hTERT-HPNE細(xì)胞

生長(zhǎng)狀態(tài)： 貼壁生長(zhǎng)

運(yùn)輸方式： 凍存運(yùn)輸

數(shù)量： 大量

器官來(lái)源： 胰腺

是否是腫瘤細(xì)胞： 0

物種來(lái)源： 人

ATCC Number： CRL-4023?

組織來(lái)源： duct

細(xì)胞形態(tài)： 上皮樣

年限： 52 years

Designations: hTERT-HPNE

Depositors: M Ouellette

hTERT-HPNE細(xì)胞Biosafety Level: 2 [cells containing SV40 viral DNA sequences ]

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Homo sapiens

Morphology: epithelial-like

Source: Organ: pancreas

Tissue: duct

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Restrictions: This material requires that the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations be signed and returned to ATCC before shipment. The price listed above is for noncommercial and academic organizations only. hTERT-HPNE細(xì)胞Commercial and for-profit organizations should call for pricing.

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].

Antigen Expression: Positive for epithelial marker pan-cytokeratin (immunocytochemistry); positive for nestin (flow cytometry).

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

Cytogenetic Analysis: hTERT-HPNE細(xì)胞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.

Age: 52 years

Gender: male

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.

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%

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.

Remove and discard culture medium.

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.

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: hTERT-HPNE細(xì)胞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.

Add 2.0 to 3.0 ml of complete growth medium and aspirate cells by gently pipetting.

Transfer cell suspension to a centrifuge tube and spin at approximately 125 X g for 5 to 10 minutes. Discard supernatant.

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.

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

Preservation: Freeze medium: 95% FBS; 5% DMSO

Doubling Time: about 31 hours

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