Ect1/E6E7 (ATCC® CRL-2614)

Organism: Homo sapiens, human  /  Cell Type: epithelial HPV-16 E6/E7 transformed  /  Tissue: ectocervix; cervix  / 

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Organism Homo sapiens, human
Tissue ectocervix; cervix
Cell Type epithelial HPV-16 E6/E7 transformed
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2  [Cells contain human Papilloma viral sequences]
Age 43 years
Gender female
Applications

These cell lines may provide the basis for valid, reproducible in vitro models for studies on cervicovaginal physiology and infections and for testing pharmacological agents for intravaginal application.


Storage Conditions liquid nitrogen vapor phase
Derivation

The ectocervical Ect1/E6E7 (ATCC CRL-2614) and endocervical End1/E6E7 (ATCC CRL-2615) cell lines were established in 1996 from normal epithelial tissue taken from a premenopausal woman undergoing hysterectomy for endometriosis.

The VK2/E6E7 (ATCC CRL-2616 cell line was established in 1996 from the normal vaginal mucosal tissue taken from a premenopausal woman undergoing anterior-posterior vaginal repair surgery.


Clinical Data
female
43 years
Genes Expressed
cytokeratins 8 (CK8), 10 (CK10), 13 (CK13), 18 (CK18) and 19 (CK19),macrophage colony-stimulating factor (M-CSF); transforming growth factor beta1; interleukin 8 (IL-8); prostaglandin E2; the secretory leukoproteinase inhibitor; polymeric immunoglobulin receptor,Stimulation with interferon gamma and tumor necrosis factor alpha (TNF alpha) induces or significantly up-regulates expression of several of the cytokines and chemokines as well as major histocompatibility complex (MHC) class II antigens
The Cyt c-/- cell line provides a unique resource of mammalian cells lacking cytochrome c.
Comments

The endocervical cell line expresses characteristics of simple columnar epithelium, whereas the ectocervical and vaginal cell lines express characteristics of stratified squamous nonkeratinizing epithelia.

Without stimulation, all three cell lines produce macrophage colony-stimulating factor (M-CSF), transforming growth factor beta1, interleukin 8 (IL-8), prostaglandin E2, the secretory leukoproteinase inhibitor, and the polymeric immunoglobulin receptor.

The endocervical cell line (End1/E6E7), but not the others, also produce the lymphopoietic cytokines IL-6, IL-7, and consistently detectable levels of the chemokine known as "regulated-upon-activation, normal T cell expressed and secreted" (RANTES).

Stimulation with interferon gamma and tumor necrosis factor alpha (TNF alpha) induces or significantly up-regulates expression of several of the cytokines and chemokines as well as major histocompatibility complex (MHC) class II antigens in the lines.

Piliated, but not nonpiliated, Neisseria gonorrhoea strain F62 variants actively invade these epithelial cell lines. Invasion of these cells by green fluorescent protein-expressing gonococci is characterized by colocalization of gonococci with F actin.

Complete Growth Medium Keratinocyte-Serum Free medium (GIBCO-BRL 17005-042) with 0.1 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract, and additional calcium chloride 44.1 mg/L (final concentration 0.4 mM)
Subculturing Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: The cells should not be allowed to become confluent, subculture at 60 to 90% of confluence.

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.03% (w/v) EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. 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: 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 37°C to facilitate dispersal.
  4. Neutralize the trypsin by adding 6.0 to 8.0 mL of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium containing 10% fetal bovine serum. Aspirate cells by gently pipetting. 
  5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
  6. Discard supernatant and resuspend cells in fresh serum-free growth medium.  Add appropriate aliquots of cell suspension to new culture vessels.
  7. Place culture vessels in incubators at 37°C. Cells will not attach well for 24 hours after subculture.

Subcultivation Ratio: 1:4 to 1:6
Medium Renewal: Every 2 to 3 days.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994

Cryopreservation
Freeze medium: A 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium, 85%; fetal bovine serum, 10%; DMSO, 5%
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Population Doubling Time 24 hrs
Name of Depositor D Anderson, RN Fichorova, JG Rheinwald
Passage History
Cells at passage 3 were immortalized by transduction with the retroviral vector LXSN-16E6E7 in the presence of polybrene. Clones were selected in medium containing G418.
Year of Origin 1996
References

Fichorova RN, et al. Generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins. Biol. Reprod. 57: 847-855, 1999. PubMed: 9314589

Fichorova RN, Anderson DJ. Differential expression of immunobiological mediators by immortalized human cervical and vaginal epithelial cells. Biol. Reprod. 60: 508-514, 1999. PubMed: 9916021

Fichorova RN, et al. Distinct proinflammatory host responses to Neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells. Infect. Immun. 69: 5840-5880, 2001. PubMed: 11500462

Fichorova RN, et al. The molecular basis of nonoxynol-9-induced vaginal inflammation and its possible relevance to human immunodeficiency virus type 1 transmission. J. Infect. Dis. 184: 418-428, 2001. PubMed: 11471099

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.
Basic Documentation
Restrictions

The line is available with the following restrictions: 1. This cell line was deposited at the ATCC by Dr. Deborah Anderson of The Brigham and Women's Hospital, Inc. and is provided for academic research purposes only. Neither the cell line nor products derived from it may be sold or used for commercial purposes, including screening of compounds. Nor can the cells be distributed to third parties for purposes of sale, or producing for sale, cells or their products. The cells are provided as a service to the research community. They are provided without warranty of merchantability or fitness for a particular purpose or any other warranty, express or implied. 2. All products derived from the cells or genetically altered forms of the cells cannot be commercialized without express written permission from the The Brigham and Women's Hospital, Inc. 3. Any proposed commercial manufacture, use, or sale of the these cells, or their products must first be approved and the terms for such commercial manufacture, use, or sale must first be negotiated with TheBrigham and Women's Hospital, Inc., Office of Corporate Sponsored Research and Licensing, 75 Francis Street, Boston, MA 02115 (617) 525-6010.

References

Fichorova RN, et al. Generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins. Biol. Reprod. 57: 847-855, 1999. PubMed: 9314589

Fichorova RN, Anderson DJ. Differential expression of immunobiological mediators by immortalized human cervical and vaginal epithelial cells. Biol. Reprod. 60: 508-514, 1999. PubMed: 9916021

Fichorova RN, et al. Distinct proinflammatory host responses to Neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells. Infect. Immun. 69: 5840-5880, 2001. PubMed: 11500462

Fichorova RN, et al. The molecular basis of nonoxynol-9-induced vaginal inflammation and its possible relevance to human immunodeficiency virus type 1 transmission. J. Infect. Dis. 184: 418-428, 2001. PubMed: 11471099