I have been trying to culture MCF7 spheroids by seeding 5000 cells in DMEM, in a low-adhesion 96 well-plate (Corning) and then centrifuging at 1000g for 10 mins. However, I am only able to get irregular cell clusters. The method has previously worked for me in obtaining uniform spheroids with other cell lines and I have also come across papers who use a similar method for MCF7. Could someone please tell me how the protocol can be improved for the generation of MCF7 spheroids?
We had consistent problems with MCF-7 (not for the procedure you indicate, but other procedures we put cells through) and it is perhaps that they are closer to "normal " than other equivalent lines (like T47D, MDA 231 etc.) . Their secreted matrix is presumably different. and so they don't conform as well as seeming equivalents (like T47D, MDA-231 etc..). Other groups may have introduced some additives to aid spheroid formation. Checkout DNase (and make sure you distinguish between the Mg++ dependent and indepent forms). It may be if you treat the cells quickly (2-5 mins max) with DNase before subjecting them to your procedure, it might work.
Thank so much for your response Pauline. I don't remember seeing any additives used in the other papers, but I will look into the possibility of such additives and treatments aiding spheroid formation. Thanks!
May be if you use 10000 cells/well, it might work, it worked for me using the same method u described. Good luck.
Thank you so much for your response, Walid. I've tried different cell seeding densities (including 10000 cells/well) but I encountered the same problem of irregular clusters. May I know how long it took for your spheroids to start appearing uniform? Thank you!
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