Roto therm

Tumor tissue was cut into 2 mm pieces in RPMI on ice with the connective tissue, blood vessels and blood clots, and necrotic tissue discarded. These pieces were placed into 50 mL Falcon tubes of RPMI (+2% penicillin/streptomycin), collagenase V (Worthington; 1 mg/mL), neutral protease (Worthington; 0.5 U/mL), and DNase (Roche; 1 μg/mL). The tissue was digested while rotating at 37°C (Benchmark Scientific Roto-Therm) until digestion was complete. Digested tissue was strained through a 200 μm strainer (Pluriselect) using a syringe plunger on the remaining undigested pieces. The digested tissue was then pushed through a 100 μm strainer (Thermo Fisher Scientific), and the cells were spun down at 300g for 5 minutes at 4°C. The supernatant was removed, and the pellet was depleted of RBCs via 10-second resuspension in 1 mL of water followed by 49 mL of 1× PBS. The cells were then spun down at 300g for 5 minutes at 4°C, resuspended in RPMI (+2% penicillin/streptomycin), and counted using a hemocytometer. The cells were then used for either a drug response screening, implantation into mice, or for in vitro experiments. Cells derived from PDX and used for in vitro experiments were subjected to mouse cell depletion according to the manufacturer’s instructions (Miltenyi Biotec).