Screening and culture of human vaginal mucosa stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Nowadays stem cells have been isolated successfully from the epithelial tissue of human and rodents such as skin, hair follicle, cornea, oral mucous, intestinal mucosa dental epithelial cells, and salivary glands by the Chinese or foreign researchers. But according to the vaginal mucosa stem cells (VMSCs), it is not known in the field of isolation and application.OBJECTIVE: To find out the suitable method of isolation, identification, and culture of human VMSCs.DESIGN: Observational basic study. SETTING: Department of Gynaecology and Obstetrics, the Third Affiliated Hospital of Guangzhou Medical College. MATERIALS: This study was performed in the Zhongshan Ophthalmology Center of Zhongshan Medical College from October 2004 to August 2005. Two-week gestation mice were provided by the Experimental Animal Center of Guangzhou University of Chinese Medicine. The sterile human vaginal mucosa was collected from five patients (25-38 years old) after the plastic operation of vagina from the 3rd Affiliated Hospital of Guangzhou Medical College. METHODS: Vaginal mucosa cells could be harvested from human vagina mucosa treated with trypsin-collagenase digesting. The VMSCs were selected by adhesion to type Ⅳ collagen for 20 minutes. The cell cycles of the VMSCs and primary human vaginal mucosa cells were analyzed by flow cytometry. VMSCs were cultured in vitro with 4 different media, including feeder layer cells plus epidermal total culture media, feeder layer cells plus DMEM/F12 (3:1), epidermal total culture media and DMEM/F12 (3:1). At the 12th day, their colony forming efficiency (CFE), CK19 and CK10 positive rates were compared. Then VMSCs were continuously cultured and passaged till they were apoptosis.MAIN OUTCOME MEASURES: ① Positive rate of CK19 and CK10 and G0/G1 proportion; ② Positive rate of CK19 and CK10, the colony forming efficiency (CFE) and the times be passaged when primary VMSCs were cultured in different media after 12 days. RESULTS: The cells at the G0-G1 phase cell cycle, CK19 and CK10 positive rates between the VMSCs and primary human vaginal mucosa cells were statistical different (P < 0.05). When cultured at 12th day, the VMSCs cultured with feeder layer and epidermal total culture media had the highest CFE and CK19 positive rate and lowest CK10 positive rate. They could be passaged over 15 times in vitro. CONCLUSION: It is an effective way that select VMSCs by adhesion to type Ⅳ collagen for 20 minutes. In this research, culturing VMSCs in vitro with feeder layer and epidermal total culture media was the best way to keep VMSC characteristics.