In vitro production of dendritic cells from human blood monocytes for therapeutic use

Dendritic cells (DC) are professional antigen-presenting cells that are pro mising adjuvants for clinical immunotherapy. Methods to generate in vitro l arge numbers of functional human DC using either peripheral blood monocytes or CD34(+) pluripotent hematopoietic progenitor cells have been now develo ped. For this purpose, their in vitro production for further clinical use n eed to fit good manufacturing practice (GMP) conditions. In the present rev iew, we give our experience of such a procedure: it includes collection of mononuclear cells by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + human serum (autologous patient 's serum or AB serum) or in a serum-free medium (AIM V). The characteristic s of monocyte-derived DC grown in these various conditions varied mainly re garding their phenotype and their morphology in confocal microscopy, wherea s no significant differences were found in their capacity to phagocytize la tex particles and to stimulate allogeneic (MLR) or autologous lymphocytes ( antigen-presentation tests). The DC were also cryopreserved in bags (either by putting the bags directly in a -80 degreesC mechanical freezer or using a classical liquid nitrogen controlled-rate freezer at -1 degreesC/min) in a solution containing 10% dimethyl sulfoxide (Me2SO) and 2% human albumin in doses of DC available for several infusions. The mean recoveries after f reezing and thawing were not statistically different (around 70%). The immu nophenotype of DC, as well as the T lymphocyte-stimulating capacity, were n ot modified by the freezing-thawing procedure. The results obtained demonst rate that the experimental conditions we set up are easily applicable in cl inical trials and lead to large numbers of well-defined DC. Clinical trials using DC already published will be discussed.