Since not too many human uterus cervical squamous cell carcinoma (CSCC) cell lines in existance, efficient isolation, culture and purification protocols for primary CSCC cells were optimized as a tool for the study of uterus CSCC.
The protocols for partial multiple enzymatic digestion and explant cell culture were combined and then the resulting mixed cell component cultures were purified by magnetic activated cell sorting. Colony-forming assay was utilized for detection of cell carcinogenesis potential and immunofluorescence were used to detect protein expression of CSCC. Finally, flow cytometry (FCM) was performed to analyze cancer stem cells (CSCs) phenotypic markers as well as programmed cell death ligand 1(PD-L 1).
Freshly isolated cells containing tumor cells and cancer-associated fibroblasts (CAFs) efficiently proliferate to 85% confluence on a 6 cm petri dish in 5-7 days. Anti-Epithelial Cell Adhesion Molecule antibody (EpCAM) microbeads were used to successfully separate a homogeneous subpopulation of epithelial tumor cells. Both EpCAM+ and EpCAM- cell subpopulations were able to be passaged more than 30 times. Proportions of tumor cell populations expressed CSCs markers such as CD133, CD24, aldehyde dehydrogenase 1 (ALDH1) and CD44. The vimentin+&EpCAM- population, defined with CAFs, could express CD146 mesenchymal stem cells marker. Meanwhile, PD-L 1 was identified in most subpopulation of CD44+ cells at low passage numbers.
Efficient isolation, culture and purification protocols for primary CSCC cells were successfully built. Additionally, the profiling of CSCs cell markers might provide promising therapeutic targets and clinic strategies.

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