A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use

Dendritic cells (DC) are increasingly used as a vaccine. Unfortunately, a s atisfactory cryopreservation of DC in the absence of FCS is not yet availab le, so that laborious repeated generation of DC from fresh blood or frozen peripheral blood mononuclear cells for each vaccination has been required t o date. We now aimed at developing an effective cryopreservation method, an d by testing several variables found that it was crucial to combine the mos t advantageous maturation stimulus with an improved freezing procedure. We generated monocyte-derived DC from leukapheresis products by using GM-CSF a nd IL-4 and showed that amongst several known maturation stimuli the cockta il consisting of TNF-alpha+IL-1 beta+IL-6+PGE(2) achieved the highest survi val of mature DC. We then systematically explored cryopreservation conditio ns, and found that freezing matured DC at 1 degreesC/min in pure autologous serum+10% DMSO+5% glucose at a cell density of 10x10(6) DC/ml gave the bes t results. Using this approach 85-100% of the frozen DC could be recovered in a viable state after thawing (Table 1). The morphology, phenotype, survi val 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 dem onstrate that DC can effectively be loaded with antigens (such as Tetanus T oxoid, influenza matrix and melan A peptides) before cryopreservation so th at it is now possible to generate antigen-preloaded, frozen DC aliquots tha t after thawing can be used right away. This is an important advance as bot h the generation of a standardized DC vaccine under GMP conditions and the carrying out of clinical trials are greatly facilitated. (C) 2000 Elsevier Science B.V. All rights reserved.