Objective   To investigate an effective method to enrich tumor stem cells by comparing the efficiency of the 3 cell isolation methods.    Methods   CW-2 stem cells were enriched with 3 different methods

including simple serum-free medium

serumf

ree medium combination with oxaliplatin

and the second one further combined with flow cytometry (multi-combination). The enrichment efficiency of the 3 methods was then analyzed and compared with flow cytometry

tumorigenicity assay in NOD-SCID mice and Transwell invasion assay in vitro.  The expressions of CD44 and EPCAM were determined by immunohistochemistry method.    Results   The frequency of CD44

+

EPCAM

+

cells were 57.39%

73.85 % and 89.57% separately in cells enriched by simple serum-free medium

serum-free medium combination with oxaliplatin and multi-combination; The tumor xenograft of cells enriched by multi-combination were formed earliest and grew fastest than the tumor xenograft of cells enriched by pure serum-free medium and serum-free medium combination with oxaliplatin. The volume of transplantation tumor from cells enriched by multi-combination were (209.25±31.54)

(500.00±72.90)

(852.75±157.51)

(2 280.00±440.91)

(4 897.00±1 154.15)

(11 070.00±3 559.86) mm

3

separately on the 4th

7th

9th

14th

21st and 28th day. Tumor-forming capability of the 3 group cells had statistically significant differences (P0.05). The number of cells past through man-made GBM to down-room were (98±7) cells per field view

(135±7) cells per field of view

and (188±6) cells per field of view separately in cells enriched by simple serum-free medium

serum-free medium combination with oxaliplatin and multi-combination. Invasion capability of the 3 group cells had statistically significant differences (P0.05). So the cells enriched by multi-combination had most tumor stem cells than the cells enriched by other 2 methods. CD44 and EPCAM all expressed  in each group of tumor xenograft.     Conclusion   Multi-combination is highest than pure serum-free medium and serum-free medium combination with oxaliplatin in enrichment capability of tumor stem cells and is a better method to enrich tumor stem cells.