Beads coated with anti cd3 and anti cd28

PBMCs from healthy donors or NSCLC patients were isolated by Ficoll-Paque Plus (GE Healthcare). Isolated cells were grown for 24 h or 5 days, in complete medium composed by RPMI 1640 containing human AB serum (10%), Ultraglutamine I (1%), penicillin and streptomycin (1%) along with beads coated with anti-CD3 and anti-CD28 (Life Technologies) at a ratio of 1 bead per 10 cells. Cells were cultured in presence or absence of MEK-I selumetinib at 0.01 uM concentration.

Human samples and biopsies were collected after obtaining a written informed consensus from patients in accordance with the Declaration of Helsinki. The use of these samples for research purposes was approved by our local Ethical Committee and all patients gave their written informed consent to the use of the tumor sample. All below described methods were performed in accordance with guidelines and regulations. PBMCs from NSCLC patients were isolated by Ficoll-Paque Plus (GE Healthcare). Isolated cells were grown for 24 h or 5 days in complete medium composed by RPMI 1640 containing human AB serum (10%), Ultraglutamine I (1%), penicillin and streptomycin (1%) along with beads coated with anti-CD3 and anti-CD28 (Life Technologies) at a ratio of 1 bead per 10 cells. We developed a protocol to generate ex-vivo 3D cultures from lung cancer patient samples. All fresh tumor tissue samples were kept on ice and processed in sterile conditions on the day of collection. Tissue fragments were digested as previously described [6 (link)] in a 37 °C shaker at low to moderate speed (e.g. 200 rpm) for incubation time for 1-2 h and cells were separated with serial centrifugation. For 3D cultures, cells were seeded in complete medium with 5% matrigel in order to preserve three-dimensional structure.