Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use

In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8 °C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs.

Flow cytometric dot plots of Ad-RTS-hIL-12-transduced DCs following 3 h of incubation with vector, washing, and 18-20 hour incubation with activator ligand. Shown is a dot plot of CD11c x intracellular IL-12 staining.Highlights► Averaged a 7.7% decrease in viability compared to before cryopreservation. ► Statistically identical in antigenicity in MLR; phenotype and activation. ► Have statistically identical transgene expression based on IL-12 secretion. ► Can be held in cryoprotectant for up to 75 min. at 2-8 °C before freezing.