Complete Growth Medium
The base medium for this cell line (BEBM) along with all the additives can be obtained from Lonza/Clonetics Corporation as a kit: BEGM, Kit Catalog No. CC-3170. ATCC does not use the GA-1000 (gentamycin-amphotericin B mix) provided with the BEGM kit.
Note: Do not filter complete medium.
These cells should be subcultured before reaching confluence since confluent cultures rapidly undergo squamous terminal differentiation. Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Add 2.0 to 3.0 mL of 0.25% Trypsin - 0.53mM EDTA solution containing 0.5% polyvinylpyrrolidone (PVP) to flask and observe cells under an inverted microscope until cell layer is dispersed (usually with 5 to 10 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes
- Discard supernatant and resuspend cells in fresh growth medium. Inoculate new flasks at 1500 to 3000 cells per cm2. The culture flasks used should be pre-coated with a mixture of 0.01mg/ml fibronectin, 0.03 mg/ml bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in BEBM.
- Place culture flasks in incubators at 37°C.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.
Interval: Subcultured before reaching confluence.
Medium Renewal: Every 2 to 3 days
- Prepare a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin (BSA) dissolved in culture medium. Store pre-prepared Coating Solution at 4°C in cold room for up to 3 months.
- For a growth area of 75 cm2, add 4.5 mL of the fibronectin/collagen/BSA solution and rock gently to coat the entire surface.
- Incubate the freshly coated vessel(s) in a 37°C incubator overnight (it is preferable to use tissue culture vessels with tightened, plug-seal caps to prevent evaporation during the coating process).
- Store coated flasks with solution at room temperature, light protected, up to 1 month. Suction off solution before plating cells.
Freeze medium: Complete growth medium supplemented with 1% PVP and 7.5% DMSO
Storage temperature: liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Growth Conditions: The culture flasks used should be pre-coated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in BEBM medium (see references: U.S. Pat. 4,885,238 and Lechner, J.F. and LaVeck, M.A. A serum-free method for culturing normal bronchial epithelial cells at clonal density. J. Tissue Culture Methods 9: 4348, 1985)