Isolation and Culture of Triple-Negative Breast Cancer Cells

Triple-negative human breast cancer cells were isolated and cultured as previously reported (53 (link)). Briefly, tumor tissue was mechanically cut into small pieces (<1 mm³) using scalpels and enzymatically dissociated in a mixture of collagenase/hyaluronidase (STEMCELL Technologies, 07912) for 16 hours at 37°C, followed by further digestion with trypsin (0.25%) for 2 minutes and then 5 units/mL of dispase (STEMCELL Technologies, 07913) and 0.05 mg/mL of DNase I (STEMCELL Technologies, 07900) for 1 minute. After centrifugation at 150g for 5 minutes, cells were seeded at a density of 1 × 10⁵/well onto Geltrex-coated (23 μg of protein per 1 cm²; Thermo Fisher Scientific, A1413202) 6-well plates in human complete EpiCult-B medium (STEMCELL Technologies, 05602, 05630, and 07925). Cells were either further cultured or passaged using trypsin-EDTA (0.25%) for downstream usage.