In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy.
DC are professional APC 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 HPC have been developed recently. However, the various steps of their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. Our study focused on setting up such a full procedure, including collection of mononuclear cells (MNC) by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + autologous serum (SAuto) in sterile Teflon bags. The procedure was first developed with apheresis products from 7 healthy donors. Its clinical feasibility was then tested on 7 patients with breast cancer. The characteristics of monocyte-derived DC grown with SAuto (or in some instances with a pooled AB serum) were compared with those obtained in the presence of FBS by evaluation of their phenotype, their morphology in confocal microscopy, and their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). 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 SAuto-derived DC as efficient as those derived with FBS.
Cao
H
H
Inserm U76, Laboratoire de Thérapie Cellulaire et de Radioprotection Accidentelle, Paris et Fontenay Aux Roses, France.
Vergé
V
V
Baron
C
C
Martinache
C
C
Leon
A
A
Scholl
S
S
Gorin
N C
NC
Salamero
J
J
Assari
S
S
Bernard
J
J
Lopez
M
M
D017951:Antigen Presentation / Q000187:drug effects; D015703:Antigens, CD / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D018949:Antigens, CD1 / Q000096:biosynthesis / Q000187:drug effects; D001943:Breast Neoplasms / Q000097:blood; D002451:Cell Compartmentation / Q000276:immunology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects* / Q000276:immunology; D003470:Culture Media / Q000494:pharmacology; D016238:Cytapheresis; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000637:transplantation*; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006684:HLA-DR Antigens / Q000096:biosynthesis / Q000187:drug effects; D006801:Humans; D018793:Interleukin-13 / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation; D008297:Male; D018613:Microscopy, Confocal; D008863:Microspheres; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D010587:Phagocytosis / Q000187:drug effects; D010641:Phenotype; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism; D060149:Tetraspanin 30; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D015703:Antigens, CD; D018949:Antigens, CD1; C554586:CD63 protein, human; D003470:Culture Media; D006684:HLA-DR Antigens; D018793:Interleukin-13; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor