Primary culture of rat vaginal epithelial cells

Female SD rats were euthanatized by CO₂ asphyxia and the freshly excised vaginal tissues were washed and cut into small pieces under antiseptic conditions. The finely minced tissues were subsequently digested enzymatically twice (1 h for each time) with type I collagenase (0.5 mg/mL, C0130, Sigma-Aldrich, USA) dissolved in DMEM/F12 (Gibco, USA) at 37°C, with gentle agitation. The digested tissues were further dissociated and dispersed by repeatedly pipetting and centrifuged at 500 g for 30 s. Afterward, the isolated cell clusters were resuspended and washed twice with DMEM/F12 (Gibco, USA) medium containing 10% FBS (Gibco, USA). The fragments were collected and placed in the six-well plate, then humidified in keratinocyte serum-free medium (K-SFM, Gibco, USA) supplemented with bovine pituitary extract (50 μg/mL, Gibco, USA), recombinant epidermal growth factor (5 ng/mL, Gibco, USA), 100× insulin-transferrin-selenium solution (1×, Gibco, USA), cholera toxin (Sigma Aldrich, USA) and penicillin/streptomycin (100 U/mL/100 μg/mL, Hyclone, USA), in an atmosphere of 5% CO₂ at 37°C. After 24 h, the fragments were washed and cells were cultured with the supplemented K-SFM medium. On day 5, the cells were used for immunofluorescence staining or incubated with live T. vaginalis (1×10⁶) for Western blot assay.