Tumor Tissue Dissociation and Cell Isolation

Tumor tissues were obtained within 1 to 2 h after surgical removal, washed in sterile Dulbecco’s phosphate-buffered saline (PBS) (L1825-BC – Merck Millipore, Italy), and mechanically minced into small pieces (2 to 4 mm). Minced samples were digested using a tumor dissociation kit in a disposable gentle MACS™ C-Tube (Miltenyi Biotec, Italy) according to the manufacturer’s instructions. Samples were digested for 60 min at 37°C in a gentle MACS Octo dissociator, filtered through 70-μm sterile cell strainers, centrifuged at 300 × g for 5 min, and resuspended in a DMEM and HAM’S F12 media mixture (2:1) (Gibco) containing 50 IU/mL penicillin-streptomycin and 4 mM glutamine. Viable cells were counted using an optic phase-contrast microscope [7 (link)].