T HESCs (ATCC® CRL-4003)

Organism: Homo sapiens, human  /  Cell Type: Fibroblast immortalized with hTERT  /  Tissue: Uterus; Endometrium  /  Disease: Non-malignant myomas

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Organism Homo sapiens, human
Tissue Uterus; Endometrium
Cell Type Fibroblast immortalized with hTERT
Product Format frozen
Morphology Fibroblast-like
Culture Properties Adherent
Biosafety Level 2 [Cells contain CMV viral DNA sequences]
Disease Non-malignant myomas
Age Adult
Gender Female
Storage Conditions Liquid nitrogen vapor phase
Karyotype This is a diploid cell line of female origin.
Overall, the karyology is stable with a modal chromosome number of 46 in 57% of the examined cells and a low rate of polyploidy.
No consistent structural chromosomal aberrations were found in any of the cells examined.
Derivation

This endometrium cell line was derived from the stromal cells obtained from an adult female with myomas. 

The primary stromal endometrium cells were immortalized by infection with supernatant from the packaging cell line pA317-hTERT (Geron Corp.; Menlo Park, CA) which expressed the hTERT and the puromycin resistance genes.

Antigen Expression The cell line expresses vimentin (Homo sapiens) a fibroblast marker, as determined by flow cytometry, and fibroblast surface protein (FSP, Homo sapiens) as determined by immunocytochemistry.
Complete Growth Medium A 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium with 3.1 g/L glucose and 1mM sodium pyruvate and without phenol red (Sigma Cat# D 2906) supplemented with 1.5 g/L sodium bicarbonate, 1% ITS+ Premix (BD Cat# 354352), 500ng/mL puromycin, 90%; charcoal/dextran treated fetal bovine serum (HyClone Cat# SH30068.03), 10%
Subculturing

Volumes are given for T-75 flasks; proportionally reduce or increase the amount of dissociation medium for culture vessels of other sizes.

  1. Remove and discard culture medium.
  2. Add 2.0-3.0 mL of 0.25% Trypsin-0.53mM EDTA solution to the flask and observe cells under an inverted microscope until the 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 to detatch may be placed at 37°C to facilitate dispersal.
  3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  4. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
  5. Discard supernatant and resuspend cells in fresh growth medium.
  6. Add appropriate aliquots of cell suspension to new culture vessels. An inoculum of 3 X 103 to 4 X 103 viable cells/cm2 is recommended. Subculture when cell concentration reaches between 2 x 104 and 3 x 104 cells/cm2.
  7. Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 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 Aminal Cells: A Manual of Basic Techniques by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.
Cryopreservation
Complete growth medium 95%; DMSO 5%. 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
Amelogenin: X
CSF1PO: 10,12
D13S317: 12,13
D16S539: 12,14
D5S818: 8
D7S820: 10
THO1: 7,8
TPOX: 9,11
vWA: 16,18
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 GG Mor, G Krikun, CL Lockwood
References

Krikum G, et al. A novel immortalized human endometrial stromal cell line with normal progestational response. Endocrinology 145: 2291-2296, 2004. PubMed: 14726435

Krikun G, et al. Endometrial angiopoietin expression and modulation by thrombin and steroid hormones: a mechanism for abnormal angiogenesis following long-term progestin-only contraception.. Am. J. Pathol. 164: 2101-2107, 2004. PubMed: 15161644

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

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
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.

References

Krikum G, et al. A novel immortalized human endometrial stromal cell line with normal progestational response. Endocrinology 145: 2291-2296, 2004. PubMed: 14726435

Krikun G, et al. Endometrial angiopoietin expression and modulation by thrombin and steroid hormones: a mechanism for abnormal angiogenesis following long-term progestin-only contraception.. Am. J. Pathol. 164: 2101-2107, 2004. PubMed: 15161644

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.