title: A modified method for the culture of naturally HPV-infected high-grade cervical intraepithelial neoplasia keratinocytes from human neoplastic cervical biopsies.
abstract: Few studies on cervical intraepithelial neoplasia (CIN) keratinocyte cultures are available due to the numerous technical and methodological problems associated with thein vitrocultivation of these cells. The present study investigated an applicable and effective method for thein vitrocultivation of high-grade CIN keratinocytes from human neoplastic cervical biopsies. Human neoplastic cervical tissue sections were obtained and digested using type I collagen in order to dissociate the cells. The cells were seeded in tissue culture plastic plates that were coated with rat tail collagen type I and contained modified keratinocyte serum-free medium (K-SFM) supplemented with 5% fetal bovine serum. The medium was replaced with K-SFM on days 3, 5 and 7, respectively. The unattached cells were recovered and the cell viability was determined accurately using the Trypan Blue exclusion method. The expression of keratin 14 (K14), keratin 19 (K19), keratin 17 (K17) and P63 was assayed using immunofluorescence in order to identify the presence of CIN keratinocytes. The present results indicated that the attachment rate of CIN keratinocytes significantly increased between 56.75±1.76% on day 3 and 77.09±3.55% on day 5, and became relatively stable between days 5 and 7. The cell viability significantly decreased between 83.00±0.50% on day 5 and 68.17±1.04% on day 7. The passaged CIN keratinocytes maintained the original unequally sized, abnormally shaped morphology and did not undergo differentiation. In addition, the passaged CIN keratinocytes exhibited the same human papilloma virus (HPV) genotype that was detected in the original primary cells. K14 and K19 were expressed in the majority of the normal and CIN keratinocytes, whereas K17 and P63 were expressed only in high-grade CIN keratinocytes. The present study proposes a simple and practical method for rapidly obtaining highly purified naturally HPV-infected high-grade CIN keratinocytes from small neoplastic cervical tissues, and provides an appropriate first medium change time for the primary culture of CIN keratinocytes.
authors.affiliation: Department of Obstetrics and Gynecology, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261041, P.R. China.
authors.forename: Yu-Zhen
authors.initials: YZ
authors.lastname: Liu
authors.affiliation: Department of Obstetrics and Gynecology, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261041, P.R. China.
authors.forename: Tian-Tian
authors.initials: TT
authors.lastname: Wang
authors.affiliation: Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China.
authors.forename: You-Zhong
authors.initials: YZ
authors.lastname: Zhang
keywords: cell attachment; cell viability; cervical intraepithelial neoplasia; epithelial cells; keratinocyte; primary cell culture