This protocol will demonstrate how differentiated induced pluripotent stem cells can be seeded on an organ-on-chip to generate a fully human, personalized, microfluidic blood-brain barrier, which can be used to predict central nervous system drug permeability and to study neurological disorders. Using a commercially available organ-on-chip, we will demonstrate how any biologically oriented lab can use this technology to generate a personalized, microfluidic blood-brain barrier-on-chip. Accumulating evidences suggest that BBB plays a role in neurological disease by generating personalized BBB chips derived from iPSCs of individuals with a genetic neurological disease.
It will be possible to study the role of the BBB in health and disease. This method can also be useful for pharma companies, which can use this human BBB platform to screen for the penetrability of candidate neurological drugs into the human brain. The application of organ-on-chip technologies usually requires specialized engineering skills.
Using commercially available platforms enable the application of this technology in any biologically oriented lab. 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 one times 10 to the six cells per milliliter to the top channel inlet, and gently release the tip from the pipette.
Take an empty P200 pipette, depress 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 and homogenous 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 pulling the liquid via 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 times 10 to the six 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 chip.
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-degree 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 three 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 six 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.
Immunocytochemistry on the iPSC-based blood-brain barrier chip seven days post-seeding shows the EZ spheres differentiated into a mixed neural cell population in the top brain side channel, including S100 beta-positive astrocytes, Nestin-positive neural progenitor cells, as well as GFAP-positive astrocytes, and beta III tubulin-positive neurons. IPSC-derived brain microvascular endothelial cells seeded in the bottom blood side channel expressed GLUT-1 and PECAM-1. Evaluation of the blood-brain barrier chip permeability demonstrates that the organ chip seeded with iPSC-derived brain microvascular endothelial cells and EZ spheres had a tight barrier compared to organ chips seeded with iPSC-derived brain microvascular endothelial cells alone.
Organ chips seeded with EZ spheres alone did not display any barrier properties. From the initial surface treatment of the channel to the medium switching each day, avoid bubble formation in the channel. Throughout the step in the protocol in which surface activation reagent, extracellular matrix, or cell-containing medium needs to be inserted through the channels, it is extremely important to carefully verify that no bubble are found.
The permeability assay can be performed to assess the human brain penetrability of candidate drugs. This approach can serve for testing potential therapeutics. The application of the iPSC-based BBB chip allows studying the role of the BBB in various neurological diseases.
In the future, such platform may be useful for predictive, personalized medicine applications.
