| Applications |
NL20 (ATCC CRL-2503) is an immortalized, nontumorigenic human bronchial epithelial cell line derived from normal bronchus taken from an accident victim at autopsy. The cell line was established by transfection with the origin of replication-defective SV40 large T plasmid, p129. The non-tumorigenic NL20 cell line and the tumorigenic NL20-TA cell line form a pair of immortal cell lines that can be used to study tumor progression. |
| Derivation |
NL20 (ATCC CRL-2503) is an immortalized, nontumorigenic human bronchial epithelial cell line derived from normal bronchus taken from an accident victim at autopsy.
The cell line was established by transfection with the origin of replication-defective SV40 large T plasmid, p129.
NL20 cells at passage 183 were inoculated into nude mice and a small slowly growing subcutaneous tumor developed from a minor clone in this otherwise stable cell line.
After three months the tumor was removed and placed in culture. At passage 3, these cells were re-injected into nude mice.
One of the resulting tumors was dissociated, placed in culture and designated NL20-TA. This cell line (ATCC CRL-2504) remains tumorigenic up to at least passage 250. |
| Comments |
NL20 (ATCC CRL-2503) is an immortalized, nontumorigenic human bronchial epithelial cell line derived from normal bronchus taken from an accident victim at autopsy. The cell line was established by transfection with the origin of replication-defective SV40 large T plasmid, p129. NL20 cells at passage 183 were inoculated into nude mice and a small slowly growing subcutaneous tumor developed from a minor clone in this otherwise stable cell line. After three months the tumor was removed and placed in culture. At passage 3, these cells were re-injected into nude mice. One of the resulting tumors was dissociated, placed in culture and designated NL20-TA. This cell line (ATCC CRL-2504) remains tumorigenic up to at least passage 250. Neoplastic transformation of the NL20 cell line was associated with loss of chromosome 18 together with acquisition of multiple copies of 9q21.2-->34. The non-tumorigenic NL20 cell line and the tumorigenic NL20-TA cell line form a pair of immortal cell lines that can be used to study tumor progression. |
| Subculturing |
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 10 to 15 mL dissociation solution (0.02% EDTA and 5% dialyzed fetal bovine serum in Ca-Mg free Hanks' BSS) and allow the flask to sit at 37°C for 12 minutes or until the cells detach.
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.
- Add appropriate aliquots of the cell supension to new culture vessels
- Place culture vessels in incubators at 37°C.
Subcultivation ratio: 1:50 to 1:500 is recommended. Subculture weekly.
Medium Renewal: Every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005. |
| References |
Schiller JH, et al. Phenotypic, molecular and genetic characterization of transformed human bronchial epithelial cell strains. Int. J. Oncol. 4: 461-470, 1994.
Schiller JH, Bittner G. Loss of the tumorigenic phenotype with in vitro, but not in vivo, passaging of a novel series of human bronchial epithelial cell lines: possible role of an alpha 5/beta 1-integrin-fibronectin interaction. Cancer Res. : 6215-6221, 1995. PubMed: 8521416
Schiller JH, et al. Karyotypic changes associated with spontaneous acquisition and loss of tumorigenicity in a human transformed bronchial epithelial cell line: evidence for in vivo selection of transformed clones. In Vitro Cell. Dev. Biol. Anim. 34: 283-289, 1998. PubMed: 9590501
Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo, J. L., 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.
|
