We are developing a tissue chip model of the blood-brain barrier to understand the impact of sepsis on the brain and the contribution of bloodborne factors. Using human induced pluripotent stem cells, we plan on evaluating the contribution of genetic factors to the susceptibility of certain patient populations. Current experimental challenges within the tissue chip community are the reproducibility of results and the adoption of techniques by outside labs.
Unique tissue chip designs, small volumes within tissue chip, and lack of standard functional assays all contribute to these challenges and often limit users to fluorescence based assays as their primary output. microSIM devices offer modularity for versatile in vitro modeling, a glass-like surface ideal for high resolution imaging, and an ultrathin 100 nanometer, highly porous nanomembrane, ensuring seamless soluble factor crosstalk and precise permeability assays. With the microSIM, we have seen that specific cell types tend to produce more of the matrix proteins that provide a supporting scaffold around brain blood vessels.
Now we can ask whether inflammation causes these same cells to strengthen or break down their matrices, thereby altering barrier integrity. In the future, we will add another compartment to the model with green cells such as astrocytes and microglia to study how those cells interact with the blood-brain barrier in cases of systemic inflammation like sepsis. We also plan to investigate additional causes of systemic inflammation like postoperative delirium superimposed on dementia.
To begin, place all the materials required for microSIM device assembly in a biosafety hood. Using chip tweezers, place the membrane chip onto the center of fixture A1.The flat surface of the chip faces down and the trench area faces up. Tilt the fixture with a chip to show reflection from the trench.
Peel off the blue protective layers on both sides of component one using straight tweezers and place it in fixture A1 with the top chamber facing down. Then gently press component one until the PSA touches the chip. Place fixture A2 onto fixture A1 and apply firm pressure at different corners to ensure a tight fit of the chip and component one.
For bonding component two to component one with the chip, grab one corner of component two with straight tweezers and peel it off from the sheet. Then grab the non-PSA or the triangle region of component two and remove the blue layers of component two, exposing the PSA surface. Place component two in fixture B1 with PSA side up.
Then place component one along with the membrane chip in fixture B1 with the top chamber facing up. Place fixture B2 onto component one and apply firm pressure at different corners. Remove the assembled device from the fixture and use straight tweezers to seal the edges of the channel.
Before using the device for cell culture, sterilize it using ultraviolet light for 20 minutes. To begin, place the assembled microSIM device in sterile hose clamps. Culture the device in a large sterile Petri dish.
For extra humidity, place a 50 milliliter conical tube lid filled with sterile water. To prepare the device for cell culturing, rinse the top chamber with 100 microliters of water. Next, prepare a coating solution by mixing collagen four, bovine fibronectin, and sterile water.
Remove the water from the top chamber and add 100 microliters of coating solution. Incubate the chamber at 37 degrees Celsius for two to four hours. After incubation, replace the coating solution in the top chamber with 100 microliters of room temperature hECSR.
Add 20 microliters of hECSR solution in the bottom chamber. To passage EECM-BMEC-like cells, add a cell detachment enzyme mixture to the cells and incubate at 37 degrees Celsius for five to eight minutes. Next, pipette the cell suspension and add it to four times the volume of the endothelial medium in a centrifuge tube.
Centrifuge at 200 G for five minutes and resuspend the cells in one milliliter of hECSR. Seed the cells at a density of 40, 000 cells per square centimeter in the top chamber and incubate at 37 degrees Celsius for two to four hours to facilitate cell adhesion. After incubation, exchange the medium with fresh hECSR in both chambers.
Fix the cells by adding 20 microliters of 100%methanol, chilled at minus 20 degrees Celsius, into the bottom chamber and 50 microliters into the top chamber. Incubate the device at room temperature for two to 10 minutes. Next, wash the cells by adding 20 microliters of PBS into the bottom chamber and 100 microliters into the top chamber.
After each wash, confirm the lack of bubbles in the bottom chamber. Block the cells for 30 minutes by adding 20 microliters of the blocking solution into the bottom chamber and 50 microliters into the top chamber. After blocking, replace the volume in the top chamber with 50 microliters of the primary antibody solution and incubate for one hour at room temperature.
After PBS washing, add 50 microliters of the secondary antibody solution into the top chamber and incubate for one hour, protected from light. After PBS washing, add 50 microliters of Hoechst to the top chamber and incubate for three minutes. Then add 20 microliters of PBS to the bottom chamber and 100 microliters to the top chamber.
Image the device immediately on a confocal microscope. Locate the membrane window using a wet long working distance 40x objective and switch to fluorescence channels to check Hoechst and junction staining. Optimal seeding densities for hiPSC cell culture and underseeded BPLC are demonstrated here.
The hiPSC-derived co-culture was easier to distinguish in phase contrast imaging than primary co-cultures. Low BPLC seeding results in poor pericyte coverage and clumping, while overseeding leads to the pericyte layer peeling off the membrane. In immunostained hiPSC derived cell co-culture, two layers of cells were seen in close proximity, separated only by a thin silicon nitride nanomembrane.
In the permeability assay, high variability in the permeability of the endothelial cells cultured for two days indicates that two days of culture was insufficient for barrier maturation. Further, there were no significant differences in permeability between the labs upon barrier maturation from four days on.
