hTERT-HPNE E6/E7/st (ATCC® CRL-4037)

Organism: Homo sapiens, human  /  Tissue: pancrease, duct  / 

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Organism Homo sapiens, human
Tissue
pancrease, duct
Product Format frozen
Morphology epithelial-like
Culture Properties adherent
Biosafety Level 2
Age 52 years
Gender male
Storage Conditions liquid nitrogen vapor phase
Karyotype This is a pseudodiploid human cell line of male origin with a der(21)t(17;21)(q21.3;p13). Another subclone, present at earlier passages, may contain the additional derivative chromosome: der(3)t(3;18)(p21.1;q11.2). Overall, the cell line has a relatively stable karyotype.
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Derivation
Cells were derived by infection of hTERT-HPNE E6/E7 cells (ATCC CRL-4036) with retroviral vector (pBabeZeo) carrying the SV40 small t antigen
Clinical Data
male
Antigen Expression
positive for nestin (flow cytometry) (verified at ATCC)
Genes Expressed
positive for nestin (flow cytometry)(verified at ATCC)
Tumorigenic NO
Complete Growth Medium The base medium for this cell line is:
  • 75% DMEM without glucose (Sigma Cat#. D-5030 with additional 2 mM L-glutamine and 1.5 g/L sodium bicarbonate)
  • 25% Medium M3 Base (Incell Corp. Cat# M300F- 500)
To make the complete growth medium, add the following components to the base medium:
  • fetal bovine serum 5% (final conc.)
  • 10 ng/ml human recombinant EGF
  • 5.5 mM D-glucose (1g/L)
  • 750 ng/ml puromycin

Subculturing
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. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which 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. Add 2.0 to 3.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 X g for 5 to 10 minutes. Discard supernatant.
  6. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 4 X 103 to 6 X 103 viable cells/cm2 is recommended.
  7. Incubate cultures at 37°C. Subculture when cell density reaches between 5 X 104 and 6 X 104 cells/cm2.
Subcultivation ratio: 1:8 to 1:12 twice weekly
Medium renewal: every 2 to 3 days
Cryopreservation
Freeze medium: fetal bovine serum (FBS), 90%; DMSO, 10%
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
STR Profile
CSF1PO: 12
D13S317: 12, 13
D16S539: 12, 13
D5S818: 11
D7S820: 9, 10
TH01: 8, 9
TPOX: 8, 11
vWA: 17
Amelogenin: XY
Population Doubling Level (PDL)

Longevity: >15 PDLs post-cryopreservation recovery

Population Doubling Time approximately 29 hours
Name of Depositor M Ouellette
Year of Origin July 2002
References

Lee KM. et al. Notch2-positive progenitors with the intrinsic ability to give rise to pancreatic ductal cells. Lab. Invest. 85 (8): 1003-1012, 2005. Pubmed: 15924149

Lee KM. et al. Immortalization with telomerase of the Nestin-positive cells of the human pancreas. Biochem. Biophys. Res. Commun. 301(4):1038-1044, 2003. Pubmed: 12589817

Campbell PM, et al. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling. Cancer Res. 67(5): 2098-2106, 2007. PubMed: 17332339

Campbell PM, et al. Ras-driven transformation of human nestin-positive pancreatic epithelial cells. Methods Enzymol. 439: 451-465, 2008. PubMed: 18374182

Basic Documentation
Other Documentation
Restrictions

This material is subject to claims under U.S. Patent Nos. 6,261,836 and 6,337,200, other pending patent applications, and foreign counterparts thereof. It is required that either the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations, as appropriate, be signed and returned to ATCC before shipment.

References

Lee KM. et al. Notch2-positive progenitors with the intrinsic ability to give rise to pancreatic ductal cells. Lab. Invest. 85 (8): 1003-1012, 2005. Pubmed: 15924149

Lee KM. et al. Immortalization with telomerase of the Nestin-positive cells of the human pancreas. Biochem. Biophys. Res. Commun. 301(4):1038-1044, 2003. Pubmed: 12589817

Campbell PM, et al. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling. Cancer Res. 67(5): 2098-2106, 2007. PubMed: 17332339

Campbell PM, et al. Ras-driven transformation of human nestin-positive pancreatic epithelial cells. Methods Enzymol. 439: 451-465, 2008. PubMed: 18374182