Umbilical cord blood-derived natural killer cells (Celgene)

Umbilical cord blood (UCB) is obtained by cannulation of the umbilical vein and collection into a bag containing citrate-phosphate-dextrose and processing by red blood cell depletion using Hetastarch, followed by volume reduction. The sample is then washed with RPMI 1640 (without phenol red) containing fetal bovine serum (FBS; 5% v/v). Cell pellets are then resuspended at 5x10⁷ cells/ml in RoboSep buffer. DNase I (0.1mg/ml solution) is added to the cell suspension to a final concentration of 100μl/ml, mixed gently by pipetting and incubated for 15min at room temperature (RT) prior to isolation of NK cells using the EasySep® NK Cell Enrichment Kit. Human NK Cell Enrichment Cocktail (containing monoclonal antibodies to human cell surface antigens CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA-DR, and glycophorin A) is added to the cell suspension at a final concentration of 50μl/ml and incubated for 10min at RT. After incubation with EasySep® Magnetic Microparticles (final concentration of 100μl/ml) for 5min at RT, enrichment of NK cells is performed according to the protocol provided by the manufacturer. A CD56+CD3− population is collected and ready for further analysis or cultivation.