|| BEAS-2B cells were derived from normal bronchial epithelium obtained from autopsy of non-cancerous individuals. Cells were infected with a replication-defective SV40/adenovirus 12 hybrid and cloned. Squamous differentiation can be observed in response to serum. This ability can be used for screening chemical and biological agents inducing or affecting differentiation and/or carcinogenesis. The cell line has been applied for studies of pneumococcal infection mechanisms. BEAS-2B was described to express keratins and SV40 T antigen. Subculturing the cells before confluency is necessary as confluent cultures rapidly undergo squamous terminal differentiation. This material is cited in a U.S and / or other Patent (U.S. Pat. 4,885,238) and may not be used to infringe the patent claims.
||This cell line is a suitable transfection host.
The cells retain the ability to undergo squamous differentiation in response to serum, and can be used to screen chemical and biological agents for ability to induce or affect differentiation and/or carcinogenesis.
|| 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. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
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.
||Atmosphere: air, 95%; carbon dioxide (CO2), 5%
||95% FBS + 5% DMSO (Dimethyl sulfoxide)
|Thawing Frozen Cells:
||95% FBS + 5% DMSO (Dimethyl sulfoxide)
||Reddel RR, et al. Immortalized human bronchial epitherial mesothelial cell lines. US Patent 4,885,238 dated Dec 5 1989
Lechner JF, LaVeck MA. A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J. Tissue Culture Methods 9: 43-48, 1985.
Sakamoto O, et al. Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility. J. Clin. Invest. 99: 701-709, 1997. PubMed: 9045873
Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988
Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.
||Marize Campos Valadares