Mammocult medium

The human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the Korea Cell Line Bank (KCLB, Seoul, Korea). Both cell lines were grown in Dulbecco’s Modified Essential Medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (HyClone), and were maintained at 37 °C in a humidified incubator containing 5% CO₂. We used breast cancer cell lines up to 15 passages after purchasing cell lines from KCLB and the cell lines were authenticated by the KCLB and showed >90% similarity in a short tandem repeats (STR) DNA fingerprint profile when compared with the American Type Culture Collection (ATCC) and Korean Cell Line Bank (KCLB) databases. Cells were plated at a density of 1 × 10⁶ cells in 10-cm culture dishes. To establish primary mammospheres, single-cell suspensions of MCF-7 and MDA-MB-231 cells were seeded at a density of (3.5∼4) × 10⁴ and (0.5∼1) × 10⁴ cells/well, respectively, in ultralow attachment six-well plates containing 2 mL of complete MammoCult^TM medium (StemCell Technologies; Vancouver, BC, Canada), which was supplemented with 4 μg/mL of heparin, 0.48 μg/mL of hydrocortisone, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. The cells were incubated for seven days in a 5% CO₂ incubator at 37 °C.

MCF-7 and MDA-MB-231 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, CA, USA), 1% penicillin and streptomycin in a humidified 5% CO₂ incubator at 37 °C. Breast cancer cells were cultured at a concentration of 3.5 × 10⁴ or 0.5 × 10⁴ cells/well in an Ultralow Adherent plate containing MammoCult^TM medium (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with heparin and hydrocortisone in a humidified 5% CO₂ incubator at 37 °C. A 6-well plate was scanned, and mammosphere counting was performed using the NICE program [17 (link)]. A mammosphere formation assay was determined by evaluating mammosphere formation efficiency (MFE) (%) as previously described [18 (link)].

Cancer cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). We followed a previously described method [18 (link)] and the breast cancer cells were incubated in Dulbecco’s modified essential medium (DMEM; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Hyclone, Logan, UT, USA). Breast cancer cells were incubated at 37 °C in 5% CO₂ incubator. Cancer cells were plated with 2 × 10⁶ cells in 10 cm culture dishes. For mammospheres culture, cancer cells were cultured with 3.5 × 10⁴ and 0.5 × 10⁴ cells/well with MammoCult^TM medium (StemCell Technologies, Vancouver, BC, Canada). The cells were incubated in a 5% CO₂ incubator. The complete MammoCult™ medium was supplemented with 4 μg/mL heparin, 0.48 μg/mL hydrocortisone, 100 U/mL penicillin, and 100 μg/mL streptomycin. At 7 days of culture, a 6-well plate was scanned and counted using the software program NICE [19 (link)]. For mammosphere formation assay, MFE (%) was determined as previously described [20 (link)].