We are isolating single cell suspension from breast tumor tissues by enzymatic digestion (trypsin digestion). But I am getting low cell count after passing the suspension through 40 micron filter, also alive cells are interfered by dead cells. I am interested in sorting cancer stem cells directly from breast tumor tissues without establishing primary cell cultures.
Looking forward for your valuable suggestions.
This is a very innovative idea, but I have not seen relevant literature reports. Generally, the tumor tissue is digested into single cells, and serum-containing cell culture medium is added for adherent growth. After the cells are attached, replacing with CSC culture medium. The obtained cells are subjected to microsphere culture, and the finally obtained microspheres are CSCs.
To isolate single cells from tissue is tricky, need to keep a good balance between yield and viability. We spent a lot of time to optimize the enzyme conc, digestive time and buffer, digestive condition(such as shakers temperature, time, speed) stoppage buffer, et al.
Other peoples reports are good a start, validation and optimization is tedious but necessary.
Cheers
Hi,
if you know surface markers of your target cells, you can isolate them with labeled magnetic beads. Miltenyi MACS or similar technique. Then you can grow these cells directly, without detachment from the beads. This can also can help to concentrate cells and also separate dead and live cells. Miltenyi has special kits for that. Low cell count probably means that your tissue digestion is not complete. Trypsin usually is not sufficient, try other enzymes, like pronase or some others.
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