Hello everyone,
I would like to know about the formation of spheroids (for cancer stem cells) in cancer cell lines using B27 supplement. If someone has succeeded in developing spheres in cancer cell lines using B27 supplement, kindly share the protocol or the related link for the same.
thank you,
I used to grow tumorspheres with B27.
I seed between 10.000-15.000 cells/ml in ultra low attachment cell plates (or agarose coated if not available) with B27 and EGF 20ng/ml.
Some suggestions:
- Wash serum, even small amounts of serum will reduce chances of sphere formation.
- Not every cell line form spheres, or at least not solid ones.
- Keep EGF in the freezer aliquoted for few uses, 2 or max 3 experiments. Freeze and thaw cycles will reduce EGF activity and spheres will not form.
- I used low attachment plates from Greiner bio one, those are not treated for cell attachment growth, however, some cell lines did attach. For those cell lines I used ultra low attachment or polyhema or agarose coated plates.
- If you use agarose coating (2% agarose), beware that sometimes cells are able to migrate through the agarose and attach to the bottom of the plate.
- You will see the spheres in few days, 2 or 3 days they will appear and you can count them after 5 to 8 days in general.
- Look the plates after seeding them and check that they are a single cell suspension.
I believe that it all I remember. You can check my papers for more info or pictures.
Hope it help!
i never used B27. If you like a protocol for cancer stem cells sphere formation (human melanoma in my case) we published this. It quite detailed, maybe could help
https://bio-protocol.org/e2233
Good luck, Michela
There are many methods simplest is over hanging drop, cell in inverted position using petri plate or 6 well plate. I recommend you 6 well plate. 20uL drop with 5000 cells. Spheroids will form After 2 days 48 hours. 3rd day it will proper and stablE. It will work.
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