Cell Culture and Spheroid Formation

The colon carcinoma cell line HCT 116 was purchased from ATCC (Manassas, VA) and maintained in McCoy’s 5A cell culture media (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Thermo Scientific, Gaithersburg, MD) and 1% L-glutamine (Invitrogen, San Diego, CA). Cells were grown in 5% CO₂ at 37°C and passed every four days. Cell lines were used within three months after resuscitation of frozen aliquots thawed from liquid nitrogen. The cells were verified by Short Tandem Repeat (STR) sequencing in 2016.

Spheroids were prepared in an agarose-coated 96-well plate as previously described.^27–29 Cells were seeded into each well at a density of 7000 cells/well, incubated at 37°C and 50% of cell culture media was changed every 48 hours after four days in culture. After 13 days in culture, spheroids reach their maximum growth and were dosed with liposomal doxorubicin, free doxorubicin, or control media.