Culture of Primary Human Tracheobronchial Epithelial Cells


 This protocol is intended for culture of primary human tracheobronchial epithelial cells (pHBEC) obtained by brush biopsy during clinical bronchoscopy, or purchased commercially, using Lonza-based medium.Note: This is a historical protocol. At the time of publication, this protocol has been superseded by a different version in the McCullough lab; however, it is being published to support the transparency and reproducibility of other studies by which it is referenced.Disclaimer: The information presented here has been reviewed and approved for publication by the US Environmental Protection Agency do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

Portable pipet-men, single and multi-channel pipettors, repeat pipettors and appropriate tips Equipment Laminar ow hood (any manufacturer) Biosafety Level II equipped with vacuum connection.
Tissue culture incubator capable of maintaining 37 °C and 5% CO 2 (any manufacturer) Olympus CK2 (or comparable) inverted microscope with 4x, 10x and 20x objectives Light microscope for cell counting (any brand) Hemocytometer for cell counts Procedure Thawing Cells DAY 1: 1. If not already prepared, make BEGM by thawing growth supplements and adding to the basal medium and sterile lter (0.22 um pore).
2. Generally, one vial of cryopreserved cells is thawed into one T75 ask.
3. Add 18 mL of growth medium to the ask and pre-equilibrate in the incubator for approximately 30 minutes.
4. To thaw cells, place the vial(s) in a 37 °C water bath. Swirl frequently to check if the contents have thawed. It usually takes less than three minutes. Upon thawing, spray the vial with 70% ethanol to reduce surface contaminants.
5. Using aseptic technique, pipet the vial contents up and down to resuspend the cells. Add cells to the pre-equilibrated T75 ask.
6. Alternatively, instead of adding thawed cells directly to the ask, you can add the cells to a tube of pre-equilibrated medium, centrifuge cells at 1000 RPM for ve minutes, resuspend the pellet in fresh medium then add to the ask. This removes the DMSO in which the cells were cryopreserved. Place asks in a CO 2 incubator (5% CO 2 , 37 o C).
OPTIONAL : Check cell viability by adding trypan blue to some cells on a microscope slide. About 50% or more of the cells should exclude the dye. a. This will remove the dead cells and any left-over DMSO if cells were added directly to the ask without a prior rinse.
3. Place asks back into the incubator. DAY 4 and LATER: 1. Aspirate media and replace with 18 mL of BEGM.
2. Replace media every two days.
2. Add 4 mL of trypsin-EDTA solution (room temperature) to each T75 ask. Incubate at 37 o C for 2-3 minutes.
3. 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 ask bottom.
a. This aids mixing of the trypsin and SBTI, and helps remove some of the still adherent cells.
4. Transfer the cells to a sterile centrifuge tube. Cells from individual asks can be combined. 5. Centrifuge cells at 1000 RPM for ve minutes at room temperature.
6. Aspirate off the supernatant. Agitate the pellet by tapping to break up the pellet. Add 10 mL BEGM, and vigorously and quickly pipet the cells to create a homogeneous suspension.
7. Transfer a 10 μL aliquot to a hemocytometer and count the cells. A con uent T75 ask typically has 2-4 million cells.
8. Add 5 x 10 5 cells/T75 ask. Final volume of BEGM should be 18ml/ ask. i. OPTIONAL: Additionally, some cells can be set aside for cryopreservation at this point. (See below). a. Culture cells as above, changing the media every two days until cells reach con uence.
b. When asks reach con uence: 1) cryopreserve the cells, 2) transfer to Transwells or other plastic ware for studies, and/or 3) further passage cells.
c. After cells have been removed by trypsin, the trypsin neutralized with SBTI, and cells are counted, adjust cell density to the desired one by either adding more media and/or centrifuging and re-suspending in less media. Usually we adjust the density to 10 6 cells/ml.

For Transwells:
Determine the number of inserts to be seeded and adjust cell density and volume as outlined in the table below: Suggested Cell Seeding Densities/Volumes are given in Table 1 Example: seed twelve 24 mm inserts at 300,000 cells per insert. Include a fudge factor so you do not run out of cells. In this case, calculate as if you were seeding 14 inserts. Fourteen x .75 mL = 10.5 mL nal volume. Fourteen x 300,000 cells = 4.2 million cells.
If your cells were resuspended at a concentration of 1 million/ml, you would add 4.2 mL of the cell suspension to 6.3 mL of additional medium to get the desired plating density. (300,000 cells/0.75 mL added to each insert).
Let cells adhere for 16-24 hours. After the adhesion period, aspirate off nonadherent cells, and then add fresh medium apically. Once the cells reach con uence, apical medium is removed and cells are fed basolaterally every other 48 hours, until used.
For Plasticware: Lonza suggests seeding cells at 1 x 10 5 /cm 2 in 1 mL BEGM/5cm 2 growth area. Cells should be used at 90-100% con uence for experiments. As cells near con uence, cells may need to be fed daily as they quickly deplete the nutrients in the medium.