Recombinant human gm csf rhgm csf

Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors by density gradient centrifugation, and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) containing 10% FBS and penicillin/streptomycin. Following 4 h of culture, the suspended cells (T cells) were grown to generate CIK cells in RPMI-1640 with 10% FBS containing 500 ng/mL anti-CD3 antibody, 100 U/mL IFN-γ (Servicebio, Wuhan, China) and 10 μg/mL polyhydroxyalkanoates (Solarbio, Beijing, China). In addition, adhered cells were used for dendritic cell differentiation via culturing in RPMI supplemented with 1000 U/mL recombinant human GM-CSF (rhGM-CSF; R&D, MN, US) and 500 U/mL rhIL-4 (R&D, MN, US) for 7 days. Next, A549 and HepG2 cells were lysed (Repeated freeze-thaw procedure), and the supernatant was obtained as the tumor antigen. On day 8, the supernatant and 10 ng/mL TNF-α and 10 ng/mL IL-1β were added to the DCs medium, after which the culture was maintained for two more days. Then, DCs and CIKs were co-cultured at a ratio of 1:10 for two days. On day 12, we added the Nb36 to DC-CIK cells to mediate CTLA-4 blockade. The study were approved by the local ethics committee of Hainan Medical University.