PL.84 Short-Term Culture of Human Ecto-Cervical Epithelial Cells for Genomic, Proteomic and Functional Studies. (26th April 2013) - Lashkari, BS
Abstract : Background: Understanding cell physiology is limited by reliance on tumour-derived immortalised cell lines. Primary cell culture models may offer more relevant mechanistic insight into cell physiology but are often difficult to establish and maintain. Aims: We sought to develop an optimal method for the isolation and short-term culture of Human primary Ecto-Cervical Epithelial Cells (HECECs). Methods and Material: Fresh ecto-cervical tissues were obtained at hysterectomy and epithelia isolated and cultured (using MEM D-Valine media to prevent fibroblast proliferation) using three explants methods: i) tiny fragments of epithelium; ii) dissociated cells cultured after digestion using Collagenase IV and trypsin; and iii) digested tissue clumps. The epithelial phenotype of cultured cells was verified by double immunofluorescence sequential staining to detect cytokeratin, specific antigen for epithelial cells. The expression of oestrogen (ERα, ERβ) and progesterone receptors (mPRα, mPRβ, PRγ and nPRA&B) genes were investigated by RT-PCR. Flow cytometry was employed to detect TLR2 and TLR4, receptor targets for our proposed of pattern recognition in the cervix. Results: Cultures were successfully established using all three methods but cell growth was best from digested tissue clumps, which was employed for subsequent experiments. Primary cells were sub-cultured at least twice. Exclusion of fibroblasts from cultures was confirmed by absence of staining to CD90. We confirmed the expression of all ER and PR genes, as well as TLR2, TLR4 in HECECs. Conclusion: HECECs cultured from explants of digested tissue clumps, employing our protocol, yield pure epithelial cell populations, uncontaminated by stromal fibroblasts, suitable for molecular investigations.