title: Establishment of a novel method for primary culture of normal human cervical keratinocytes.
abstract: BACKGROUND
  Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS). However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro.
  
  
  METHODS
  Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity.
  
  
  RESULTS
  Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%.
  
  
  CONCLUSIONS
  A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.
authors.affiliation: Department of Obstetrics and Gynecology, Qilu Hospital, Shandong Univeristy, Jinan, Shandong 250012, China.
authors.forename: Yu-Zhen
authors.initials: YZ
authors.lastname: Liu
authors.forename: Xiu-Ping
authors.initials: XP
authors.lastname: Lü
authors.forename: Zi-Xuan
authors.initials: ZX
authors.lastname: Pan
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Zhao-Ri
authors.initials: ZR
authors.lastname: Chen
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Liu
authors.forename: You-Zhong
authors.initials: YZ
authors.lastname: Zhang
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002584:Cervix Uteri / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology; D005260:Female; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*