TIME (ATCC® CRL-4025)

Organism: Homo sapiens, human  /  Cell Type: Endothelial cells immortalized with hTERT  /  Tissue: Foreskin; Dermal microvascular endothelium  /  Disease: Normal

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Organism Homo sapiens, human
Tissue Foreskin; Dermal microvascular endothelium
Cell Type Endothelial cells immortalized with hTERT
Product Format frozen
Morphology Endothelial-like
Culture Properties Adherent
Biosafety Level 2 [Cells contain EMCV viral DNA sequences]
Disease Normal
Age Neonatal
Gender Male
Applications

The cells represent an effective cell model for studying endothelial cell biology including signal transduction and angiogenesis.

Storage Conditions Liquid nitrogen vapor phase
Karyotype 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.
Images
Derivation

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. 

Antigen Expression Positive for integrin alpha v beta 3 RefVenetsanakos 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 and CD31 (flow cytometry) RefVenetsanakos 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
Receptor Expression The cells express the low density lipoprotein (LDL) receptor and are capable of acetylated LDL uptake. RefVenetsanakos 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
Comments

The immortalized cells do not undergo growth arrest in culture due to the exogenous hTERT expression.

When plated on Matrigel, TIME cells undergo tubule formation exhibiting capillary-like structures.

Complete Growth Medium The base medium for this cell line is Vascular Cell Basal Medium (ATCC PCS-100-030), supplemented with Microvascular Endothelial Cell Growth Kit-VEGF (ATCC PCS-110-041). To make the complete growth medium, add the following components to a 500 mL bottle of Vascular Cell Basal Medium (ATCC PCS-100-030): Microvascular Endothelial Cell Growth Kit-VEGF (ATCC PCS-110-041) and 12.5 μg/mL blasticidine. Note: Do not filter complete medium
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.
Note: Subculture when cultures are about 80% confluent.

  1. 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°C, 5% CO2, humidified incubator for at least 30 minutes. If not using vented caps, loosen caps of flasks.
  2. Remove and discard spent medium.
  3. Briefly rinse the cells with Dulbecco's Phosphate Buffered Saline (D-PBS, ATCC 30-2200) and discard rinse solution.
  4. Add 2.0 to 3.0 mL room temperature Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) to the flask. Incubate at 37°C for 5 min (until cells have detached).
  5. Neutralize trypsin by adding an equal volume of room temperature 2% FBS in D-PBS.
  6. Transfer cells to a centrifuge tube. Rinse the flask with an additional room temperature 2% FBS in D-PBS and pool into centrifuge tube with cells.
  7. Centrifuge cells at 250 x g for 10 min at room temperature.
  8. Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
  9. Count cells, and seed 5 x 103 to 8 x 103 viable cells/cm2 to new culture vessels. Subculture when cells become 80 to 90% confluent, which normally yield approximately 3.0 x 104 viable cells/cm2.
  10. Incubate cultures at 37°C in a 5% CO2 humidified incubator.
Subcultivation ratio: A subcultivation ratio of 1:4 to 1:6 is recommended.
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.

Cryopreservation
Fetal bovine serum, 90% (v/v); DMSO, 10% (v/v). Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Culture Conditions
Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO2), 5%
STR Profile
D5S818: 11
D13S317: 9, 11
D7S820: 8, 9
D16S539: 9, 12
vWA: 16, 18
THO1: 6, 7
TPOX: 8
CSF1PO: 11, 12
Amelogenin: XY
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 McMahon
Year of Origin June 2001
References

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

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

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

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

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
  • License agreement required for commercial customer uses.
  • This material is distributed for research purposes only. A signed addendum to the ATCC Material Transfer Agreement must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
Restrictions

This material is subject to claims under U.S. Patent Nos. 6,261,836 and 6,337,200, other pending patent applications, and foreign counterparts thereof. It is required that either the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations, as appropriate, be signed and returned to ATCC before shipment.

In addition to the foregoing, any proposed commercial use of ATCC® CRL-4025™ must also be negotiated with University of California, San Francisco, Office of Technology Management, 3333 California Street, Suite S-11, San Francisco, CA 94143-1209, Tel: 415.502.1585, Fax: 415.502.1661.

References

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

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

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

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