Unused tumour samples were also minced to small pieces and cryopreserved in DMSO, like PBMC [21]. The

establishment of cell lines that divided at least 20 times was successful only with samples from patients who had not yet received chemotherapy or radiation therapy. All cell lines originated from Caucasian patients. Isolation of immune cells.  PBMC were isolated from venous blood puncture or leukapheresis samples by density gradient centrifugation as described previously [21] using lymphocyte separation medium (LSM; PAA). Immune cells were either BMN 673 molecular weight used immediately or cryopreserved and stored in the nitrogen gas phase. Isolation, cryopreservation and thawing procedures as well as the use of optimized culture conditions (38.5 °C, 6.5% CO2) have been described in detail [21]. Activation of T cells in PBMC bulk cultures: CD3 activation and CAPRI cell generation.  Both methods started with the activation of T cells in PBMC bulk cultures using the CD3 monoclonal antibody OKT3 (Orthoclone; Cilag, Sulzbach/Taunus, Germany), which

binds to signaling pathway the non-polymorphic ε-chain of the CD3 molecule, and the addition of interleukin 2 (IL-2; Proleukin; Chiron, Ratingen, Germany). CD3 antibodies were immobilized at a concentration of 1 μg/ml in 0.05 M borate buffer pH 8.6 and distributed in 50-ml tissue culture flasks (Greiner cAMP Bio-One, Frickenhausen, Germany). Coated flasks were kept at 4 °C at least overnight and washed twice with phosphate-buffered saline prior to incubation with

PBMC. PBMC were added at a concentration of 2 × 106 cells/ml in a total volume of 10 ml, and IL-2 was added within 2–12 h at a concentration of 20 U/ml. CD3-activated cells were expanded on day 4 with IL-2 (20 U/ml) and harvested on day 7 for immediate use or cryopreservation. For the generation of CAPRI cells, CD3-activated PBMC were removed from the flask after 4–6 h, washed and then cocultured in a second CD3 ‘antibody-free’ flask with an equal number of unstimulated autologous PBMC, which contained naïve/resting T cells, at a concentration of 2 × 106/ml in a total volume of 10 ml. Cells were expanded on day 1 with IL-2 (20 U/ml) and harvested on day 4. Microscopic classification, preparation of tumour target cells and quantification of cancer cell destruction using the Cr51-release assay.  Cancer cells were removed from flasks by trypsinization, resuspended in culture medium (RPMI 1640 with l-glutamine; PAA) supplemented with 10% FCS and washed twice. Cancer cells were counted and distributed in different concentrations into 96-well flat-bottom culture plates (Falcon; Becton Dickinson, Heidelberg, Germany) either for microscopic evaluation of lysis or for the Cr51-release assay.