Long-term stable culture of canine vaginal epithelial cells and smooth muscle cells in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND:In vitro culture of sufficient vaginal epithelial cels and smooth muscle cels is the key for vaginal tissue engineering. However, the culture, purification and passage of vaginal epithelial celsin vitro are difficult. Primary culture and passage of vaginal epithelial cels from large animals such as canines has not been reported. OBJECTIVE:To establish a stable method of culturing canine vaginal epithelial cels and smooth muscle cels. METHODS: Vaginal epithelial cels were isolated from the vaginal specimens by enzymatic digestion with Dispase and trypsin separately, and cultured in keratinocyte serum-free medium. Vaginal smooth muscle tissue were minced and digested with colagenase type II; the colected smooth muscle cels were cultured in DMEM culture medium containing 10% fetal bovine serum. The cultured cels were passaged regularly. Cel morphology and proliferation characteristics were observed and cel phenotypes were confirmed by morphology and immunohistochemistry staining. RESULTS AND CONCLUSION: Primary vaginal epithelial cels began to adhere after 24-36 hours, grew logarithmicaly after 4-5 days, and reached 70% confluence after 7-8 days; the epithelial cels showed a typical cobblestone, with no fibroblasts. Cultured epithelial cels passaged every 4-5 days and subcultured to 6-7 generations continuously. Immunohistochemical staining confirmed a positive staining for anti-pancytokeratin (AEl/AE3). Primary cultured smooth muscle cels adhered and grew after 24 hours. The smooth muscle cels were spindle-shaped and proliferated logarithmicaly. After 4 days, primary cultured smooth muscle cels were confluent and showed a typical shape of “peaks and valeys”, and then the cels could be passaged every 3-4 days and passaged 7-8 generations. Immunohistochemistry staining showed α-actin staining was positive. These findings indicate that canine vaginal epithelial cels and smooth muscle cels could have a long-term stable culture and proliferation, to provide adequate seed cels for vaginal tissue engineering.