Hello,
I would be very grateful if anyone could help me with a problem described below:
I have looked through publications on culturing cancer cell lines with a drug to obtain drug-resistant cells. Hovewer, there are no information on how to deal with tumor initiating cells in this matter. Some companies provide cell lines which are positive for cancer stem markers, but they claim that this "stemness" lasts only for a few passages. Should I culture those cells with a drug (of course concentrations similar to those in plasma etc.) in 3D? How will I assess that they are resistant? - and which method is the best in your opinion?
Thank you again very much in advance for your help.
Hello,
I think you should maintain your cell lines as spheroids in cancer stem cell medium. Drug resistant cell lines will slowly lose their resistance if you passage them without selection stress. I will normally add drug to ~1-2x IC50(sensitive lines) to maintain resistant cells. If you are not sure, you can keep your cells with or without drug at the same time and then compare their resistance after several passages.
Hello,
I'm afraid that this is not the exact answer - I was asking how do you create resistant cancer 'stem' (stem-like) cells taking into consideration that they loose their stemness after only a few passages? Do you mean that you culture them in colonies/spheroids so that you do not passage them? Nonetheless, there is a definite amount of doublings after which those cells will loose their 'stem' markers. And for how long do you culture those cells? When exactly are you sure that they are resistant? Which tests do you perform to confirm that cells are drug-resistant? Thank you again in advance for your help.
In our lab, we did selected several drug resistant stem cells in vitro. We start with cells cultured in stem cell medium and increase the concentration of drugs slowly. Cells are first treated with drugs killing 50-80% cells. After cells growing up, maintain cell at this concentration for at least one passage before using higher concentration. Do not forget to freeze cells at every drug concentration. After several months of selection, we can get the resistant cells. I know you consider the lost of stemness. However, drug selection will select for very small population of cells even you culture them in stem cell medium. If you need "pure" stemness cells, you could sorting them out with specific stemness marker after you get the resistant cells. Survival and proliferation under higher dosage is the major phenomenons of resistant cells. To test the resistance of cells, I did IC50 assay or dose-response assay with AnnexinV-PI staining. If I am not answer your question right, please let me know.
Thank you very much for your answer! It has helped me a lot.
Kind regards,
ZFB
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