This protocol highlights the key steps for amplifying, differentiating, and cryopreserving primary nasal epithelial cells. In our culture conditions, primary nasal epithelial cells mimic the lung epithelium. This allows patient derived cultures and personalized medicine studies from fresh or biobanked cells.
The patient-derived nasal epithelial cultures can be used to evaluate CFTR activity and help with cystic fibrosis therapy by evaluating the patient's individual response to new therapies. Demonstrating the procedure will be Aurelie Hatton, an engineer from my laboratory. To begin, grow nasal epithelial or HNE cells, in 10 milliliters of amplification medium.
Incubate at 37 degrees Celsius and 5%carbon dioxide in a 75 square centimeter collagen coated flask until it reaches 80 to 90%confluency. Change the medium every 48 to 72 hours. Later, wash the cells with 10 milliliters of magnesium and calcium free DPBS.
Aspirate and discard the saline. Before adding two milliliters of 0.25%trypsin and returning the flask to the incubator for eight to 12 minutes. Later, tap the flask firmly with a palm to help cell detachment.
Add 10 milliliters of the amplification medium to stop the enzymatic reaction. Vigorously rinse the flask using a 10 milliliter pipette to draw up and expel the amplification medium over the flask surface to rinse and detach cells and collect the cells in a 15 milliliter tube. Centrifuge the cell suspension at 500 times G for five minutes at four degrees Celsius and discard the supernatant.
Re-suspend the pellet in five to 10 milliliters of amplification medium. Then count the cells on a hemocytometer. After cell counting, centrifuge the cells at 500 times G for five minutes at four degrees Celsius and discard the supernatant.
Then re-suspend the pellet in the enriched freezing medium to obtain three to five times 10 to the six cells per milliliter and place in a cryo vial. Slowly freeze the cells by reducing the temperature by approximately one degree Celsius per minute in an appropriate cryofreezing container at minus 80 degree Celsius. The next day, move the preserved sample to a nitrogen storage container for longterm storage.
Warm the freshly prepared amplification medium in a water bath set to 37 degrees Celsius. Remove cryo vials from the nitrogen storage tank and rapidly place them in the water bath, taking care not to submerge the whole vial in water. Remove the cryo vials from the water bath when only a small frozen droplet remains.
Wipe the vials with 70%alcohol and place them under the hood. Using a one milliliter pipette, transfer the thawed cells to a 15 milliliter tube. Then add one milliliter of warm amplification medium in a drop-wise manner.
After one minute, add another one milliliter of amplification medium and wait for a minute. Add 10 milliliters of amplification medium and centrifuge at 500 times G for two minutes at four degrees Celsius. Aspirate and discard the supernatant.
Re-suspend the pellet in a volume of amplification medium required to achieve a cell density of one times 10 to the six cells per milliliter. After seeding the cells onto a 25 square centimeter collagen coated flask, containing amplification medium, incubate the cells at 37 degrees Celsius and 5%carbon dioxide. Visually observe cell expansion over two to three days before performing cell amplification as demonstrated earlier.
Seed the cells at a density of 3.3 times 10 to the fifth cells per filter on 0.33 square centimeter collagen coated porous filters, supplemented with 300 microliters of amplification medium at the apical side and 900 microliters of air liquid medium at the basolateral side. After three days, aspirate the apical medium and culture the cells at the air-liquid interface for three to four weeks in the air-liquid medium to establish a differentiated epithelium. Change the basal medium every 48 to 72 hours.
Fresh HNE cells cultured at the air-liquid interface displayed a typical features of the polarized and differentiated respiratory epithelium. In the 78 HNE cell samples, success rates were compared in nasal brushing seeded immediately and after one to seven days of transport in a flushing medium. The initial mean percentage of successful cultures was 82%and reached 87%in samples seeded between zero to five days.
83%of the samples that differentiated successfully in fresh conditions also were successful in cryopreserved samples. Similarly, in samples that failed to differentiate in fresh conditions, 71%were also unsuccessful in freeze-thaw conditions. Moreover, 50%of samples frozen between two to three times 10 to the six cells per cryo vial failed to grow when thawed, reaching 80%for below two times 10 to the six cells.
For short circuit current change in response to forskolin and short circuit current change in response to CFTR inhibitor 172 in DMSO treated HNE cells, no significant difference was observed between fresh or frozen thawed HNE. Upon treatment, both cell types displayed significant correction with an increase in short circuit current change in response to forskolin and a decrease in short circuit current change in response to CFTR inhibitor 172. the corrective response of CFTR function dual therapies were assessed as compared to DMSO treated HNE cells short circuit current change in response to forskolin and in response to CFTR inhibitor 172 were significantly improved by both VX-809 plus VX-770, and VX-661 plus VX-770.
Three different CFTR modulator therapies in HNE from six F508del homozygous patients were compared. When used in combination with a CFTR corrector and potentiator, both vertex and a CFTR modulators, corrected short circuit current change in response to forskolin and short circuit current change in response to CFTR inhibitor 172 to a similar extent. When freezing HNE cells, it is important to have at least 3 million cells per cryo vial to increase the chances of a good outcome upon thawing.
CFTR activity in HNE cells has been shown to be a good predictive model to evaluate and adjust personalized therapies in cystic fibrosis.
