Dendritic cells (DC) are increasingly used as a vaccine. Unfortunately, a satisfactory cryopreservation of DC in the absence of FCS is not yet available, so that laborious repeated generation of DC from fresh blood or frozen peripheral blood mononuclear cells for each vaccination has been required to date. We now aimed at developing an effective cryopreservation method, and by testing several variables found that it was crucial to combine the most advantageous maturation stimulus with an improved freezing procedure. We generated monocyte-derived DC from leukapheresis products by using GM-CSF and IL-4 and showed that amongst several known maturation stimuli the cocktail consisting of TNF-alpha+IL-1 beta+IL-6+PGE(2) achieved the highest survival of mature DC. We then systematically explored cryopreservation conditions, and found that freezing matured DC at 1 degrees C/min in pure autologous serum+10% DMSO+5% glucose at a cell density of 10x10(6) DC/ml gave the best results. Using this approach 85-100% of the frozen DC could be recovered in a viable state after thawing (Table 1). The morphology, phenotype, survival as well as functional properties (allogeneic mixed leukocyte reaction, induction of influenza matrix or melan A peptide-specific cytotoxic T cells) of these thawed DC were equivalent to freshly prepared ones. The addition of CD40L or TRANCE/RANKL further improved DC survival. Importantly, we demonstrate that DC can effectively be loaded with antigens (such as Tetanus Toxoid, influenza matrix and melan A peptides) before cryopreservation so that it is now possible to generate antigen-preloaded, frozen DC aliquots that after thawing can be used right away. This is an important advance as both the generation of a standardized DC vaccine under GMP conditions and the carrying out of clinical trials are greatly facilitated.