I am growing cancer stem cells in the form of spheroid culture using ultra low attachment dish and cancer stem cell specific media. I generally grow it for 14 days and then harvest the cells. On the 6th day, I give it a spin at 49g for 5 minutes to exclude the dead cells. While harvesting the cells, centrifugation at 49g is not sufficient to exclude the entire dead cell population. I have also tried using Histopaque but even this method doesn't appear very effective. It would be very helpful if I could get some suggestions on harvesting spheroid cells by effectively removing the dead cells. Thanks.
Hi, Ananya.
Any cancer cells continuously die during proliferation on plate as well as spheroid culture. That is a spontaneous cell death occurring in vitro situation. So spheroids grown in culture already contain many dead or dying cells. If you can dissociate spheroids into single cell population (that is not an easy task, too), spread the single cells suspension onto gelatin-coated culture dish for 1, 2, 3, 4h experimentally. The selected cell attachment will be as a routine procedure. Observe the extents of cell attachment, tapping the dish after 3~4 times of gentle wash will help detach or disconnect the loosely attached dead or dying cells to eliminate them by washing cells again. You can collect cells again for your analysis.
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