In Vitro Technologies

Permits and Restrictions	View Permits
Organism	Homo sapiens, human
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.
Age	Batch-specific
Gender	Batch-specific
Ethnicity	Batch-specific
Applications	Microbial (e.g., viral, bacterial) infection and pathogenesis; airway inflammation and wound healing; asthma; pulmonary fibrosis, chronic obstructive pulmonary disease; emphysema; toxicology/other testing of pharmaceuticals.
Product Format	frozen 1 mL
Storage Conditions	-130°C or below

Comments	Human Primary Small Airway Epithelial Cells are cryopreserved at passage 2 (cells have been isolated, plated, and expanded in culture vessels twice prior to cryopreservation) to ensure the highest viability and proliferation efficiency. These cells, when transiently transfected using TransfeX Transfection Reagent (ATCC^® ACS-4005™), express high levels of GFP.

Comments

Human Primary Small Airway Epithelial Cells are cryopreserved at passage 2 (cells have been isolated, plated, and expanded in culture vessels twice prior to cryopreservation) to ensure the highest viability and proliferation efficiency. These cells, when transiently transfected using TransfeX Transfection Reagent (ATCC^® ACS-4005™), express high levels of GFP.

Complete Growth Medium

Obtain one Bronchial Epithelial Growth Kit (ATCC^®PCS-300-040) from the freezer; make sure that the caps of all components are tight.
Thaw the components of the growth kit just prior to adding them to the basal medium. If the growth kit contains L-glutamine, warm the L-glutamine component in a 37°C water bath and shake to dissolve any precipitates prior to adding to the basal medium.
Obtain one bottle of Airway Cell Basal Medium (485 mL) from cold storage.
Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
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 or 2, to the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. When using the Bronchial Epithelial Cell Growth Kit (ATCC^® PCS-300-040), add the indicated volume for each component:

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 each optional component to be added to the complete media is summarized in Table 2.

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

Component	Volume	Final Concentration
Gentamicin-Amphotericin B Solution	0.5 mL	Gentamicin: 10 µg/mL Amphotericin B: 0.25 µg/mL
Penicillin-Streptomycin-Amphotericin B Solution	0.5 mL	Penicillin: 10 Units/mL Streptomycin: 10 µg/mL Amphotericin B: 25 ng/mL
Phenol Red	0.5 mL	33 µM

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.
Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete media is stable for 30 days.

Subculturing

1. Passage normal small airway cells when the culture has reached approximately 70% to 80% confluence. 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 the 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. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium. 5. Add pre-warmed trypsin-EDTA solution (1 to 2 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, and then aspirate the excess fluid off of the monolayer. 7. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 1 to 3 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 appear to have detached, quickly add an equal volume of the 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 culture flask. 10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that might have been left behind. 11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-dissociated cells. 12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask. 13. Centrifuge the cells at 150 x g for 3 to 5 minutes. 14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium. 15. Count the cells and seed new culture flasks at a density of 5,000 viable cells per cm². 16. Place newly seeded flasks in a 37°C, 5% CO₂ incubator for at least 24 to 48 hours before processing the cells further. Refer to Maintenance>/i> for guidelines on feeding.

Volume	1 mL
Cells per Vial	One vial contains a minimum of 5 x 10⁵ viable cells.
Sterility Tests	Bacteria and Yeast: NegativeMycoplasma: Negative
Viral Testing	Hepatitis B: Negative Hepatitis C: Negative HIV: Negative
Viability	≥ 70% when thawed from cryopreservation.
Population Doubling Time	≥ 15 in complete growth medium
C of A	Certificate of Analysis

Permits	These permits may be required for shipping this product to Australia: DAFF Import Permit formerly known as AQIS Import Permit must be obtained and a copy of the permit must be sent to ATCC in advance of shipment.
Basic Documentation	Product Sheet Certificate of Analysis SDS

Permits

These permits may be required for shipping this product to Australia:

DAFF Import Permit formerly known as AQIS Import Permit must be obtained and a copy of the permit must be sent to ATCC in advance of shipment.

Basic Documentation

Product Sheet Certificate of Analysis SDS

E: care@invitro.com.au
P: 1300 552 003

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Primary Small Airway Epithelial Cells; Normal, Human Item ID: ATCPCS301010

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