Culture and Differentiation of Primary Human Tracheobronchial Epithelial Cells Using STEMCELL Technologies Pneumacult Media

NOTE: A PDF methods document is attached in the supplementary materials. SCOPE OF APPLICATION (LIMITATIONS) This method describes the culture and differentiation of primary human tracheobronchial epithelial cells (pHBEC). Cells used for this method can be obtained by brush biopsy during clinical bronchoscopy or purchased commercially. This method replaces the previous version described in (Dailey and McCullough, 2021a; b). Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.


Introduction
Reagents Supplies · Primary bronchial epithelial cells obtained by brush biopsy or commercially.
NOTE: Companies will supply vessels already plated with cells, or cryopreserved cells. Follow supplier's instructions on how to handle plated cells, which will then go into an incubator. Upon receiving frozen cells, make sure contents are still frozen. Store cells immediately in liquid nitrogen.
Prepare Pneumacult Ex-Plus Growth Medium 1. Thaw the 50X supplement at room temperature. Mix by gently inverting the bottle.
2. Add the contents of the 50X supplement to a bottle of Ex-Plus Basal medium.
3. Add 0.5 mL of hydrocortisone stock solution to the above supplement-containing medium.
4. Sterile lter with a 0.22 mm vacuum ltration bottle and store at 4 °C for up to one month.
Prepare Pneumacult ALI Differentiation Medium 1. Prepare complete base medium by adding 1 bottle of 10X Supplement (thawed overnight at 4 °C) to a bottle of ALI base medium.
2. Prepare maintenance medium as follows: starting with 100 mL of complete base medium, add 1 vial of 100X Maintenance supplement (1 mL), 0.2 mL of heparin solution, and 0.5 mL of hydrocortisone stock solution. Sterile lter with a 0.22 mm vacuum ltration bottle and store at 4 °C for up to 2 weeks.
3. Prepare the required volume of ALI maintenance medium by adjusting the above volumes accordingly.
NOTE: Prepare aliquots of ALI media to avoid repeated warming and cooling.
Thawing Cryopreserved Cells 1. Warm Pneumacult Ex-Plus growth medium to 37 °C and prepare materials prior to obtaining a vial of cells from cryostorage.
a. It is important that cells are thawed quickly to reduce the duration of exposure to the high concentration of DMSO in the freezing medium, which will negatively impact cell viability.
3. Add 24 mL of growth medium to a 50 mL conical tube then transfer the thawed cells from the cryovial into the growth medium using a P1000 pipette and gently mix by inverting 10 times. NOTE: do not vortex 4. Pellet cells via centrifugation at 1,000 x g for 4 minutes at room temperature in centrifuge with a swinging bucket rotor. 5. Carefully aspirate the supernatant.
6. Gently resuspend the cells in pre-warmed growth medium and transfer to a ask. Distribute cell suspension on the dish through a combination of gentle rocking and swirling of the dish for approximately 15 seconds. Some investigators prefer to thaw cells into a collagen coated vessel, although this is not necessary. All subsequent passages use non-coated vessels.
NOTE: If a vial containing 1.0x10 6 cells is thawed, then the cells should be plated in a 75 cm 2 ask. 7. Place in a tissue culture incubator overnight.
8. Check cells the following day for attachment to the dish. Aspirate and replace medium.

Passaging and Expansion of pHBEC
It is recommended to passage the cells when they are >50-70% con uent.
1. Pre-warm Pneumacult ALI growth medium and DPBS in a 37 °C water bath and a trypsin aliquot at room temperature for approximately 30 minutes.
2. Aspirate growth medium from asks to be passaged. Cells may be rinsed once with DPBS but this is not required.
3. Add trypsin-EDTA solution and place in a tissue culture incubator for 2-3 minutes.
a. 500 mL/well of 12 well plate. b. 4.0 mL/T75 ask. 4. When approximately 80-90% of the cells are detached (which can be facilitated by tapping sides and bottom of the ask), add a half volume of SBTI to the ask, pipet up the trypsin/SBTI mixture, and rinse the vessel bottom. This aids mixing of the trypsin and SBTI, and helps remove remaining adherent cells.

Transfer the cells to a sterile centrifuge tube.
NOTE: Cells from the same donor source plated over individual wells or asks can be combined.
6. Pellet cells at 200-600 x g for 5 minutes at room temperature in a centrifuge with a swinging bucket rotor.
7. Aspirate the supernatant. Note the size of the cell pellet before tapping the tube to break up the pellet. Ideally, the pellet should be resuspended in a volume of medium that will yield a cell density of 1.0x10 6 cells/mL. Triturate the cells with a serological pipet to create a homogeneous suspension. NOTE: With experience, you will be able to judge this volume based on the pellet size. For a barely visible pellet, start with 1 mL medium; for a pellet of 1 cm, start with 10ml medium (generally, resuspension volumes will range from 5-10 mL).
8. Transfer a 10 mL aliquot of the cell suspension to a hemocytometer and count the cells. Viability can be determined, if desired, by diluting a sample of the cells with an equal volume of trypan blue.
9. Adjust the number of cells to approximately 1.0x10 6 /mL. If the cell density is less than 1.0x10 6 /mL, re-pellet the cells and suspend in the appropriate volume. If the cells density is greater than 1.0x10 6 /mL, dilute the cell suspension with the appropriate volume of medium.
10. Plating density will be based on how quickly you want the cells to be ready for subsequent passage/plating. Cells are typically seeded at 0.5 x 10 4 cells/cm 2 but can be seeded more heavily if desired.
11. Change the medium every other day until cells reach approximately 70% con uence. Denser cultures may require more frequent medium replacement.
12. Upon approaching con uence cells can be: Cells should be used at 90-100% con uence for experiments.
Differentiation of pHBEC Cultures at ALI 1. Trypsinize and collect cells from expansion asks as described in steps #1-9 of the "Passaging and Expansion of pHBEC" section above.
2. Starting with a cell density of 1.0x10 6 cells/mL, prepare a volume and dilution of cell suspension according to the size and number of Transwell inserts needed based on the seeding density and volume table below.
NOTE: Prepare a volume of diluted cell suspension that is greater than the exact amount needed for the desired plating to account for volume loss and/or slight variations in pipetting.
Example: If seeding 12 24mm inserts at 3.0x10 5 cells per insert then prepare su cient cell suspension for seeding 14 inserts to allow for volume lost during handling.
1. Trypsinize and collect cells from expansion asks as described in steps #1-8 of the "Passaging and Expansion of pHBEC" section above.
2. Pellet cells again at 200-600 x g for 4 minutes at room temperature in centrifuge with a swinging bucket rotor.
3. Resuspend the pellet in CryStor10 Freezing Medium at a minimum of 1.0x10 6 cells/mL. 4. Gently mix by pipetting. Transfer 1.0 mL of the cell suspension to each cryovial. 5. Place cryovials in a Mr. Frosty freezing container ( lled to indicated line with isopropyl alcohol) and place in a -80 °C freezer overnight.
a. NOTE: Isopropanol should be changed after every three freezing cycles. b. NOTE: Do not leave vials at -80 °C longer than overnight as it will impact viability after thawing.
Transfer the frozen vials in liquid nitrogen storage.