This protocol is helpful because it provides easy to follow steps that allow for the generation of functional airway epithelial cells, which can then be used to study various aspects of airway biology. The main advantage of this protocol is the ability to generate many functional airway epithelial cell cultures while simultaneously avoiding the difficulties of obtaining and culturing primary airway cells. Demonstrating the procedure will be Taylor Matte, a graduate student from our laboratory.
Begin by thawing a sufficient volume of 3D matrix on ice. Keep it on ice until ready for use. Thaw a vial of previously cryopreserved single cell suspensions of iBCs by incubating it in a 37 degree Celsius water or bead bath until there are no visible frozen media.
Using a five milliliter serological pipette, transfer the cell suspension to a 15 milliliter conical tube. Add six to 10 milliliters of DMEM/F12 dropwise to the cell suspension. Gently mix and centrifuge at 300 RCF for five minutes to pellet the cells.
Aspirate the supernatant and resuspend the cell pellet in one milliliter of Basal Cell Medium with a P1000 micropipette. Prepare a 10 microliter aliquot and perform a cell count. Centrifuge the cells at 300 RCF for five minutes.
Aspirate the supernatant and resuspend the cells at a density of 4, 000 cells per microliter in the previously thawed at 3D matrix with a P1000 micropipette. With a P200 micropipette, add a droplet of the matrix solution corresponding to approximately 25 to 50 microliters to the base of each well of a 12-well tissue culture-treated plate. Incubate the plate at 37 degrees Celsius for approximately 15 minutes.
Then add enough basal cell medium to each well to fully submerge the droplet using a five milliliter serological pipette. Return the plate to a humidified incubator. Feed cells every two days using fresh Basal Cell Medium.
Add fresh medium to the side of the well with a five milliliter serological pipette, taking care not to disturb the droplet of cells. Thaw a sufficient volume of the 3D matrix on ice. Keep it on ice until ready for use.
After approximately five to seven days of incubating culture plates, aspirate the medium from each well, then using a P1000 micropipette, add one milliliter of dispase II directly onto the spheroids to dissociate from the droplet of the matrix. Place the plate in 37 degrees Celsius incubator for 10 to 15 minutes. Using a P1000 micropipette, mix the dispase by pipetting up and down twice, breaking up large clumps of the 3D matrix.
Return the plate to 37 degrees Celsius for an additional 30 to 40 minutes, allowing the matrix to dissolve completely. When the droplet of the 3D matrix is no longer visible under the light microscope, add the freely floating spheroids to a 15 milliliter conical tube using a five milliliter serological pipette tip. Add DMEM/F12 for a final volume of 10 milliliters per conical tube and centrifuge at 200 RCF for three minutes to pellet the spheroids.
Aspirate the supernatant and add one milliliter of 0.05%trypsin for every initial droplet dissociated. Incubate at 37 degrees Celsius, triturating it every two to three minutes. Evaluate the solution with the light microscope every few minutes.
When more than 90%of spheroids have been dissociated to single cells, add 10%fetal bovine serum to the trypsin. Filter the cells through a 40 micrometer cell strainer and centrifuge at 300 RCF for five minutes. Perform a cell count and evenly resuspend the cells at a density of 400 cells per microliter of thawed 3D matrix.
Avoid introducing air bubbles. Resuspend as many droplets as needed for downstream application. Repeat this process for each well.
After 10 to 14 days of the most recent passage, dissociate the spheroids to a single cell suspension as demonstrated earlier and perform a cell count. Resuspend the cells in sort buffer. This will be the main population.
Transfer a small aliquot of 25 to 50 microliters to a separate tube. This will be the minor population. Add conjugated anti-NGFR antibody to the main cell population.
Add isotype control antibody to the minor cell population. Protect the cells from light and keep them on ice for 30 minutes intermittently Triturate the cells to prevent pelleting. After 30 minutes, add the sort buffer to each tube of cells.
Centrifuge cells at 300 RCF for five minutes. Aspirate the supernatant, resuspend the pelleted cells in the sort buffer and add live or dead cell stain. Using an appropriate NGFR positive gating strategy, sort enough NGFR positive cells for necessary downstream applications.
Prepare 6.5 millimeter porous membrane inserts by adding a 200 microliter matrix to the apical chamber per the manufacturer's instruction. Place it at 37 degrees Celsius for at least two hours before needed. Aspirate the coating matrix from the apical chamber of the insert.
Add 500 microliters of the Basal Cell Medium to the basolateral chamber. Resuspend at least 30, 000 sorted NGFR positive cells in 100 to 200 microliters Basal Cell Medium and transfer to the apical chamber using a P200 micropipette. Repeat for the remaining wells.
Place the plate in a humidified 37 degrees Celsius incubator. In two to three days, aspirate apical and basolateral chambers and feed with fresh Basal Cell Medium. Monitor the apical chamber daily with light microscopy.
When cells reach more than 80%confluence, replace Basal Cell Medium with ALI differentiation medium in both apical and basolateral chambers. Protect the ALI differentiation medium from light by keeping media containers wrapped in foil and by turning off overhead lights when possible. The next day, aspirate the medium from the apical chamber, thus exposing the apical surface to air.
Replace the ALI differentiation media in the basolateral chamber every two to three days. Monitor the culture appearance every one to two days. Carefully aspirate any accumulated liquid from the apical chamber without disturbing the cell layer.
Gently add 100 microliters of PBS without calcium or magnesium to the apical chamber to remove debris or mucus. Incubate at 37 degrees Celsius for 10 minutes and then carefully aspirate PBS. Assess the TEER for epithelial integrity.
After seven to 10 days of air exposure, observe for the appearance of multicilia using light microscopy. Depending on the planned experiment and readout, cells can be analyzed after 14 to 28 days of air exposure. After 10 to 14 days of the most recent 3D culture passage, dissociate the spheroids to single cell suspension as demonstrated earlier.
Perform a cell count, resuspend the cell suspension in cryopreservation medium in a cryovial. Place cryovials in a container to ensure a steady drop in temperature. And transfer to minus 80 degrees Celsius for 24 to 48 hours followed by transfer to minus 150 degrees Celsius for long-term storage.
With this gating technique, 28%of live single cells were NGFR positive. After two days, individual cells were initially easily identifiable and had an elongated spindle-shaped appearance. After two more days, the cells formed a confluent and loosely packed monolayer.
Over the subsequent days to weeks, the cells formed a tightly packed, highly cellular epithelial layer. And after seven to 10 days, there was a clear emergence of beating cilia and mucus production. The TEER of samples was measured and the results were similar to measurements of primary airway epithelial cell control samples.
Subsequent fixation with paraform aldehyde and immunolabeling for canonical airway epithelial cell markers was performed for MUC5AC and acetylated alpha-tubulin among others. Following this protocol, researchers can generate a large number of patient-derived airway epithelial cell cultures to assess various readouts, including those that test the functions of basal, secretory, and multiciliated cells in both disease and health.
