90.74 (ATCC® CRL-11654)

Organism: Homo sapiens, human  /  Cell Type: transformed with adenovirus 5 DNA  / 

Organism Homo sapiens, human
Cell Type transformed with adenovirus 5 DNA
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2 Cells contain Human adenovirus sequences
Age fetus
Applications
packaging cell line
Storage Conditions liquid nitrogev vapor phase
Derivation
This line is an amphotropic retrovirus packaging cell line derived from the human embryonic kidney line, 293 (ATCC CRL-1573). 293 cells were co-transfected with the pIK6.1MCVampac plasmid (ATCC 75483) encoding the gag and pol genes from ectropic MMLV and the envelope gene from the 4070A amphotropic MLV and MC1 neo [U.S. Pat. 5,858,740]. The line does not depend on continued G418 selection to maintain the packaging genome over time.
Comments
This line is an amphotropic retrovirus packaging cell line derived from the human embryonic kidney line, 293 (ATCC CRL-1573). 293 cells were co-transfected with the pIK6.1MCVampac plasmid (ATCC 75483) encoding the gag and pol genes from ectropic MMLV and the envelope gene from the 4070A amphotropic MLV and MC1 neo [U.S. Pat. 5,858,740]. The line does not depend on continued G418 selection to maintain the packaging genome over time.
Complete Growth Medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: bovine calf serum to a final concentration of 10%.
Subculturing
Protocol:
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  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 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.
  4. NOTE: Coat vessels with 0.1% porcine gelatin for 30 minutes to enhance attachment. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
  6. Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:3 is recommendedA subcultivation ratio of 1:4 to 1:6 is recommended
Medium Renewal: Twice weekly
Cryopreservation
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogev vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37.0°C
Growth Conditions: An inoculum of 2 X 10(3) to 6 X 10(3) viable cells/cm2 is recommended. Maintain cultures at a cell concentration between 2 X 10(3) and 2.5 X 10(5) cells/cm2.
Population Doubling Time 22 hours
Name of Depositor Cell Genesys
References

Finer MH, et al. Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells. US Patent 5,858,740 dated Jan 12 1999

Finer MH, et al. Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells. US Patent 6,506,604 dated Jan 14 2003

Basic Documentation
References

Finer MH, et al. Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells. US Patent 5,858,740 dated Jan 12 1999

Finer MH, et al. Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells. US Patent 6,506,604 dated Jan 14 2003