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hTERT-HPNE; Pancreatic Duct; Human (Homo sapiens) Item ID: ATCCRL4023

Permits and Restrictions

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Organism Homo sapiens, human
Tissue Pancreas, duct
Cell Type intermediary cells formed during acinar-to-ductal metaplasia
Product Format frozen
Morphology Epithelial-like
Culture Properties Adherent
Biosafety Level 2 [cells containing SV40 viral DNA sequences]

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Disease Normal
Age 52 years
Gender Male
Storage Conditions Liquid nitrogen vapor phase
Karyotype This is a pseudodiploid human cell line of male origin with a modal chromosome number of 46 and a low polyploidy rate. Approximately 50% of the cells contained a consistent derivative chromosome 21 with additional material at p12.
Derivation

The hTERT-HPNE cell line was developed from human pancreatic duct by transduction with a retroviral expression vector (pBABEpuro) containing the hTERT gene. The infected cells became positive for telomerase, failed to senesce, and were still proliferating after more than 150 doublings RefLee 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.
Comments

Exposing hTERT-HPNE cells to sodium butyrate and 5-aza-2'-deoxycytidine lead to the formation of pancreatic ductal cells marked by the expression of p-glycoprotein (multidrug resistance (MDR-1)), carbonic anhydrase II, and the cytokeratins 7, 8, and 19.

hTERT-HPNE cells were found to have properties of the intermediary cells formed during acinar-to-ductal metaplasia, which included their undifferentiated phenotype, expression of Nestin and, evidence of active Notch signaling RefLee KM. et al. Notch 2-positive progenitors with the intrinsic ability to give rise to pancreatic ductal cells. Lab. Investigation 85 (8): 1003-1012, 2005. Pubmed: 15924149.
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 10 3 viable cells/mL is recommended.
  7. Incubate cultures at 37°C. Subculture when reaching a concentration between 5 x 104 and 6 x 104 cells/cm2.
Subcultivation Ratio: 1:8 - 1:12 twice 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.
Cryopreservation Freeze Medium: 95% FBS; 5% DMSO
Culture Conditions Temperature: 37°C Atmosphere: Air, 95%; Carbon dioxide (CO2), 5%
STR Profile D5S818: 11 D13S317: 12,13 D7S820: 9,10 D16S539: 12,13 vWA: 17 Amelogenin: X,Y TPOX: 8,11 CSF1PO: 12 TH01: 8,9
Population Doubling Level (PDL) As part of our quality control, we have tested this cell line for its ability to grow for a minimum of 15 population doublings after recovery from cryopreservation. We have also compared its karyotype, telomerase expression level, growth rate, morphology and tissue-specific markers when first recovered from cryopreservation with that of cells at 10+ population doublings to ensure that there is no change in these parameters and that the cells are capable of extended proliferation.
Name of Depositor M Ouellette
References

Lee KM. et al. Notch 2-positive progenitors with the intrinsic ability to give rise to pancreatic ductal cells. Lab. Investigation 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):1038-44 (2003). Pubmed: 12589817

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

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

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.

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