Dear David

With however little experience I have of working on spheroids I would like to ask you your protocol to develop spheroids in brief. In case you are using 96 well ULA plates I can suggest seeding single cell per well and using the spheroids so developed to analyze the effect of drugs. Developing single cell derived spheroids is indeed a tough job but you'll find many research articles for assistance. Also, you may try using multiple cells per well say upto 25 and use a sorter to seed equal numbers. Keep the number of cells per well small and same. I am new to spheroid cultures, hope this helps.

Hi Yashika, thank you very much for your answer. Unfortunately we are generating organoids from non-disociated tissue of mice brain tumors, not from cell culture.

In brief, we induce the development of endogenous brain tumors in mice and microdisect then when reach terminal morbidity, then use pieces of this resected tumors to generate organoids. Even using a machine to slice the tumor tissue the resultant pieces vary in size enough that the resultant organoids aren't homogeneous. Since I can't control the initial cell number per organoid I would like to filter from my maintenance organoid culture the organoids with an appropriate size for the experiments while leaving the rest (bigger and smaller) in culture to continue growing/slicing them.

Thank you!

David