the culturcs of lymphoblastoid and leukemic cells of mice, respectively. We also thank Mrs. Margaret M.Rice for assistance in preparing the manuscript.

Primary human vulvovaginal fibroblast cell lines are useful for studying biological mechanisms underlying genital pain, pelvic organ prolapse, and the spread of sexually transmitted infections. However, the vaginal biopsies necessary for establishing these cell lines are invasive and relatively difficult to obtain. Primary mouse fibroblast cell lines derived from pre-clinical animal models of these conditions can be used for better controlled experiments that can help us dissect disease mechanisms. To our knowledge, there are no published protocols for establishing primary murine vaginal fibroblast cell lines to date. Here, we describe a protocol for the establishment of murine vaginal fibroblast cell lines via enzymatic digestion of vaginal canal tissue. Cell lines generated using this method can be used for in vitro studies of these important structural cells in a variety of pre-clinical mouse models; such studies are required to identify and characterize relevant regulatory and therapeutic targets in a wide array of diseases of interest. As shown in our representative data, this protocol yields viable cell lines from ND4 Swiss outbred mice. These cells bear surface markers characteristic of fibroblasts and are capable of producing inflammatory cytokines in response to treatment with bacterial and yeast antigens in vitro .

Chronic, unexplained vulvar pain or vulvodynia affects 8% of women of reproductive age, and is more common with histories of yeast infections and allergies. We have established mouse models of localized, provoked genital pain driven by repeated labiar and vaginal exposures to allergens such as oxazolone, dinitrofluorobenzene and the common chemical preservative methylisothiazolinone (MI). Pain sensitivity is concomitant with tissue mast cell increases and reduced when mast cells are reduced by chemical degranulation or topical phytocannabinoid treatment. Vulvar fibroblasts from patients show altered inflammatory responses in vitro.to repeated topical application of the allergenic cosmetic preservative methylisothiazolinone (MI) to the murine vaginal canal produces long-lasting contact hypersensitivity. Here, we have optimized methods to grow skin and vaginal canal fibroblasts from outbred Swiss mice that are responsive to lipopolysaccharide and zymosan treatment in vitro. We also show that in vitro MI treatment reduces the viability of derived fibroblasts in a dose-dependent manner, with a more robust effect on cell lines derived from MI-treated skin, indicating that previous exposure to MI in the skin primes fibroblasts to undergo cell death upon future exposures. Our data suggest that fibroblasts may become more sensitive to allergens as a result of previous exposures, which could contribute to the development of chronic allergic and pain sensitivity after repeated low dose chemical exposures.