15353-j-veluchamy

5 Towards UCB-NK cells treatment in colorectal cancer | 131 line palliative chemotherapy consisting of oral capecitabine (1000 mg/m 2 , bid, day 1-14), i.v. oxaliplatin (130 mg/m 2 , day 1) and i.v. bevacizumab (7.5 mg/kg, day 1, in 4/10 mCRC patients). PBMC were isolated using Lymphoprep™ (STEMCELL Technologies, Cologne, Germany) density gradient centrifugation. CD56 + NK cells were isolated from PBMC using a MACS Human NK cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. PBNK cells purity and viability were checked using CD3 VioBlue, CD56 APC Vio 770, and CD16 APC (Miltenyi Biotech) and 7AAD (Sigma Aldrich, Zwijndrecht, The Netherlands). Isolated PBNK cells were activated overnight with 1000U/ ml IL-2 (Proleukin®; Chiron, München, Germany) and 10ng/ml IL-15 (CellGenix) for use in cytotoxicity assays. The parameters compared before and after stimulation with cytokines were NK purity (87 ± 5 % versus 84 ± 2%), NK CD16 + , 92 ± 12 % versus 88 ± 8%) and NK viability (89 ± 5 % versus 84 ± 8%), respectively. Flow cytometry The antibody staining mix for the assessment of NK cell functionality consisted of CD45 VioGreen, CD14 VioBlue, CD19 VioBlue and SYTOX ® Blue, together with CD3 PerCP-Vio 700 and TCRγδ PerCP-Vio700 to exclude dead cells, debris and non-NK populations from PBMC. NK cells were identified by the expression of CD45 + CD3 - CD56 + cells, and further characterized for NK functionality by plotting against CD16 APC, CD25 VioBrightFITC, CD107a PE, and NKp44 PE-Vio770 and for NK cell phenotype by plotting against NKG2A PE- Vio770, NKG2C PE, NKG2D PerCP-Cy5.5 and PanKIR2D FITC. All antibodies were supplied by Miltenyi Biotec except SYTOX ® Blue (Thermo Fisher Scientific, Berlin, Germany). UCB-NK cultures Allogeneic NK cells (UCB-NK) were generated fromcryopreserved umbilical cord blood (UCB) hematopoietic stem cells as previously described 25 . CD34 +  UCB cells from six UCB-donors were plated (4 x 10 5 /ml) into 12-well tissue culture plates (Corning Incorporated, Corning, NY, USA) in Glycostem Basal Growth Medium (GBGM ® ) (Clear Cell Technologies, Beernem, Belgium) supplemented with 10% human serum (HS; Sanquin Bloodbank, Amsterdam, The Netherlands), 25ng/mL of SCF, Flt-3L, TPO, and IL-7 (CellGenix, Freiburg, Germany). In the expansion phase II, from day 9 to 14, TPO was replaced with 20ng/mL IL-15 (CellGenix). During the first 14 days of culture, low molecular weight heparin (LMWH) (Clivarin ® ; Abbott, Wiesbaden, Germany) in a final concentration of 20µg/ml and a low-dose cytokine cocktail consisting of 10pg/ml GM-CSF (Neupogen), 250 pg/ml G-CSF and 50 pg/ml IL-6 (CellGenix) were added to the expansion cultures. Cells were refreshed with new medium twice a week and maintained at 37°C, 5% CO 2 . On day 14, the NK cell differentiation process was initiated by addition of NK cell differentiation medium consisting of the same basal medium with 2% HS but with high-dose cytokine cocktail consisting of 20ng/ml of IL-7, SCF, IL-15 (CellGenix)