Efficient elutriation of monocytes within a closed system (Elutra) for clinical-scale generation of dendritic cells.
Dendritic cells (DC) are promising tools for the immunotherapy of cancer. The induction of tumor-specific T cells and clinical regressions have already been observed in early phase I/II vaccination trials. As DC vaccination is now facing trials with larger patient collectives it becomes increasingly important to obtain large numbers of cells suitable for therapeutic applications under labor- and cost-effective conditions. We describe here a procedure that uses a novel cell separator (Elutra, Gambro BCT) to enrich monocytes from an entire apheresis product within one hour. Cells are separated on the basis of size and to a lesser extent density, by elutriation in a 40-ml conical chamber. The total monocyte recovery following elutriation (n = 6) was 98.53% (+/-8.07%), the recovery in the monocyte-rich fraction 75.45% (+/-11.31%), and the mean purity 82.95% (+/-6.01%). These monocytes can be cultured either in conventional culture dishes or in closed cell culture bags and differentiated, by using GM-CSF+IL-4 followed by a maturation cocktail composed of IL-1beta+IL-6+TNF-alpha+PGE2, into fully mature DC. The Elutra separator allows for fast and easy enrichment of monocytes within a closed system. Subsequently, elutriated monocytes can be successfully cultured into phenotypically and functionally mature DC for immunotherapeutic approaches. The method neither requires a density gradient step to enrich PBMC from leucapheresis products nor does it apply (xenogeneic) antibodies to target monocytes. Isolation of monocytes with Elutra may greatly facilitate future DC-based vaccination approaches.
Berger
Thomas G
TG
Department of Dermatology, University Hospital of Erlangen, Hartmannstr. 14, D-91052 Erlangen, Germany. thomas.berger@derma.imed.uni-erlangen.de
Strasser
Erwin
E
Smith
Richard
R
Carste
Curt
C
Schuler-Thurner
Beatrice
B
Kaempgen
Eckhart
E
Schuler
Gerold
G
D002454:Cell Differentiation; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology*; D006801:Humans; D007937:Leukapheresis; D009000:Monocytes / Q000166:cytology*