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VK2/E6E7

CRL-2616

VK2/E6E7 is an epithelial cell line that was isolated in 1996 from the mucosa of a 32-year-old, female patient undergoing a hysterectomy for endometriosis. This cell line was deposited by D Anderson, RN Fichorova, and JG Rheinwald.
 
 
Product category
Human cells
Organism
Homo sapiens, human
Cell type
epithelial cell
Morphology
epithelial
Tissue
Vagina; Mucosa
Disease
Endometriosis
Applications
3D cell culture
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Cells contain Human papillomavirus (HPV) sequences

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

General

Specific 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.

Characteristics

Growth properties
Adherent
Derivation

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

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.

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.

Age
32 years
Gender
Female
Immortalization method
HPV-16 E6/E7 expression
Genes expressed
cytokeratins 8 (CK8), 10 (CK10), 13 (CK13), 18 (CK18), 19 (CK19); macrophage colony-stimulating factor (M-CSF); transforming growth factor beta1; interleukin 8 (IL-8); prostaglandin E2; secretory leukoproteinase inhibitor; polymeric immunoglobulin receptor.
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.

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete 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)
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.

  1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
  2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  3. It is recommended that the cryoprotective agent be removed immediately. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. Centrifuge the cell suspension at approximately 125 x g for 5 to 10 minutes.  Discard the supernatant and resuspend the cell pellet in an appropriate amount of fresh growth medium.
  4. Transfer the cells an appropriate size vessel.  It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
  5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.
Subculturing procedure
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 (DMEM:F-12, ATCC Catalog No. 30-2006), 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: 1:3 to 1:5
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
Reagents for cryopreservation
A 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 10% (v/v) fetal bovine serum and 5% (v/v) DMSO (ATCC 4-X)

Quality control specifications

Mycoplasma contamination
Not detected
Population doubling time
Approximately 24 hrs
STR profiling
Amelogenin: X
CSF1PO: 10,11
D13S317: 9,12
D16S539: 9
D5S818: 9,10
D7S820: 10,11
TH01: 7,9.3
TPOX: 11
vWA: 16
D3S1358: 17
D21S11: 29,31.2
D18S51: 17,19
Penta_E: 7,8
Penta_D: 9,12
D8S1179: 12,13
FGA: 19,25
D19S433: 11,12
D2S1338: 17,24

History

Deposited as
human
Depositors
D Anderson, RN Fichorova, JG Rheinwald
Year of origin
1996
Special collection
NCRR Contract

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

This material was deposited by Dr. Deborah Anderson and is distributed for research purposes only under the ATCC Material Transfer Agreement. The Brigham and Women's Hospital has released the material subject to the following:

  1. Transfers - the material may not be transferred to third parties for purposes of sale, or producing for sale.
  2. Commercial Use - all for-profit and non-profit Recipients must obtain a commercial use license prior to Commercial Use.

Any proposed commercial use of these cells, or their products, must first be negotiated with:

Brigham and Women's Hospital
Office of Corporate Sponsored Research and Licensing
75 Francis Street
Boston, MA 02115
Phone: (617) 525-6010

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

Frequently Asked Questions

References

Curated Citations

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

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. 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.

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