title: In vitro production of dendritic cells from human blood monocytes for therapeutic use.
abstract: Dendritic cells (DC) are professional antigen-presenting cells that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34(+) pluripotent hematopoietic progenitor cells have been now developed. For this purpose, their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. In the present review, 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 characteristics of monocyte-derived DC grown in these various conditions varied mainly regarding their phenotype and their morphology in confocal microscopy, whereas no significant differences were found in their capacity to phagocytize latex 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 degrees C mechanical freezer or using a classical liquid nitrogen controlled-rate freezer at -1 degrees C/min) in a solution containing 10% dimethyl sulfoxide (Me(2)SO) and 2% human albumin in doses of DC available for several infusions. The mean recoveries after freezing and thawing were not statistically different (around 70%). The immunophenotype of DC, as well as the T lymphocyte-stimulating capacity, were not modified by the freezing--thawing procedure. The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined DC. Clinical trials using DC already published will be discussed.
authors.affiliation: Inserm U76, Laboratoire de Thérapie Cellulaire et de Radioprotection Accidentelle, Paris et Fontenay Aux Roses, France.
authors.forename: L
authors.initials: L
authors.lastname: Garderet
authors.forename: H
authors.initials: H
authors.lastname: Cao
authors.forename: J
authors.initials: J
authors.lastname: Salamero
authors.forename: V
authors.initials: V
authors.lastname: Vergé
authors.forename: E
authors.initials: E
authors.lastname: Tisserand
authors.forename: S
authors.initials: S
authors.lastname: Scholl
authors.forename: N C
authors.initials: NC
authors.lastname: Gorin
authors.forename: M
authors.initials: M
authors.lastname: Lopez
mesh_terms: D000276:Adjuvants, Immunologic / Q000627:therapeutic use; D018929:Cell Culture Techniques / Q000379:methods; D015925:Cryopreservation; D016238:Cytapheresis; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000648:ultrastructure; D006801:Humans; D016130:Immunophenotyping; D007959:Lymphocyte Culture Test, Mixed; D018613:Microscopy, Confocal; D009000:Monocytes / Q000166:cytology*; D010587:Phagocytosis
chemical_list: D000276:Adjuvants, Immunologic