Method for enriching and screening tumor stem cells
A technology for cancer stem cells and screening methods, which is applied in the fields of tumors/cancer cells, biochemical equipment and methods, animal cells, etc. Effect
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Embodiment 1
[0053] Example 1 Combined sorting of renal cancer cell line ACHN tumor stem cells (CD133 / CD24 positive double selection)
[0054] 1. Multi-generation sphere culture
[0055] 1) Cultivate ACHN tumor cells until the bottle is 80-90% full, digest and collect the cells, and the single cell suspension is 5×10 4 cells / ml were inoculated into 6-well ultra-low adsorption cell culture plates, and each well was cultured with 2 mL of SFMD medium;
[0056] 2) 37°C, 5% CO 2 Cultivate for 96 hours, add 1mL culture medium every other day;
[0057] 3) After 96 hours, at 37°C, 5% CO 2 Digest with 0.05% trypsin in the cell culture incubator for 5 minutes, the tumor spheres become loose, add 2 mL of SFMD medium to stop digestion, pipette evenly, take 10 μL of uniform cell suspension for cell counting, continue to press 5×10 4 Cells per ml were planted in a new 6-well ultra-low adsorption cell culture plate to start P2 generation sphere culture;
[0058] 4) Repeat the above steps 1) to 3) to c...
Embodiment 2
[0130] Example 2 Joint sorting of bladder cancer cell line 5637 tumor stem cells (CD133 / CD44 positive double selection)
[0131] 1. Multi-generation sphere culture
[0132] 1) Cultivate 5637 tumor cells until the bottle is 80-90% full, digest and collect the cells, and press 5×10 single-cell suspension 4 cells / ml were inoculated into 6-well ultra-low adsorption cell culture plates, and each well was cultured with 2 mL of SFMD medium;
[0133] 2) 37°C, 5% CO 2 Cultivate for 96 hours, add 1mL culture medium every other day;
[0134] 3) After 96 hours, at 37°C, 5% CO 2 Digest with 0.05% trypsin in the cell culture incubator for 5 minutes, the tumor spheres become loose, add 2 mL of SFMD medium to stop digestion, pipette evenly, take 10 μL of uniform cell suspension for cell counting, continue to press 5×10 4 Cells per ml were planted in a new 6-well ultra-low adsorption cell culture plate to start P2 generation sphere culture;
[0135] 4) Repeat the above steps 1) to 3) to c...
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