My drugs are insoluble in water and 5% alcohol, 5% DMSO as well. It fully dissolves in ethyl acetate however it is toxic for cells.
There are several organic solvents that have been used in in vitro mammalian cell assays. Typically 1% final concentration of the solvent in the culture medium is used, but you should validate the maximum percentage that is compatible with your cells and assay system. Try dissolving your drug in the solvent before adding to the culture medium rather than attempting to dissolve it in diluted solvent. You can try DMSO, ethanol, methanol, THF, acetone, and others that you can find using an internet search. Also check other questions on Researchgate, such as this one.
https://www.researchgate.net/post/In_cell_culture_what_is_the_appropriate_solvent_for_a_drug_other_than_DMSo
Good luck, Peter
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