Designations: | hTERT-HPNE |
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Depositors: | M Ouellette |
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Biosafety Level: | 2 [cells containing SV40 viral DNA sequences ] |
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Shipped: | frozen |
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Medium & Serum: | See Propagation |
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Growth Properties: | adherent |
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Organism: | Homo sapiens |
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Morphology: | epithelial-like
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Source: | Organ: pancreas
Tissue: duct |
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Permits/Forms: | In addition to theMTAmentioned above, otherATCC and/or regulatory permitsmay be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Pleaseclick herefor information regarding the specific requirements for shipment to your location. |
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Restrictions: | This material requires that theAddendum for Commercial and For-Profit Organizationsor theAddendum for Noncommercial and Academic Organizationsbe signed and returned to ATCC before shipment. The price listed above is for noncommercial and academic organizations only. Commercial and for-profit organizations should call for pricing. |
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Applications: | s 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.
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 [Pubmed: 15924149].
The infected cells became positive for telomerase, failed to senesce, and were still proliferating after more than 150 doublings [Pubmed: 12589817]. |
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Antigen Expression: | Positive for epithelial marker pan-cytokeratin (immunocytochemistry); positive for nestin (flow cytometry). |
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DNA Profile (STR): | D5S818: 11
D13S317: 12,13
D7S820: 9,10
D16S539: 12,13
vWA: 17
Amelogenin: X,Y
TPOX: 8,11
CSF1PO: 12
TH01: 8,9 |
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Cytogenetic Analysis: | 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. |
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Age: | 52 years |
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Gender: | male |
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Comments: | The hTERT-HPNE (Human Pancreatic Nestin Expressing cells) 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 [Pubmed: 12589817].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 [Pubmed: 15924149].
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. |
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Propagation: | ATCC 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
Temperature: 37.0°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
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Subculturing: | Protocol: Volumes used in this protocol are for 75 sq cm 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 37C 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 10(3) to 6 X 10(3) viable cells/sq. cm is recommended. 7.Incubate cultures at 37C. Subculture when reaching a concentration between 5 X 10(4) and 6 X 10(4) cells/sq. cm.
Subcultivation Ratio: 1:8 - 1:12 twice weekly
Medium Renewal: Every 2 to 3 days |
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Preservation: | Freeze medium: 95% FBS; 5% DMSO |
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Doubling Time: | about 31 hours |
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References: | 16172577: 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
16172578: 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 |
