This protocol is for a robust and cost-effective method that can be used to address a variety of research questions related to the function and the role of airway epithelial cells in health and disease. The main advantage of this technique is that it results in a cell layer that's a good representation of the human epithelial cell layer, including mucus production and ciliary activity. This technique is versatile and can be used to study disease-related insults, or therapies.
To begin, rinse the bronchial ring isolated from human lung tissue with 10 milliliters of sterile PBS in a 10 centimeter Petri dish. While holding the ring with tweezers, use small scissors to remove any excess connective tissue and blood remnants. Cut the ring in two, and submerge two halves in 10 milliliters of pre-warmed Protease 14 solution in HBSS containing Primocin in a closed sterile container.
Incubate the bronchial ring pieces at 37 degrees Celsius for two hours. After incubation, transfer the pieces to a Petri dish containing 10 milliliters of warm PBS, and using bent tweezers, scrape the inside of the ring to obtain a cell solution. Discard the ring.
Transfer the cell solution to a 50 milliliter tube, and add warm PBS to obtain a final volume of 50 milliliters. Centrifuge the cell solution, and aspirate the supernatant before re-suspending the pellet in 10 milliliters of warm PBS. Make up the volume to 50 milliliters with PBS, and repeat the centrifugation.
Re-suspend the pellet in warm complete KSFM containing Primocin. Next, replace the coating solution from the six-well plate with two milliliters of cell suspension per well. Allow the cells to grow until 80 to 90%confluency is reached, For cryopreservation of PBECs, aspirate the medium from the wells and wash the cells once with two milliliters of warm PBS per well.
Trypsinize the cells by adding 500 microliters of soft trypsin per well, and incubate for five to 10 minutes at 37 degrees Celsius. Swirl the trypsin solution, and release the cells by gently tapping the plate. Transfer the detached cells to a 50 milliliter centrifuge tube containing soybean trypsin inhibitor.
Centrifuge the suspension, and discard the supernatant before re-suspending the pellet in 10 milliliters of KSFM containing penicillin and streptomycin. After counting the cells on an automated cell counter, re-suspend the cells to a concentration of 400, 000 cells per milliliter in a freezing medium, and add one milliliter of this suspension in a cryovial. Transfer the vials to a cool cell container, and place it at minus 80 degrees Celsius.
After 24 hours, transfer the vials to minus 196 degrees Celsius liquid nitrogen for long-term storage. To coat a T75 cell culture flask, add 10 milliliters of coating solution in PBS, and close the lid tightly before placing the flask in an incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, replace the coating solution from the flask with 10 milliliters of complete KSFM.
Allow the medium to warm to 37 degrees Celsius in the incubator with a slightly opened lid to let the incubator air in. Quickly thaw the cryopreserved PBECs in a 37 degree Celsius bead bath. Add the entire content to the cryovial to the pre-warmed T75 flask and distribute the cells evenly.
After ensuring that the cells are firmly attached, replace the medium with 10 milliliters of fresh and warm complete KSFM, and grow them until 80 to 90%confluency is reached. Coat cell culture inserts by adding 400 microliters of coating solution per insert, and incubate overnight at 37 degrees Celsius. Trypsinize the PBECs in the flask by adding two milliliters of soft trypsin, and incubate for five to 10 minutes.
Facilitate cell detachment by swirling and gently tapping the flask. Next, add four milliliters of soybean trypsin inhibitor to the flask, and transfer the cell suspension to a 25 milliliter tube. Centrifuge the suspension and re-suspend the pellet in six milliliters of complete BD medium.
Count the cells on an automated cell counter. After coating, remove the coating solution from the inserts. Dilute the cell suspension with complete BD medium supplemented with one nanomole EC 23 and add 500 microliters onto the top of the membrane of the inserts.
Add 1.5 milliliters of complete BD medium to the well underneath the insert. When the cells are ready for transfer to the Air-Liquid Interface, or ALI, remove the medium from the inserts and the well, and add new complete BD medium supplemented with 50 nanomolar EC 23 only to the well. Change the medium in the wells three times a week.
To remove excess mucus and cellular debris, gently add 200 microliters of warm PBS on the apical side of the cell layer inside the insert, and incubate at 37 degrees Celsius for 10 minutes. After incubation, aspirate the PBS containing excess mucus and cellular debris. TEER was measured to assess the quality of ALI PBEC cultures.
At seven days, the electrical resistance of the cell layer higher than 300 ohms is considered a successful culture. Further, inter-donor variability in the electrical resistance reduced over time between day seven and day 14 of culture at the Air-Liquid Interface, and is markedly influenced by the origin of DMEM used. All the major different cell types, such as basal, ciliated, goblet, and club cells, were observed upon 14 days of ALI culture, and the levels of expression were donor-dependent.
Among the different concentrations of tested EC 23, maximal TEER was observed at 50 nanomolar. A comparison of retinoic acid and its synthetic analog, EC 23, confirms that gene expression of markers for ciliated and goblet cells were similar between 50 nanomolar EC 23 and retinoic acid. Using medium from different suppliers resulted in substantial differences in cellular composition, whereas differences in TEER were less pronounced.
On the other hand, using inserts from different suppliers did not result in substantial differences in cellular decomposition. Moreover, a difference in TEER values and cellular composition of the ALI PBEC and the PneumaCult medium compared to the complete BD medium was also observed. When the cells are ready to transfer to ALI, increase the EC 23 concentrations.
Also, do not centrifuge the cells after thawing, and quickly remove the cells from cell culture plastics after adding SBTI. Using cultures of airway epithelial cells the way provided in this protocol, you can follow that up using a variety of analysis methods to look at, for instance, the function, gene expression, mediator production of the airway epithelial cells, and thus, for instance, get insight into the consequences of exposures to, for instance, cigarette smoke. The next steps for this cell culture protocol is the integration of additional cell types, such as immune cells, or vascular cells, and this will help to increase tissue representation.
