【Title】

Isolation and identification of renal carcinoma stem cells in serum free medium

【Source】

Journal of Clinical Rehabilitative Tissue Engineering Research  August 6, 2009  Vol.13 No. 32，6284-6288

【Author】

Pan Peng, Guo Tao, Tian Fu-qi, Sun Hao, Ma Ke-jun, Xu Qing

【Setting】

Department of Urology Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang  212001, Jiangsu Province, China

【Found】

Supported by: the Scientific and Technological Planning Project of Zhenjiang City, No.SH2007024*

【Abstract】

BACKGROUND: The proposition of renal cell carcinoma (RCC) stem cell theory put forward the study underlying genesis, development and pathogenesis of tumor, which provide a newly approach to tumor research. Specific tumor stem cell has been separated and identified with serum-free culture. Presently, serum-free has been the classical method for tumor stem cell culture.

OBJECTIVE: To obtain renal cell carcinoma (RCC) cells line with serum free medium (SFM) culture, in addition, to identify the cells.

DESIGN, TIME AND SETTING: The in vitro cytology experiment was conducted at the Jiangsu University between February  2008 and February 2009.

MATERIALS: RCC cells line OS-RC-2 was provided by Cell Bank of Chinese Academy of Sciences.

METHODS: RCC cells was collected in logarithmic growth phase and dissociated into single cell, then they was seeded in DMEM/F12 serum free medium containing 20 μg/L epidermal growth factor (EGF), and 20 μg/L basic fibroblast growth factor (bFGF). After generation, the growing process of cells was observed, and the expression of CD133 and CD34 in cells from SFM was detected by flow cytometry at days 7 after culture. RCC cells seeded with serum free DMEM/F12 medium were served as control group. At last the cells from SFM were recultivated in 10% fetal bovine serum (FBS) medium, and the growth state was observed.

MAIN OUTCOME MEASURES: The growth of RCC stem cells was observed by an inverted microscopy. The expression of CD133 and CD34 was detected by flow cytometry. Meantime, differentiation of conventional cultured RCC cells after 7 days of SFM culture was observed.

RESULTS: ①The RCC cell spheres formed at 2 days after culture in SFM with cell factors, and the spheres volume became larger and their numbers became increased after 7 days. In the control group, RCC cells grew adhesion to walls without cell spheres formed. ②The flow cytometry revealed that expressions of CD133⁺CD34^- were (1.24±0.36)% in RCC cells, which reached to (8.33±1.26)% in the control group (t =-19.71, P < 0.05). ③After the cell spheres were recultivated in 10% FBS supplemented medium and they were induced to differentiate and adherent to the bottom of the culture bottles after 2 days, and the expression of CD133⁺CD34 was identical to the control group.

CONCLUSION: RCC stem cells can be enriched from RCC cells cultured with SFM containing EGF and bFGF.

【Number of pictures】

2 figures

【Full text】

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