Antigen expression: This cell line is positive for epithelial marker pan-cytokeratin as assessed by immunocytochemistry.

 
Note: The culture flasks should be pre-coated with 60µg/ml solution of Human Placental Collagen Type IV. (Sigma, Cat. No. C-7521) at least 18 hours in advance then air-dried and rinsed 2-3 times with Dulbecco's Phosphate Buffered Saline.

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Remove and discard culture medium.
2. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 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.
3. To remove Trypsin-EDTA solution, add 2.0 to 3.0 mL of 1% FBS in Dulbecco's Phosphate buffered Saline and aspirate cells by gently pipetting.
4. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
5. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 1 x 10⁴ to 2 x 10⁴ viable cells/cm² is recommended.
6. Incubate cultures at 37°C.
7. Subculture when cell concentration is between 4 x 10⁴ and 5 x 10⁴ cells/cm².

Zabner J, et al. Development of cystic fibrosis and noncystic fibrosis airway cell lines . Am. J. Physiol. Lung Cell Mol Physiol. 284: L844-L854, 2003. PubMed: 12676769

Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

Halbert CL, et al. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65: 473-478, 1991. PubMed: 1845902

Kiyono T, et al. Both Rb/p161NK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396: 84-88, 1998. PubMed: 9817205

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.