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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 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.

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.

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.

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