Primary Lobar Epithelial Cells (ATCC^® PCS-300-015^™)

One bottle of Airway Epithelial Cell Basal Medium (ATCC PCS-300-030) plus Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040)

1. Obtain one Bronchial/Tracheal Epithelial Cell Growth Kit from the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. Warm the L-glutamine component in a 37°C water bath and shake to dissolve any precipitates prior to adding to the basal medium.
3. Obtain one bottle of Airway Epithelial Cell Basal Medium (485 mL) from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the indicated volume of each growth kit component, as indicated in Table 1, to the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. Brochial/Tracheal Epithelial Cell Growth Kit Components 

Component	Volume	Final Concentration
HLL Supplement	1.25 mL	HSA 500 mg/mL Linoleic Acid 0.6 mM Lecithin 0.6 mg/mL
L-Glutamine	15 mL	6 mM
Extract P	2.0 mL	0.4%
Airway Epithelial Cell Supplement	5.0 mL	Epinephrine 1.0 mM Transferrin 5 mg/mL T3 10 nM Hydrocortisone 5 mg/mL rh EGF 5 ng/mL rh Insulin 5 mg/mL

 

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of either of the optional components to be added to the complete growth media is summarized in Table 2.

Table 2. Addition of Antimicrobials/Antimycotics and Phenol Red (Optional)

Component	Volume	Final Concentration
Penicillin-Streptomycin-Amphotericin B Solution	0.5 mL	Penicillin: 10 Units/mL Streptomycin: 10 µg/mL Amphotericin B: 25 µg/mL
Phenol Red	0.5 mL	33 µM

6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for 30 days.

1. Passage normal lobar epithelial cells when culture has reached approximately 80% to 90% confluence, and are actively proliferating.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC® PCS-999-003) and the Trypsin Neutralizing Solution (ATCC® PCS-999-004) to room temperature prior to dissociation. Warm complete growth medium to 37°C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Briefly rinse the cell layer with 3 to 5 Ml/25 cm2 DPBS (ATCC® 30-2200) to remove residual traces of serum and then aspirate and discard the DPBS.
5. Add pre-warmed trypsin-EDTA solution (3-4 mL for every 25 cm2) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells.
7. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 3 to 5 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.
8. When the majority of cells are detached, quickly add an equal volume of Trypsin Neutralizing Solution (ATCC® PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the flask.
10. Add 3 to 4 mL Trypsin Neutralizing Solution to the flask to collect any remaining dissociated cells. Transfer remaining cells into the centrifuge tube.
11. Repeat steps 10 as needed until all cells have been collected from the flask.
12. Centrifuge the cells at 150 x g for 3 to 5 minutes.
13. Carefully aspirate the neutralized dissociation solution from the cell pellet and re-suspend the cells in 5 to 8 mL fresh, pre-warmed, complete growth medium.
14. Count the cells and seed new flasks at a density of 5,000 to 8,000 cells per cm2.
15. Place freshly seeded flasks in a 37°C, 5% CO₂ incubator for at least 24 to 48 hours or when cells are 80-90% confluent before subculturing further. Refer to Maintenance for guidelines on feeding.

Eisenhauer P, et al. Endogenous distal airway progenitor cells, lung mechanics, and disproportionate lobar growth following long-term postpneumonectomy in mice. Stem Cells 31(7):1330-9, 2013. PubMed: 23533195

Roberts JK, et al. Regulation of CYP3A genes by glucocorticoids in human lung cells. F1000Res 2:173, 2013. doi: 10.12688/f1000research.2-173.v2 PubMed: 24555085