Isolation and subculture of human esophageal squamous epithelial cells

    ZHANG Ru, GONG Jun, WANG Hui, WANG Li, LEI Juan, RAN Liª²Wei

    Department of Gastroenterology, Second hospital, Xi¬ðan Jiao Tong University, Xi¬ðan 710004, Shaanxi Province, China

    ¡¾Abstract¡¿ AIM: To investigate the optimal method for the culture of highly purified human normal esophageal epithelial cells and to yield significant cell numbers. METHODS: Cells were isolated from tissues by dispase digestion andtrypsinization and then primarily cultured and subcultured in serumª²free keratinocyte medium (Kª²SFM) or fetalª²bovineª²serum (FBS)ª²supplementary medium (Kª²SFM with 100 ml/L FBS). The biological characteristics of the cells were observed through cell attachments and growth kinetic curves and were confirmed immunohistochemically using antiª²human monoclonal antibody of the cytokeratin 14 (CK14), cytokeratin 13 (CK13) and vimentin. RESULTS: Fibroblasts were not seen in all the cultures. Population doubling time (PDT) in Kª²SFM was (51.5¡À11.5) h (n=3), but it could not be calculated in Kª²SFM with 100 ml/L FBS. The adherent cells significantly increased in Kª²SFM (P<0.01). The percentages of CK14ª²positive cells in Kª²SFM were significantly higher at any comparable periods (P<0.05), but decreased with the prolongation of the growth time in these 2 kinds of media. Contrarily, the percentages of CK13ª²positive cells were lower and increased. Vimentin was negative in all the cultures. CONCLUSION: To culture and proliferate human normal esophageal squamous epithelial cells productively in vitro, dispase digestion and serumª²free keratinocyte medium are feasible and credible methods. Serum may induce the differentiation of the sells. ......