Chinese Journal of Tissue Engineering Research ›› 2015, Vol. 19 ›› Issue (1): 96-100.doi: 10.3969/j.issn.2095-4344.2015.01.017

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Long-term stable culture of canine vaginal epithelial cells and smooth muscle cells in vitro

Shen Fu-jin, Luo Ruo-yu, Liang Hua, Jiang Yan-ping, Cao Lai-ying   

  1. First Department of Gynecology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
  • Revised:2014-11-19 Online:2015-01-01 Published:2015-01-01
  • About author:Shen Fu-jin, M.D., Associate chief physician, First Department of Gynecology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
  • Supported by:

    the Youth Project of National Natural Science Foundation of China, No. 81100395

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Abstract:

BACKGROUND: In vitro culture of sufficient vaginal epithelial cells and smooth muscle cells is the key for vaginal tissue engineering. However, the culture, purification and passage of vaginal epithelial cells in vitro are difficult. Primary culture and passage of vaginal epithelial cells from large animals such as canines has not been reported.
OBJECTIVE: To establish a stable method of culturing canine vaginal epithelial cells and smooth muscle cells.
METHODS: Vaginal epithelial cells 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 collagenase type II; the collected smooth muscle cells were cultured in DMEM culture medium containing 10% fetal bovine serum. The cultured cells were passaged regularly. Cell morphology and proliferation characteristics were observed and cell phenotypes were confirmed by morphology and immunohistochemistry staining.
RESULTS AND CONCLUSION: Primary vaginal epithelial cells began to adhere after 24-36 hours, grew logarithmically after 4-5 days, and reached 70% confluence after 7-8 days; the epithelial cells showed a typical cobblestone, with no fibroblasts. Cultured epithelial cells 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 cells adhered and grew after 24 hours. The smooth muscle cells were spindle-shaped and proliferated logarithmically. After 4 days, primary cultured smooth muscle cells were confluent and showed a typical shape of “peaks and valleys”, and then the cells 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 cells and smooth muscle cells could have a long-term stable culture and proliferation, to provide adequate seed cells for vaginal tissue engineering.


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


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Key words: Vagina, Epithelial Cells, Myocytes, Smooth Muscle

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