iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

My research focuses on understanding human airway remodeling. The human airway serves as a barrier against environmental toxins, including viruses, air pollution, and tobacco smoke. These toxins can injure the airway, making them change or remodel, making it difficult to breathe.

The model system includes not only the epithelial cells that form the barrier, but other cells that are important in airway homeostasis, including blood vessel cells and immune cells called macrophages. Current experimental challenges include modeling the human lung in an in vitro model system. Obtaining primary lung samples is difficult and they're also very difficult to culture.

Thus using stem cell-derived lung airway cells invested with endothelial cells and macrophages is the next step to overcome these challenges. To begin, coat the apical side of 3-micrometer pore polyester cell culture inserts with 4 milligrams per milliliter of extracellular matrix solution, or ECM. Pipette out the residual ECM solution.

Then place the plate in the incubator for 1 hour at 37 degrees Celsius. Using clean tweezers, sterilely transfer the cell culture inserts from the 12-well plate to a large Petri dish. Next, coat the basolateral side of the cell culture inserts with ECM solution.

After pipetting off the residual ECM solution, place the Petri dish in a 37 degree Celsius incubator to dry overnight. Wash the T75 flask containing iPSC-derived endothelial cells with PBS. After aspirating the solution, add 5 milliliters of trypsin-like protease to the flask and incubate at 37 degrees Celsius for 8 minutes.

Tap the flask to ensure the endothelial cells detach. Then transfer the cells into a 15 milliliter conical tube. Add 5 milliliters of stop media to halt the dissociation.

Afterward, centrifuge the cells at 300 G for 5 minutes. Once the supernatant is aspirated, re-suspend the cell pellet in 1 milliliter of endothelial culture media containing 10 micromolar of ROCK inhibitor. After aliquoting out 150, 000 cells, re-suspend in 100 microliters of media and pipette onto the inverted cell culture insert with the basolateral side facing up.

Carefully transfer the cells into the incubator and leave them undisturbed for 3 hours. Next, in a 12-well plate add 1 milliliter of endothelial culture media to each well. Remove the Petri dish with endothelial cells from the incubator.

Using clean tweezers, carefully transfer the cell culture inserts into the plate with endothelial culture media. After visually verifying that the endothelial cells have adhered, place the plate in a 37 degree Celsius incubator overnight. After isolating organoids from ECM polymer and trypsinizing them for 15-20 minutes, add 3 milliliters of stop media to stop the protease reaction.

Resuspend the solution using a P1000 pipette and centrifuge at 400 G for 5 minutes. Then, aspirate the supernatant from the airway organoids and resuspend the organoids in 1 milliliter of airway expansion media with 10 micromolar of ROCK inhibitor. Take a 10 microliter sample for a hemocytometer cell count and place the remaining cells on ice during the count.

Next seed 300, 000 iPSC-derived airway cells in 500 microliters of airway expansion media per 12 millimeter cell culture insert. Remove the plate with cell culture inserts containing endothelial cells from the 37 degrees Celsius incubator. Resuspend iPSC airway cells and pipette 500 microliters containing 300, 000 cells into the apical chamber of each cell culture insert.

Place the plate in a 37 degrees Celsius incubator for 48 hours, keeping the apical cells in liquid-liquid conditions. Following incubation, remove the media from the apical side of the cell culture insert. Change the basolateral media to a 1:1 mixture of airway differentiation media and endothelial culture media.

Return the plate to the incubator. Dissociate iPSC-derived macrophages from the flask using a cell scraper. Centrifuge at 300 G for 5 minutes.

After that, aspirate the supernatant. Then resuspend iPSC macrophages in 1 milliliter of MAC-CM2 media. Take a 10 microliter sample for cell counting and place the remaining cells on ice during the count.

After counting, transfer 300, 000 macrophages into a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 200 G for 5 minutes. After centrifugation, pipette out the supernatant from the tube.

Remove the inserts containing co-cultures of endothelial and airway cells from the incubator. Seed 300, 000 macrophages in 35 microliters of MAC-CM2 onto the apical side of the cell culture insert.