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

Organism: Homo sapiens, human  /  Tissue: Respiratory tract  / 

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Organism Homo sapiens, human
Tissue Respiratory tract
Morphology epithelial, packed cuboidal morphology
Growth Properties adherent
Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Disease normal
Age lot-specific
Gender lot-specific
Ethnicity lot-specific
Applications Normal Human Primary Lobar Bronchial Epithelial Cells provide an ideal model for the studies of toxicity, cystic fibrosis, asthma, pathogenesis, pharmacology, or airway wound healing.
Product Format frozen 1.0 mL
Storage Conditions liquid nitrogen vapor phase
Images PCS-300-015 Phase panCK
Comments A complete solution to propagate lobar epithelial cells in serum-free conditions.
Characterization: Pan-Cytokeratin (+), TE-7 (-)
Complete Growth Medium

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.
  6. 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

  7. 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.
  8. 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.
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Subculturing
  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.
Volume 1.0 mL
Cells per Vial ≥ 5.0 x 105
Sterility Tests Bacteria and yeast: No growth
Mycoplasma: No growth
Viral Testing Hepatitis B: None detected
Hepatitis C: None detected
Human immunodeficiency virus 1: None detected
Human immunodeficiency virus 2: None detected
Population Doubling Time ≥10 PDLs in complete growth media
C of A
Certificate of Analysis
Certificate of Analysis
Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
References

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