2.040 pRSV-T (ATCC® CRL-11516)

Organism: Homo sapiens, human  /  Cell Type: epithelial; transfected with a plasmid containing the SV40 early region  /  Tissue: eye, cornea  / 

Organism Homo sapiens, human
Tissue eye, cornea
Cell Type epithelial; transfected with a plasmid containing the SV40 early region
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2  [Cells contain SV-40 and Rous Sarcoma viral sequences]
Storage Conditions liquid nitrogen vapor phase
Comments A primary culture of normal corneal epithelium was immortalized by transfection with plasmid pRSV-T using lipofectamine overnight at 37C. pRSV-T contains the SV40 early region genes and the Rous Sarcoma virus long terminal repeat.
Complete Growth Medium Keratinocyte-Serum Free medium (GIBCO-BRL 17005-042) with 5 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract, 0.005 mg/ml insulin and 500 ng/ml hydrocortisone.
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.

Note: The flasks used should be precoated 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. Subculture when 80% confluent.

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.05% Trypsin – EDTA (GIBCO cat# 25300-054).
  3. 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 with 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.
  4. Add  fresh medium and aspirate cells by gently pipetting. 
  5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to10 minutes.
  6. Discard supernatant and resuspend cells in fresh growth medium.  Add appropriate aliquots of cell suspension to new coated culture vessels. 
  7. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: 1:3
Medium Renewal: Every 2 to 3 days.

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.  

Cryopreservation Complete growth medium, 92.5%; DMSO, 7.5%. Cell culture tested DMSO is available as ATCC Catalog No. 4-X.
Culture Conditions
Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO2), 5%
Name of Depositor T Walker, Gillette Medical Evaluation Laboratories
References

Walker TL, Kahn CR. Human corneal epithelial cell lines with extended lifespan. US Patent 5,672,498 dated Sep 30 1997

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.

Basic Documentation
References

Walker TL, Kahn CR. Human corneal epithelial cell lines with extended lifespan. US Patent 5,672,498 dated Sep 30 1997

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.