Primary Isolation,Culture And Identification Of Cancer Stem Cells From GBC-SD Cell Line

Backgrounds:Gallbladder Carcinoma is a malignant tumor of high invasiveness.Currently,there are no effective treatments.The tumor stem cell theory provides new insights into the treatment of gallbladder carcinoma.Objectives:To confirm the existence of gallbladder cancer stem cells in GBC-SD cell line;To establish an effective method in isolation of gallbladder cancer stem cells and to isolate,culture and identify gallbladder cancer stem cells.Methods:The primary GBC-SD cells were treated with anticancer agents Cisplatin and the sorted cells were transplanted into Nude mice to observe their tumorigenic power.The formed tumors suffered another treatment of Cisplatin in vivo.The tumor tissue left were cultured in serum-free medi urn with growth factors to observe sphere format ion,which was fol lowed by treatment of Cisplatin in vitro.Then,several stem cell genes both in finally selected cells in this experiment and primary GBC-SD cells were searched by RFQ-PCR and the result were compared with each other.Results:1) The survived cells included tumorigenic cells which could form tumors at 80%of the injected sites;2) The bulk of the xenograft was reduced by Cisplatin and those tumor cells left could propagate and form clonic spheres in serum-free medium;3) The spheres and suspension cells were treated with Cisplatin 30umol/L again.And spheres could still formed in serum-free medium later;4) The expression of stem cell genes in final selected cells was much higher than in GBC-SD cells.Conclusions:1) The existence of cells wi th characteristics of cancer stem cells in GBC-SD cell line could be confirmed in this experiment;2) The method that sorting out gallbladder cancer stem cells by the combination of serum-free and suspension culture and chemotherapeutic agents in both vitro and vivo is viable.