TIME細(xì)胞

數(shù)量： 大量

細(xì)胞形態(tài)： 其他

生長(zhǎng)狀態(tài)： 貼壁生長(zhǎng)

ATCC Number： CRL-4025?

年限： neonatal

運(yùn)輸方式： 凍存運(yùn)輸

器官來(lái)源： 其他

組織來(lái)源： dermal microvascular endothelium

細(xì)胞類型： 內(nèi)皮細(xì)胞

是否是腫瘤細(xì)胞： 0

物種來(lái)源： 人

Designations: TIME

Depositors: M McMahon

TIME細(xì)胞Biosafety Level: 2 [cells containing EMCV viral DNA sequences ]

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Homo sapiens

Morphology: endothelial-like

Source: Organ: foreskin

Tissue: dermal microvascular endothelium

Cell Type: endothelial

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Isolation: Isolation date: June 2001

Applications: TIME細(xì)胞When plated on Matrigel, TIME cells undergo tubule formation exhibiting capillary-like structures.

The telomerase-immortalized human microvascular endothelium cell line, TIME, was derived from a primary culture of neonatal foreskin microvascular endothelial cells (HMVEC) of the dermis.

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.

Receptors: acetylated low density lipoprotein (LDL) [16172671]

Antigen Expression: Integrin alpha v beta 3 [16172671]

CD31 [16172671]

DNA Profile (STR): D5S818: 11

D13S317: 9, 11

D7S820: 8, 9

D16S539: 9, 12

vWA: 16, 18

THO1: 6, 7

TPOX: 8

CSF1PO: 11, 12

Amelogenin: XY

Cytogenetic Analysis: TIME細(xì)胞This is a diploid cell line of male origin with a modal chromosome number of 46 and a low rate of polyploidy. The line shows some karyotypic instability at later passages.

Age: neonatal

Gender: male

Comments: The telomerase-immortalized human microvascular endothelium cell line, TIME, was derived from a primary culture of neonatal foreskin microvascular endothelial cells (HMVEC) of the dermis. The primary HMVECs were immortalized by infection with the retrovirus WZLblast3:hTERT and cultured in complete growth medium containing blasticidin . The immortalized cells do not undergo growth arrest in culture due to the exogenous hTERT expression.

TIME cells express a panel of characteristic endothelial cell surface marker proteins including CD31/PECAM-1 and integrin alpha v beta 3. The cells also express the low density lipoprotein (LDL) receptor and are capable of acetylated LDL uptake. When plated on Matrigel, TIME cells undergo tubule formation exhibiting capillary-like structures. The cells represent an effective cell model for studying endothelial cell biology including signal transduction and angiogenesis. [16172671]

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 Endothelial Cell Basal Medium-2 (EBM-2), which is supplied as part of the Microvascular Endothelial Cell Growth Medium-2 Bullet Kit (EGM-2-MV Bullet Kit) available from Lonza/Clonetics Inc., Catalog No. CC-3202. To make the complete growth medium, add the following components to 500 ml of the base medium:

additives that are supplied with the kit (ATCC does not use gentamycin-amphotericin B)

blasticidine to a final concentration of 12.5 ug/ml

Note: TIME細(xì)胞Do not filter complete medium

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.

Subculture when cultures are about 80% confluent

Prior to subculturing, determine the number of flasks needed. Add the appropriate volume of medium to each flask and allow the flasks to equilibrate in a 37.0°C, 5% CO2, humidified incubator for at least 30 minutes. If not using vented caps, loosen caps of flasks.

Remove and discard culture medium.

Briefly rinse the cell layer with room temperature HEPES Buffered Saline Solution (Clonetics/Lonza CC-5024).

Add 5.0 to 6.0 ml of room temperature Trypsin/EDTA Solution (Clonetics/Lonza CC-5012) to the flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 4 to 8 minutes). Do not over-trypsinize; shake remaining cell loose.

To inactivate the Trypsin/EDTA Solution, add 5.0 to 6.0 ml of room temperature Trypsin Neutralizing Solution (Clonetics/Lonza CC-5002) and aspirate cells by gently pipetting.

Transfer cell suspension to a centrifuge tube and spin at approximately 125 xg for 5 to 10 minutes.

Discard supernatant and resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to culture vessels prepared in step 1. TIME細(xì)胞An inoculum of 5 X 10(3) to 7 X 10(3) viable cells/sq. cm is recommended.

Incubate cultures at 37C. Subculture when cultures reach a cell concentration between 2 X 10(4) and 3 X 10(4) cells/sq. cm.

Subcultivation ratio: A subcultivation ratio of 1:2 to 1:3 is recommended.

Medium renewal: Every 2 to 3 days

Preservation: Freeze medium: fetal bovine serum, 90% (v/v); DMSO, 10% (v/v)

Storage temperature: liquid nittrogen vapor phase

Doubling Time: approximately 34 hours

Related Products: Recommended serum: ATCC 30-2020

Cell culture tested DMSO: ATCC 4-X

Erythrosin B vital stain solution: ATCC 30-2404

References: 44175: Yi X, et al. Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels. Mol. Cell. Biol. 19(6): 3989-3997, 1999. PubMed: 10330139

47354: Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

16172671: Venetsanakos E, et al. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells. Exp. Cell Res. 273(1):21-33, 2002. PubMed 11795943

16172672: Lagunoff M, et al. De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells. J. Virol. 76(5):2440-2448, 2002. PubMed: 11836422