a homogeneous cell suspension. Using a P200 pipette, draw up 30 to 100 microliters of the iPSC-derived brain microvascular endothelial cell suspension at the concentration of 14 to 20 x 10⁶ cells per milliliter, and place the tip into the bottom channel inlet.

The most critical step to achieve functional barrier properties is the seeding of brain microvascular endothelial cells. It is crucial to seed cells at proper density to avoid bubble formation to verify homogeneous distribution within the organ sheath.

Depress the plunger on a P200 pipette with an empty tip, insert into the bottom channel outlet, and slowly release the pipette plunger to carefully pull the single-cell suspension through the bottom channel. Aspirate the excess cell suspension from the surface of the chip. Avoid direct contact with the inlet and outlet ports to ensure that no cell suspension is aspirated out of the channels. Transfer the chip to the microscope to check the seeding density.

The bottom channel is filled with cells with no observable gaps in between. After confirming the correct cell density, seed the cells in the remaining chips. To attach the cells onto the porous membrane, which is located on top of the bottom channel, invert each chip, and let them rest in a chip cradle. Place a small reservoir containing sterile DPBS inside the 150-millimeter dish to provide humidity for the cells.

Incubate the chips at 37 degrees Celsius for approximately three hours or until cells in the bottom channel have attached. Once the iPSC-derived brain microvascular endothelial cells have attached, flip the chips back to an upright position to allow cell attachment to the bottom portion of the bottom channel. 48 hours post-seeding of the iPSC-derived brain microvascular endothelial cells, equilibrate the temperature of the endothelial cell medium by warming in a 37 degrees Celsius water bath for one hour.

Then, degas the medium by incubation under a vacuum-driven filtration system for 15 minutes. Next, sanitize the exterior of the portable module packaging and trays with 70% ethanol, wipe, and transfer them to the biosafety cabinet. Open the package, and place the modules into the tray. Orient them with the reservoirs toward the back of the tray. Pipette 3 milliliters of the pre-equilibrated warm media to each inlet reservoir. Add endothelial cell culture medium to the inlet reservoir of the bottom channel and neural differentiation medium to the top channel inlet reservoir of the top channel.

Then, pipette 300 microliters of the pre-equilibrated warm media to each outlet reservoir directly over each outlet port. Place up to 6 portable modules on each tray. Bring trays to the incubator, and slide completely into the culture module with the tray handle facing outward. Select and run the prime cycle on the culture module. Close the incubator door, and allow the culture module to prime the portable modules for about one minute.

The priming cycle is completed when the status bar reads "Ready." Remove the tray from the culture module and bring it to the biosafety cabinet. Inspect the underside of each portable module in the biosafety cabinet to verify that the portable modules were successfully primed. Look for the presence of small droplets at all four ports. If any portable module does not show droplets, rerun the prime cycle on those modules. If any media dripped onto the tray, which occurs more often by the outlet ports, clean the tray with 70% ethanol.

Next, gently wash both channels of each chip with warm equilibrated cell-specific culture medium to remove any possible bubbles in the channel, and place small droplets of media on the top of each inlet and outlet port. Insert the chips with carriers into the portable modules, and place up to six on each tray. Insert the trays into the culture module. Program the appropriate organ chip culture conditions, such as flow rate and stretch on the culture module. As soon as the Regulate cycle is complete, which takes approximately two hours, the programmed conditions start. After that, the culture module begins flow at the preset organ-chip culture conditions.