hTERT-HPNE

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.

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.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

• 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)

• fetal bovine serum 5% (final conc.)
• 10 ng/ml human recombinant EGF
• 5.5 mM D-glucose (1g/L)
• 750 ng/ml puromycin

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor phase and NOT at –70°C. Storage at –70°C will result in loss of viability.

1. Prepare a 25 cm² or a 75 cm² culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point on should be carried out under strict aseptic conditions.
4. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
5. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
6. Incubate the culture at 37°C in a suitable incubator.
7. A 5% CO₂ / 95% air atmosphere is recommended if using the medium described on this product sheet.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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