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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.

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.

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.

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.

The base medium for this cell line is provided by Invitrogen (GIBCO) as part of a kit: Keratinocyte Serum Free Medium (K-SFM), Kit Catalog Number 17005-042. This kit is supplied with two additives required to grow this cell line (bovine pituitary extract (BPE) and human recombinant epidermal growth factor (EGF; do not filter EGF through a 0.22 µm filter).

To make the complete growth medium, you will need to add the following components to the base medium:

• 0.05 mg/mL BPE - provided with the K-SFM kit
• 0.1 ng/mL EGF - provided with the K-SFM kit; do not filter
• additional calcium chloride 44.1 mg/L (final concentration 0.4 mM) - not provided

NOTE:  Do not filter complete medium.

This medium is formulated for use with a 5% CO₂ in air atmosphere.

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.

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